Lung cancer is one of the most malignant cancers around the world, with high morbidity and mortality. Metastasis is the leading cause of lung cancer deaths and treatment failure. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs), two groups of small non-coding RNAs (nc-RNAs), are confirmed to be lung cancer oncogenes or suppressors. Transforming growth factor-β (TGF-β) critically regulates lung cancer metastasis. In this review, we summarize the dual roles of miRNAs and lncRNAs in TGF-β signaling-regulated lung cancer epithelial-mesenchymal transition (EMT), invasion, migration, stemness, and metastasis. In addition, lncRNAs, competing endogenous RNAs (ceRNAs), and circular RNAs (circRNAs) can act as miRNA sponges to suppress miRNAs, thereby mediating TGF-β signaling-regulated lung cancer invasion, migration, and metastasis. Through this review, we hope to cast light on the regulatory mechanisms of miRNAs and lncRNAs in TGF-β signaling-regulated lung cancer metastasis and provide new insights for lung cancer treatment. Lung cancer is the most common cancer based on current diagnoses and the leading cause of cancer-related deaths globally . AccordiMicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are small non-coding RNAs (nc-RNAs) without the potential to encode proteins . NeverthIn this review, we summarize the positive and negative roles of lncRNAs and miRNAs in TGF-β signaling-regulated lung cancer metastasis, hoping to provide new insights for the design of more efficient therapies for lung cancer treatment in the future.MiRNAs, 18~25 nucleotides in length, can bind to miRNA response elements (MREs) on the target mRNA 3′UTR and silence target genes through mRNA degradation or by suppressing mRNA translation . NotablyLncRNAs are another class of nc-RNAs with more than 200 nucleotides in length. In the cell nucleus, lncRNAs can modify chromatin structures by interacting with polycomb repressive complex 2 (PRC2), enhance or reduce gene transcription by recruiting transcription factors, and regulate miRNA processing . In the TGF-β mediates the EMT process mostly via the Smad  pathwaysTGF-β can induce Snail, Slug, and ZEB2 expression to decrease E-cadherin, resulting in breaking adherens junctions . TGF-β cInvadopodia can degrade the surrounding extracellular matrix (ECM) through matrix-degrading proteinases, such as membrane type 1 matrix metalloprotease (MT1-MMP), MMP2, MMP9, cathepsin B, and a disintegrin and metalloprotease 12 (ADAM12), thus propelling cell invasion ,45. In HLung tumor cell intravasation is involved in migrating to and then passing through the vessel wall. Tumor cells not only secrete TGF-β1 but also bind to Notch ligands on endothelial cells (ECs) by using Notch receptors, thus facilitating their transmigration through the endothelial tight junctions . TGF-β cLung CTCs can produce the immune suppressors interleukin-10 (IL-10) and TGF-β. Moreover, TGF-β can increase circulating CD4+CD25+ regulatory T cells (Tregs) via the cyclooxygenase-2 (Cox-2)/prostaglandin E2 (PGE2) signaling pathway. These Tregs can suppress the proliferation of CD4+ T cells, thus driving CTCs to escape from immune attacks  49]. In. In49]. CTCs are usually arrested in narrow capillaries, where they can be trapped because of the size restriction and then extravasate . In A549MiR-93 is up-regulated in NSCLC tissues . The neuMiR-128-3p is highly expressed in NSCLC tissues compared to adjacent normal tissues. It can inhibit SMURF2 and the catalytic subunit of protein phosphatase 1 (cPP1) expressions by targeting their mRNA 3′UTRs, eventually activating the TGF-β signaling pathways to promote EMT, metastasis, and chemoresistance. Further, high miR-128-3p expression can indicate poor NSCLC patient survival .MiR-9 is up-regulated in NSCLC cell lines and can be up-regulated by TGF-β1. MiR-9 can promote TGF-β-induced EMT by inhibiting E-cadherin in A549 cells . In addiMiR-134 and miR-487b are both located on chromosome 14q32, and both of them can be activated by TGF-β1 during EMT progression in lung adenocarcinoma. Moreover, over-expressed miR-134 and miR-487b can promote TGF-β1-induced EMT by suppressing membrane-associated guanylate kinase, WW and PDZ domain-containing protein 2 (MAGI2)  in A549MiR-330-3p is up-regulated in NSCLC tissues . In NCI-MiR-1246 expression has been shown to be markedly increased in NSCLC and SCLC patients’ serum by quantitative real-time polymerase chain reaction (qRT-PCR). It can decrease E-cadherin, as well as increase vimentin and TGF-β, contributing to promoting EMT in A549 cells .MiR-9-5p levels are higher in NSCLC tissues and cell lines compared to normal ones. It has been revealed that decreased receptor effects can abrogate TGF-β suppressive functions, thereby showing its oncogenic roles in NSCLC ,70. MiR-in vivo. Moreover, up-regulated miR-181b-5p can predict poor outcomes in NSCLC patients [Cancer stem cells (CSCs) possess self-renewal properties and have been demonstrated to be a small population of cells within lung cancer tissues, which can promote lung tumor metastasis . MiR-181patients .CDH1 (a gene that can encode E-cadherin) and then suppress E-cadherin expression, eventually enhancing TGF-β1-induced EMT in A549 cells. Further, miR-23a can also cause resistance to gefitinib [MiR-23a is up-regulated in NSCLC cell lines, and can also be up-regulated by the TGF-β1/Smad2/3 signaling pathway. MiR-23a can target the 3’ UTR of efitinib . Accumulefitinib . MiR-23aefitinib .In NSCLC tissues with lymphatic metastasis, the expression of miR-132 is found to be obviously lower than that in adjacent normal tissues. MiR-132 can suppress the TGF-β1/Smad2 signaling pathway and, moreover, reduce N-cadherin, ZEB1, Snail, and vimentin, leading to a repression of EMT, invasion, and migration in A549 cells .MiR-203 and miR-145 are reduced in NSCLC tissues and cell lines. Smad3 was predicted to be their target by PicTar and miRanda. Both miR-203 and miR-145 can interact with the 3′UTR of Smad3 mRNA to decrease its levels, ultimately inhibiting TGF-β1-induced EMT and invasion in A549 and 95C cells .MiR-205 is down-regulated in NSCLC cell lines. MiR-205 can repress Smad4 by targeting its mRNA, subsequently impairing TGF-β/Smad4 signaling pathway-induced EMT in A549 cells . In addiMiR-124 is decreased in NSCLC cell lines and tissues. TGF-β can increase DNMT3a expression, and then DNMT3a can bind to a miR-124 promoter, ultimately reducing miR-124 levels. MiR-124 can interact with Smad4 mRNA 3′UTR to repress Smad4, ultimately inhibiting TGF-β-induced EMT .MiR-422a is significantly down-regulated in NSCLC tissues compared to adjacent noncancerous lung tissues. MiR-422a can reduce sulfatase 2 (SULF2), which can inhibit the TGF-β1/Smad2/3 signaling pathway, eventually reducing EMT . MoreoveMiR-196b is diminished in NSCLC cell lines compared to normal cell lines. It can attenuate TGF-β1-induced EMT by decreasing N-cadherin, vimentin, ZEB1, and Snail in A549 cells .MiR-940 is declined in NSCLC tissues. It can target Snail mRNA 3′UTR and repress Snail expression, thereby inhibiting TGF-β1-induced EMT in A549 and H226 cells .MiR-22 is obviously down-regulated in NSCLC cell lines. MiR-22 can be decreased by TGF-β1 in Anip973 and AGZY83-a cells. It can reduce Snail expression by binding to Snail mRNA 3′UTR, thereby suppressing TGF-β1-induced EMT .miR-200a and miR-200c can be repressed by TGF-β through over-expressing JARID2 and embryonic ectoderm development (EED) during EMT progression. miR-200a and miR-200c can inhibit ZEB1 and ZEB2 expression to repress EMT in A549 cells [The miR-200 family consists of five members, including miR-200b, miR-200a, miR-429, miR-200c, and miR-141. The genes 49 cells ,86.MiR-145 and miR-497 levels are decreased in NSCLC cell lines compared to normal cell lines. MiR-145 and miR-497 can inhibit metadherin (MTDH) by targeting its mRNA 3′UTR, thereby inhibiting TGF-β-induced EMT, migration, and invasion in A549 and H1299 cells .The expression of miR-149 is higher in low-invasive NSCLC cells  than in high-invasive NSCLC cells (H1299 cells). MiR-149 can target forkhead Box M1 (FOXM1) mRNA 3’ UTR and repress its expression, eventually leading to a suppression of TGF-β1-induced EMT in A549 cells .MiR-134 expression is also higher in A549 and Calu1 cells than that in H1299 cells, and miR-134 can also repress forkhead Box M1 (FOXM1) to abrogate TGF-β1-induced EMT in A549 cells .MiR-133 is decreased in NSCLC cell lines. In A549 cells, miR-133 can down-regulate forkhead box Q1 (FOXQ1) to reduce TGF-β expression, finally suppressing EMT .MiR-29c is down-regulated in NSCLC cell lines compared to normal cell lines. MiR-29c can be reduced by TGF-β1. MiR-29c can inhibit specificity protein 1 (Sp1), thereby repressing TGF-β1-induced EMT in A549 and 95C cells .MiR-16 is decreased in human NSCLC tissues compared to adjacent normal tissues. Autophagy is a complex biological process associated with the lysosomal degradation of cellular proteins and other cytoplasmic contents; it can also suppress NSCLC metastasis ,93. MiR-MiR-129 is down-regulated in NSCLC tissues . OverexpMiR-485-5p and miR-3127-5p are both significantly lower in NSCLC tissues than in adjacent normal lung tissues. Both of them can impair TGF-β-induced EMT by preventing vimentin induction and increasing E-cadherin in A549 cells ,98.MiR-3607-3p expression is lower in NSCLC tissues than that in adjacent normal lung tissues. It can bind to the 3′UTR of both TGFBR1 and cyclin E2 (CCNE2) mRNAs and then repress their expressions, thereby inhibiting NSCLC invasion and migration .MiR-206 and miR-140 are decreased in NSCLC tissues. Both miR-206 and miR-140 can interact with 3′UTR of Smad3 mRNA to down-regulate its expression, thereby suppressing the TGF-β1 signaling pathway and ultimately abrogating A549 cell invasion, migration, and metastasis .MiR-136 has been found to be over-expressed in NSCLC tissues compared to adjacent normal lung tissues . In A549MiR-133a is also down-regulated in lung adenocarcinoma cells. MiR-133a can abrogate AKT signaling by reducing the TGF-β/TGFBR1 signal, contributing to suppressing A549 cell invasion and migration. In addition, low miR-133a expression can indicate a poor overall survival for lung cancer patients .MiR-143 is obviously reduced in NSCLC tissues compared to normal lung tissues. It can be stimulated by the TGF-β/Smads signaling pathway. MiR-143 can inhibit Smad3, CD44, and K-Ras expression, eventually repressing cell invasion and migration in A549 cells .The miR-886-3p level is low in SCLC tissues and cell lines. MiR-886-3p can reduce cell invasion and migration by repressing TGF-β1 in NCI-H446 cells. In addition, down-regulated miR-886-3p can predict a shorter survival for SCLC patients .MiR-138 can be down-regulated by TGF-β1 in LC006 and LC021 cells. It can inhibit CSC formation by suppressing TGF-β1-induced EMT in primary lung cancer cells .EGFR and KRAS. HCP5 can be induced by the TGF-β/Smad3 signaling pathway. In addition, it can augment Snail and Slug expression by attenuating miR-203, which can promote TGF-β/Smad3 signaling-induced EMT and invasion. Furthermore, HCP5 over-expression can indicate a poor prognosis for lung adenocarcinoma patients [LncRNA histocompatibility leukocyte antigen complex P5 (HCP5) is highly expressed in lung adenocarcinoma cell lines, especially in current smokers and patients with mutated patients .SNAI1 and FN1, which can encode Snail and fibronectin, respectively) expression to enhance TGF-β-induced EMT [LncRNA NORAD expression is higher in NSCLC tissues than in adjacent normal tissues . NORAD cuced EMT .LncRNA X inactivate-specific transcript (XIST) is up-regulated in NSCLC tissues. On the one hand, XIST can bind to miR-137 and decrease its expression, thereby suppressing miR-137′s inhibitory roles in TGF-β1-induced EMT . On the LncRNA linc00673 is up-regulated in NSCLC tissues and cell lines. It can also be elevated by TGF-β. MiR-150-5p can target ZEB1 mRNA 3′UTR and attenuate its expression. Linc00673 can act as a competing endogenous RNA (ceRNA) that can suppress the effect of miR-150-5p on ZEB1, ultimately potentiating TGF-β-induced EMT, invasion, and migration in A549 and H1975 cells. Furthermore, up-regulated linc00673 can indicate poor survival for NSCLC patients .CDH1, miR-200a, and miR-200c but can also increase the expression of genes ZEB1 and ZEB2 through the recruitment of JARID2 and the enhancer of zeste homologue 2 (EZH2) [LncRNA MEG3 can be overexpressed by TGF-β during EMT progression. In A549 and LC-2/ad cells, MEG3 shRNA can silence its expression, and MEG3 knockdown can abrogate TGF-β-induced actin stress fiber formation, which can inhibit EMT. MEG3 cDNA can increase its levels, and MEG3 over-expression can not only decrease the expression of genes 2 (EZH2) .CDH1 expression. At the same time, MEG8 can up-regulate SNAI1 and SNAI2 (a gene that can encode Slug) expression by repressing the miR-34a and miR-203 genes in A549 and LC-2/ad cells. It has also been revealed that neither MEG8 nor MEG3 can exclusively lead to EMT without TGF-β. However, the co-expression of MEG8 and MEG3 can induce EMT in the absence of TGF-β [LncRNA MEG8 can also be up-regulated by TGF-β during EMT progression. MEG8 can interact with EZH2 and can subsequently down-regulate of TGF-β .LncRNA activated by TGF-β (ATB) is obviously over-expressed in NSCLC tissues. ATB can accelerate NSCLC migration by abolishing E-cadherin and increasing N-cadherin . AdditioLncRNA TGF-β-induced lncRNA (TBILA) is highly expressed in NSCLC tissues and can be induced by the TGF-β1/Smad2/3 signaling pathway. TBILA can not only activate RhoA by up-regulating human germinal center-associated lymphoma (HGAL) (a TGF-β-induced gene) but can also increase S100A7/c-Jun activation domain-binding protein 1 (JAB1) activity by interacting with S100A7, eventually facilitating invasion and migration in A549 cells .Lnc-MMP2-2 is over-expressed in lung cancer tissues, and it can be obviously increased in the exosomes derived from A549 cells by TGF-β . On the LncRNA AWPPH expression was estimated to be higher in the blood of lung cancer patients than in normal people by qRT-PCR. AWPPH can stimulate TGF-β1 protein expression to promote NSCLC migration and invasion. Moreover, high AWPPH can indicate the postoperative distant recurrence of NSCLC patients .LncRNA CASC11 has been shown by qRT-PCR to be significantly increased in the plasma of SCLC patients. CASC11 can promote SCLC stemness by up-regulating TGF-β1, eventually increasing the number of lung CSCs. Further, CASC11 up-regulation can reveal the poor prognosis of SCLC patients .LncRNA HAND2-AS1 is down-regulated in NSCLC tissues. HAND2-AS1 can inhibit migration, invasion, and stemness by reducing TGF-β1 expression, as well as phosphorylated Smad2 and Smad3 levels, in NSCLC .LncRNA linc01186 is decreased in NSCLC tissues. It can be repressed by the TGF-β1/Smad3 signaling pathway. Linc01186 can inhibit cell invasion and migration by repressing EMT .LncRNA in nonhomologous end joining (NHEJ) pathway 1 (LINP1) is also reduced by TGF-β1/Smad4. LINP1 can increase E-cadherin and downregulate vimentin and Snail to abrogate TGF-β1-induced EMT and cell invasion .LncRNA ANCR has been shown by qRT-PCR to be markedly decreased in the plasma of NSCLC patients. Moreover, it can decrease TGF-β1 expression and thus inhibit NSCLC migration and invasion. Low ANCR expression can reveal the shorter overall survival time of patients than that of patients with high ANCR expression .LncRNA NF-κB Interacting LncRNA (NKILA) is down-regulated in NSCLC tissues. NKILA can be increased by TGF-β/Smads pathways. It can suppress invasion and migration by inhibiting NF-κB activation and then repressing Snail-mediated EMT .LncRNAs, ceRNAs, and circular RNAs (circRNAs) have been regarded as miRNA sponges to inhibit miRNAs in lung cancer. LncRNA has MREs, and it can influence the target gene downstream of the miRNAs by repressing them . CeRNAs The ceRNA myeloma overexpressed gene (MYEOV) is significantly over-expressed in NSCLC tissues compared to adjacent normal tissues. MiR-30c-2-3p can inhibit TGFBR2 and Ubiquitin specific protease 15 (USP15)  expression by targeting their mRNA 3′UTRs. MYEOV can bind to and then suppress miR-30c-2-3p, eventually contributing to activating TGF-β signaling and potentiating invasion and metastasis in NSCLC .CeRNA HMGA2 is up-regulated in metastatic lung adenocarcinoma. HMGA2 can increase TGFBR3 expression by repressing miRNA let-7, which can also enhance the TGF-β signaling pathway, ultimately promoting invasion and metastasis in NSCLC .The circRNA CircPTK2 level is lower in NSCLC tissues than in normal lung tissues. It can be inhibited by TGF-β1 during EMT progression. MiR-429 and miR-200b-3p can repress transcriptional intermediary factor 1 γ (TIF1γ)  expressAlthough traditional lung cancer therapies, such as surgical resection, chemotherapy, and radiotherapy, have made great progress, they still face some limitations. For example, drug resistance and lung cancer distant metastasis can usually influence the therapeutic effects of a treatment. As a result, it is necessary to develop new strategies. MiRNAs and lncRNAs participate in substantial lung cancer progression, and we may be able to use them as treatment targets.Curcuma longa, possesses anti-cancer effects and chemopreventive properties. Curcumin can significantly inhibit several miRNA-associated pathways, such as the MAPK, TGF-β, and Wnt signaling pathways, contributing to suppressing invasion and migration in A549 cells [Curcumin, isolated from 49 cells .Carboplatin can interact with ifosfamide and paclitaxel and possesses fewer non-hematologic toxicities than cisplatin (DDP). Carboplatin is a chemotherapeutic drug widely used to treat SCLC and NSCLC in order to improve overall survival . CarboplDDP is also extensively used to deal with lung cancer. MiR-181b is decreased in NSCLC cells. It can target TGFBR1 mRNA 3′UTR and repress its expression, thus abrogating the TGF-β signal and eventually increasing DDP chemosensitivity in NSCLC cells . MiR-17,ZEB1 and ZEB2 expression, leading to decreasing TGF-β1-induced EMT and metastasis in PC9 cells [Decitabine (DAC) is a deoxyribonucleotide and can abrogate DNA methylation. Functionally, DAC promotes demethylation of the miR-200 promoter to increase the levels of miR-200c and miR-200a, which can repress C9 cells .Lung cancer is the most common cancer in men and the second most common in women after breast cancer. The low overall survival rate of lung cancer patients is usually correlated with distant organ metastasis. MiRNAs and lncRNAs are demonstrated to be oncogenes or suppressors by targeting mRNA 3′UTR in lung cancer development. Currently, miRNAs and lncRNAs are widely used to explore lung cancer’s metastatic mechanisms, and these two nc-RNAs are increasingly used as biomarkers to predict the outcomes of lung cancer patients. TGF-β is involved in many cancers, and TGF-β signaling pathways have also been extensively confirmed to play pivotal roles in lung cancer metastasis. Interestingly, we found that Carboplatin, the first-line therapy used to treat metastatic lung cancer, suppresses TGF-β signaling-regulated lung cancer metastasis by repressing miR-21 . This remiR-200a, miR-200c [In the current review, we discussed how miRNAs and lncRNAs can exert positive  and negamiR-200c ,86, miR-miR-200c , miR-149miR-200c , miR-134miR-200c , miR-133miR-200c , miR-29cmiR-200c , miR-16 miR-200c , miR-129miR-200c , miR-485miR-200c , miR-312miR-200c , and LINmiR-200c  can suppmiR-200c , miR-206miR-200c , miR-136miR-200c , miR-133miR-200c , miR-143miR-200c , miR-886miR-200c , HAND2-AmiR-200c , linc011miR-200c , ANCR [1miR-200c , and NKImiR-200c  can reprmiR-200c . HAND2-AmiR-200c . During miR-200a, miR-200c [➣They can promote or suppress TGF-β/Smads pathways or other non-Smad pathways. MiR-93  and NORA➣They can promote or inhibit lung CSC formation. CASC11 can promote lung CSC formation , while m➣They can be up-regulated in TGF-β-mediated exosomes, including miR-23a  and lnc-They can be increased or decreased via TGF-β signaling. MiR-9 ,63, miR-miR-200c ,86, miR-miR-200c , linc011miR-200c , and LINmiR-200c  can be dMiRNAs and lncRNAs can be detected in biological fluids through a biopsy of liquids, such as whole blood (lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)  and miRNDue to the multiple functions of miRNAs and lncRNAs in lung cancer metastasis, these RNAs have been regarded as promising therapeutic targets. Several traditional treatments have been discovered to target miRNAs and lncRNAs. Low-dose radiation can increase miR-30a and miR-30b expression. MiR-30a and miR-30b can inhibit plasminogen activator inhibitor-1 (PAI-1) expression and then reduce EMT and migration in NCI-H460 cells exposed to high-dose radiation. Additionally, miR-30a and miR-30b also promoted sensitivity to radiotherapy in a mouse model . In LewiPresently, despite the great progress that has been made in traditional lung cancer treatments, there are still multifaceted limitations impairing these treatments’ effects, such as therapeutic resistance and drug toxicity. As mentioned above, miRNAs and lncRNAs are widely involved in TGF-β signaling-regulated lung cancer metastasis. Targeting the miRNA/lncRNA-TGF-β signaling axis might provide new insights to decrease lung cancer metastasis and improve the prognosis of lung cancer patients. However, these studies are performed mostly on NSCLC cell lines, and studies on SCLC cell lines and in lung cancer tissues are still lacking. Therefore, further explorations in these microenvironments are required to elucidate the roles of miRNAs and lncRNAs in lung cancer more completely.


In cultured human umbilical vein endothelial cells (HUVECs) high glucose (HG) stimulation will lead to significant cell death. Bardoxolone-methyl (BARD) is a NF-E2 p45-related factor 2 (Nrf2) agonist. In this study we show that BARD, at only nM concentrations, activated Nrf2 signaling in HUVECs. BARD induced Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation, increasing expression of antioxidant response element (ARE) genes. BARD pretreatment in HUVECs inhibited HG-induced reactive oxygen species production, oxidative injury and cell apoptosis. Nrf2 shRNA or knockout (using a CRISPR/Cas9 construct) reversed BARD-induced cytoprotection in HG-stimulated HUVECs. Conversely, forced activation of Nrf2 cascade by Keap1 shRNA mimicked BARD’s activity and protected HUVECs from HG. Importantly, BARD failed to offer further cytoprotection against HG in the Keap1-silened HUVECs. Taken together, Keap1-Nrf2 cascade activation by BARD protects HUVECs from HG-induced oxidative injury. Vascular endothelial cell injury is a primary medical issue in the pathogenesis and progression of cardiovascular complications in diabetes mellitus patients , 2. SustNF-E2 p45-related factor 2 (Nrf2) is a mater transcriptional factor suppressing cellular oxidative injury and other stresses –18. OnceStudies have demonstrated that forced activation of Keap1-Nrf2 cascade can efficiently protect vascular endothelial cells from HG and other oxidative injury. Tang et al., demonstrated that 4-octyl itaconate (OI) protected HUVECs from HG through activation of Nrf2 signaling cascade . A studyheme oxygenase-1 (HO1), NAD(P)H quinone oxidoreductase 1 (NQO1) and γ-glutamyl cysteine ligase catalytic subunit (GCLC) [Bardoxolone-methyl (BARD) is a synthetic triterpenoid –25. BARDt (GCLC) . The resBARD can induce Nrf2 signaling cascade activation by releasing Nrf2 from Keap1 , 22. A cHO1, NQO1 and GCLC, was significantly increased following BARD (10-100 nM) treatment in HUVECs  treatment in HUVECs can induce robust oxidative injury, responsible for following cell death and apoptosis , 28–31. Further studies demonstrated that HG stimulation for 48h led to significant viability (CCK-8 OD) reduction  and cellHO1, NQO1 and GCLC, was completely blocked with Nrf2 shRNA or KO . Importantly, BARD was completely ineffective on HG-induced cytotoxicity in sh-Nrf2-HUVECs and ko-Nrf2 HUVECs  established with puromycin selection. Furthermore, the stable HUVECs with the lenti-CRISPR-GFP-Nrf2 knockout (KO) construct  were utNA or KO , 3D. In NA or KO  and apopNA or KO  were int2 HUVECs , 3F. TheHO1, NQO1 and GCLC was augmented in Keap1-silenced HUVECs . Furthermore, BARD-induced cytoprotection against HG was completely reversed with Nrf2 silencing or KO in HUVECs. Further studies found that forced Nrf2 cascade activation by Keap1 shRNA mimicked BARD’s activity and protected HUVECs from HG-induced cell death/apoptosis. Importantly, BARD failed to offer further cytoprotection against HG in the Keap1-silened HUVECs. Therefore, Keap1 depletion not only mimicked, but also nullified, BARD-induced cytoprotection against HG in HUVECs. These results confirm that in HUVECs BARD-induced cytoprotection against HG requires Keap1-Nrf2 cascade activation.Our results implied that activation of Keap1-Nrf2 signaling is required for BARD-induced cytoprotection against HG in HUVECs. Nrf2 silencing or KO completely blocked BARD-induced increases of ARE activity and expression of ARE genes  patients have demonstrated a long-term increment in glomerular filtration by BARD administration . Howeverin vitro studies shall not be directly translated to humans. A single concentration of HG stimulation in cultured HUVECs is also not equal to vascular injury in the diabetes mellitus patients. The efficacy and safety of BARD will need further in vivo characterizations.It should be noted that the current results from Taken together, we conclude that Keap1-Nrf2 cascade activation by BARD protects HUVECs from HG-induced oxidative injury.BARD, CCK-8 dye and puromycin were provided by Sigma-Aldrich . Antibodies utilized in this study were provided by Santa Cruz Biotechnology  and Abcam . The cell culture reagents, fetal bovine serum (FBS) and others, were obtained from Hyclone Co. . TRIzol along with other PCR agents and Lipofectamine 2000 were provided by Thermo-Fisher Invitrogen . All the primers and sequences were synthesized by Shanghai Genechem Co. .HUVECs were provided by Dr. Jiang  at NanjiNrf2, HO1, NQO1, and GCLC were listed in Ct−∆∆ method, with GAPDH tested as the reference gene.Following the applied treatment, the total cellular RNA of HUVECs was extracted by TRIzol, that was reversely transcribed using a ReverTra Ace qPCR RT kit (Toyobo). qPCR was performed under an ABI Prism 7900 HT System . mRNA primers for 5 cells/well), and transfected with an ARE-inducible firefly luciferase vector . The trHUVECs with applied treatments were incubated with the described lysis buffer . For eacThe detailed protocols for Co-IP were described in elsewhere . Briefly2 HUVECs were initially seeded into 96-well tissue culture plates. Following the indicated treatments, cell viability was measured by using the Cell Counting Kit-8 . The optical density (OD) values of CCK-8 were tested at 550 nm.At the density of 30, 000 cells/cm2 HUVECs were initially seeded into six-well tissue culture plates. Following the applied HG stimulation, 25 μg of achieved total cell lysates were incubated with DEVD-AFC . The relative caspase-3 activity, reflected by the AFC absorbance, was examined through a Fluoroskan Ascent FL equipment machine at 355 nm excitation and 525 nm emission [At 30, 000 cells/cmemission .HUVECs with the applied treatments were co-incubated with 5 μg/mL of Annexin V and 5 μg/mL of propidium iodide (PI) for 20 min. A fluorescent-activated cell sorting (FACS) equipment  was utilized to sort the apoptotic cells with positive Annexin V staining. Annexin V ratios were recorded.2 HUVECs were initially seeded into six-well tissue culture plates. After the applied HG stimulation, the nuclei were stained with TUNEL  and DAPI  for 30 min under the dark. TUNEL ratio (TUNEL/DAPI×100%) was recorded by counting 500 cells from five random views (1 × 200 magnification) from each condition.At 30, 000 cells/cmAfter the applied treatment, mitochondrial depolarization (“∆Ψ”) in HUVECs was measured by a mito-dye JC-1, which shall form green monomers with mitochondrial depolarization in stressed cells . The pro2 HUVECs were initially seeded into six-well tissue culture plates. Following the applied HG stimulation, superoxide contents were tested through a superoxide colorimetric assay kit  following the attached protocols. At the wavelength of 450 nm the absorbance of superoxide was tested.At 30, 000 cells/cm2 HUVECs were initially seeded into six-well tissue culture plates. Following the applied HG stimulation, levels of reduced glutathione (GSH) and oxidized disulfide form glutathione (GSSG) were tested [At 30, 000 cells/cme tested , using ae tested . GSH/GSSThe Nrf2 short hairpin RNA (shRNA) lentiviral particles  or the Keap1 shRNA lentiviral particles  were individually added to cultured HUVECs (at 60% confluence) for 36h. Stable cells were established by culturing cells in puromycin (5.0 μg/mL)-containing complete medium for 10-12 days. In the stable cells over 95% knockdown of target protein (Nrf2 or Keap1) was achieved. Control cells were transduced with scramble non-sense control shRNA lentiviral particles (“sh-C”).The monoclonal stable HUVECs with the lenti-CRISPR-GFP-Nrf2 knockout (KO) construct, or the Nrf2 KO HUVECs, as well as control cells with an empty vector, were provided by Dr. Xu . Nrf2 KOP < 0.05 was considered statistically significant.Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed by repeated-measures analysis of variance (RMANOVA) with Dunnett’s post hoc test using a SPSS software . A two-tailed unpaired T test  was carried out to examine significance between two treatment groups.


Analysis of the vaccine candidates in rhesus macaques showed that animals developed protective humoral and cellular immune responses when challenged with the virus. Neutralizing antibody titers were also observed at levels similar to those seen in humans who have recovered from SARS-CoV-2 infection.The development of a vaccine to protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an urgent biomedical need. Yu Science, this issue p. 806 Successful protection against SARS-CoV-2 in rhesus macaques by means of a prototype DNA vaccine is described. 10 reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with viral loads in sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers at levels comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. After vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log The coronavirus disease 2019 (COVID-19) pandemic has made the development of a safe, effective, and deployable vaccine to protect against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) a critical global priority (We produced a series of prototype DNA vaccines expressing six variants of the SARS-CoV-2 S protein: (i) full length (S), (ii) deletion of the cytoplasmic tail (S.dCT) . WesternN = 4), S.dCT (N = 4), S.dTM (N = 4), S1 (N = 4), RBD (N = 4), S.dTM.PP (N = 5), and sham controls (N = 10). Animals received 5-mg DNA vaccines by the intramuscular route without adjuvant at weeks 0 and 3. After the boost immunization at week 5, we observed S-specific binding antibodies by enzyme-linked immunosorbent assay (ELISA) . Moreover, NAb titers in the vaccinated macaques  were comparable in magnitude to NAb titers in a cohort of 9 convalescent macaques (median titer = 106) and a cohort of 27 convalescent humans (median titer = 93) who had recovered from SARS-CoV-2 infection (We immunized 35 adult rhesus macaques (6 to 12 years old) with DNA vaccines in the following groups: S ( (ELISA)  and neut (ELISA)  and a li (ELISA) . As detenfection .S-specific and RBD-specific antibodies in the vaccinated macaques included diverse subclasses and effector functions, including antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), and antibody-dependent natural killer (NK) cell activation  . A trendH1)–biased cellular immune responses.We also observed cellular immune responses to pooled S peptides in most vaccinated animals by IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assays at week 5 . Intrace8 virus particles (VPs) [1.1 × 104 plaque-forming units (PFUs)] of SARS-CoV-2, administered as 1 ml by the intranasal route and 1 ml by the intratracheal route. After challenge, we assessed viral RNA levels by reverse transcription polymerase chain reaction (10 RNA copies/ml in BAL and a median peak of 6.82 (range = 5.96 to 7.96) log10 RNA copies/swab in NS (fig. S2). Lower levels of viral RNA were observed in the vaccine groups (figs. S3 and S4), including 1.92 and 2.16 log10 reductions of median peak viral RNA in BAL and NS, respectively, in S-vaccinated animals compared with sham controls  (fig. S5). Viral RNA assays were confirmed by PFU assays, which similarly showed lower infectious virus titers in S-vaccinated animals compared with sham controls  (fig. S5).At week 6, which was 3 weeks after the boost immunization, all animals were challenged with 1.2 × 1010 sgmRNA copies/ml in BAL and 6.40 (range = 4.91 to 7.01) log10 sgmRNA copies per swab in NS. Peak viral loads occurred variably on days 1 to 4 after challenge. Markedly lower levels of sgmRNA were observed in the vaccine groups  , which are believed to reflect viral replication cellular intermediates that are not packaged into virions, and thus putative replicating virus in cells  with a me groups , includiy tests) . Reduced10 pseudovirus NAb titer at week 5 inversely correlated with peak log10 sgmRNA in both BAL  and NS   and NS   (fig. S6). Vaccine-elicited ELISPOT responses (fig. S7), CD4+ intracellular cytokine staining (ICS) responses (fig. S8), and CD8+ ICS responses (fig. S9) did not correlate with protection.The variability in protective efficacy in this study facilitated an analysis of immune correlates of protection. The log−0.4162) . Similar−0.3360) . These d10 sgmRNA levels in BAL , RBD-specific ADCD responses (P = 0.0001), S-specific RBD responses (P = 0.0010), and RBD-specific ADCP responses (P = 0.0005) were higher in completely protected animals than in partially protected animals  with those in vaccinated animals that were partially protected . Log animals .All animals exhibited anamnestic humoral and cellular immune responses after challenge, including increased ELISA titers (fig. S10), pseudovirus NAb titers (fig. S11), live virus NAb titers (fig. S12), and IFN-γ ELISPOT responses (fig. S13) on day 14 after challenge. These data suggest that vaccine protection was probably not sterilizing  but rather was likely mediated by rapid virologic control after challenge.10 reductions in median viral loads in BAL and NS, respectively, in S-immunized animals compared with sham controls. Protection was likely not sterilizing but instead appeared to be mediated by rapid immunologic control after challenge.A safe and effective SARS-CoV-2 vaccine may be required to end the global COVID-19 pandemic. Several vaccine candidates have initiated clinical testing, and many others are in preclinical development (Our data extend the findings of previous studies on SARS and Middle East respiratory syndrome (MERS) vaccine protection in mice, ferrets, and macaques , as a significant correlate of protection against disease of both the lower and upper respiratory tracts. It is likely that protection in both anatomic compartments will be necessary for pandemic control, although protection in the upper respiratory tract may be more difficult to achieve. If this NAb correlate proves generalizable across multiple vaccine studies in both nonhuman primates and humans, then this parameter would be a simple and useful benchmark for clinical development of SARS-CoV-2 vaccines. Innate immune effector functions such as ADCD may also contribute to protective efficacy. In summary, we demonstrate effective vaccine protection against SARS-CoV-2 in rhesus macaques and define NAb titers as an immune correlate of protection, which will accelerate the development of SARS-CoV-2 vaccines for humans.


Chlamydomonas reinhardtii as a model, we isolated and characterized two novel Chlamydomonas PIH preassembly mutants, mot48-2 and twi1-1. A new allele of mot48 (ida10), mot48-2, shows large defects in ciliary dynein assembly in the axoneme and altered motility. A second mutant, twi1-1, shows comparatively smaller defects in motility and dynein assembly. A double mutant mot48-2; twi1-1 displays greater reduction in motility and in dynein assembly compared to each single mutant. Similarly, a double mutant twi1-1; pf13 also shows a significantly greater defect in motility and dynein assembly than either parent mutant. Thus, MOT48 (IDA10), TWI1 and PF13 may define different steps, and have partially overlapping functions, in a pathway required for ciliary dynein preassembly. Together, our data suggest the three PIH proteins function in preassembly steps that are both common and unique for different ciliary dyneins.Ciliary dyneins are preassembled in the cytoplasm before being transported into cilia, and a family of proteins containing the PIH1 domain, PIH proteins, are involved in the assembly process. However, the functional differences and relationships between members of this family of proteins remain largely unknown. Using  Chlamydomonas reinhardtii, we assessed the functional relationship between three PIH proteins with respect to dynein preassembly and motility. We found that these PIH proteins have complicated and related roles in dynein assembly, possibly with each playing common and unique roles in dynein assembly. Our results provide new information on each conserved PIH protein for dynein assembly and provide a new understanding of PCD caused by PIH mutations.Motile cilia are hair-like organelles that protrude from many eukaryotic cells, and play vital roles in organisms including cell motility, environmental sensing and removal of infectious materials. Motile cilia are driven by gigantic motor protein complexes, called ciliary dyneins, defects in which cause abnormal ciliary motility, ultimately resulting in human diseases collectively called primary ciliary dyskinesia (PCD). Ciliary dyneins are preassembled in the cytoplasm before being transported into cilia, and preassembly requires a family of potential co-chaperones, the PIH proteins. Mutations in the PIH proteins cause defective assembly of ciliary dyneins and can result in PCD. However, despite their importance, the precise functions, and functional relationships, between the PIH proteins are unclear. In this study, using  Paramecium and Trypanosoma, these organelles play an indispensable role in cell motility. In higher eukaryotes including humans, cilia are essential for proper development, fertilization, and homeostasis. Defects in ciliary motility cause various symptoms including situs inversus, infertility, congenital heart disease and hydrocephalus in humans, collectively called as primary ciliary dyskinesia (PCD) . Im. ImChlamof MOT48  [13]. Thng HSP90  [13, 17,ed MOT48 . The Mot 21 μm/s).mot48-1 cilia lacked a subset of dynein species [mot48-2 mutants. To semi-quantitatively estimate the amount of each dynein species in the mot48-2 cilia compared to wild-type (CC-124), spectral counting experiments were performed on isolated axonemes (Chlamydomonas genome [mot48-2 mutants (50% ~ 80% of the levels of wild type)  shoaxonemes . The HCs mutants .Chlamydomonas [pf13 [twi1 strain (LMJ.RY0402.076787) was recently identified in the CliP library [twi1 progeny (twi1-1) phenotype was characterized. Compared to wild-type Chlamydomonas (CC-125)(~ 136 ± 21 μm/s), the twi1-1 swims at a slightly reduced rate (~ 103 ± 14 μm/s) after the 3-day liquid culture, suggesting twi1-1 had subtle defects in assembly of ciliary dyneins (Chlamydomonas and human proteins). Surprisingly, pf22 (CC-1382) and pf23 (CC-1383 and CC-3660) strains completely lack TWI1 (pf22 (CC-2495), pf22A (CC-2493), and pf23 (5–4) strains have normal levels of TWI1 (pf22 (CC-1382) and pf23 (CC-1383 and CC-3660) strains occurs because of an additional mutation. TWI1 sequence in these mutant strains revealed the transposon MRC1  [TWI1 gene  and pf23 (CC-1383 and CC-3660)) are actually double mutants lacking their respective proteins (Chlamydomonas PF22 or PF23) and TWI1. Other pf strains from Luck laboratory might also carry this twi1 (twi1-2) background. We carried out spectral counting experiments  and e (DHC8) were modestly reduced compared to wild-type (CC-125). This observation is consistent with the mild motility phenotype of twi1-1.To test if the IFT-related or preassembly-related mutations affect the stability of the TWI1 proteins, we performed the immunoblots using the TWI1 antibody on the whole cell samples from various IFT-related and preassembly-related mutants. Immunoblots reveal the TWI1 protein is present in most IFT-related and preassembly-related mutants (e.g. and oda5)  insertedWI1 gene . Since teriments  of the cChlamydomonas genetics by isolating double PIH mutants  from crosses between single PIH mutants . The predictions included that if deleted proteins function together in the same path and a phenotype of one mutant is similar or the same as the other, then the double mutant phenotype would nearly match the phenotype of the single mutants. Also, if deleted proteins function together in the same path but a phenotype of one mutant is more severe to the other, then the double mutant phenotype would match the phenotype of the more deleterious single mutant. Alternatively, if the deleted proteins operate in different pathways, or have some overlapping function but do not function together in the same path, then the double mutants would have a more severe phenotype than the single mutants.We took advantage of mot48-2; twi1-1 double mutant is worse than the mot48-2 single mutant . Furthermore, about half of the double mutant cells have completely non-motile cilia while the other half of the cells display a slow swimming phenotype  is much lower than the pf13 (~ 58%) single mutant. These observations strongly suggest that TWI1 protein is involved in ciliary dynein preassembly.The motility phenotype of the henotype . In addimot48-2; pf13 double mutant grew extremely short cilia or was missing cilia. This severe phenotype hindered comparison of the amount of ciliary dynein assembled in this strain. This short cilia phenotype was also previously observed in the mot48-1; pf13 mutant [To further test the idea that dynein assembly is more defective in the double mutants, we performed spectral counting experiments on dyneins in isolated axonemes from the double and single PIH preassembly mutants to compare the amount of ciliary dyneins assembled . The motmot48-2; twi1-1 double mutant are greatly reduced compared to mot48-2  in the pf13; twi1-1 double mutant are modestly reduced compared to the pf13 mutant, indicating that PF13 and TWI1 both function in the preassembly of these dyneins possibly in different steps, and/or have some overlapping function in the same step (see Discussion).The peptide numbers for a subset of ciliary dyneins in the  mot48-2 . In partmot48-2; twi1-1 double mutant, and DHC11 and DHC12 in the pf13; twi1-1 double mutant) are much greater in the double PIH mutants than in the single PIH mutants (It is intriguing that peptide numbers of some minor dyneins (DHC12 in the d twi1-1). This remot48 background  and ciliary dynein assembly  and pf22A (CC-2493)(which contain a wild-type TWI1 gene) and compared to pf23 (CC-1383) and pf22 (CC-1382). The dynein defects in pf23 (CC-1383) are more profound than pf23 (5–4)(pf23 (CC-1383) than in pf23 (5–4) [pf22A (CC-2493) and pf22 (CC-1382) are relatively similar to each other (pf22 mutants have large defects (< 50% of wild-type) in IDAs a (DHC6) and e (DHC8) and minor dyneins DHC3 and DHC4. Also, the axonemal amount of one minor dynein DHC12 is increased in the pf22 mutants  may indicate that Chlamydomonas TWI1 is needed for efficient function of the PF23 protein in dynein preassembly. Additionally, the swimming phenotype of the pf23 (CC-1383) strain, rescued with the wild-type PF23 gene, which also harbored the twi1-2 mutation, was indistinguishable from wild-type [twi1-1 mutant.In addition to the PIH mutants, we also performed spectral counting of dyneins in isolated axonemes from f23 (5–4). Particu23 (5–4) . On the ch other . In addi mutants . Given t mutants  and DNAA mutants  in mammaild-type , consistChlamydomonas reinhardtii, mot48-2 and twi1-1. Although recent studies reveal a conserved role(s) of PIH proteins in ciliary dynein preassembly [Chlamydomonas PIH proteins, MOT48, TWI1, and PF13. Accordingly, we have updated our previous model [mot48-2; twi1-1, pf13; twi1-1 and pf23; twi1-2 (CC-1383)) than a single mutant . In addition, dynein f/I1, and possibly IDA a, do not require PIH proteins for assembly , and DNAAF2/KTU (PF13 orthologue), MOT48 groups near DNAAF2/KTU and PIH1D1 in a phylogenetic tree [https://blast.ncbi.nlm.nih.gov/Blast.cgi) also revealed that among the four PIH proteins in vertebrates, PIH1D1 showed the highest similarity to MOT48, although the E-values were relatively low (< 7E-15). In addition, recently MOT48 has been reported to interact with RPAP3 and RuvBL1 [Chlamydomonas cytoplasm  for identification of interacting partners, we failed to identify chaperone related proteins. This negative result could indicate weak and/or transient interactions of the PIH proteins with chaperones and/or interacting partners of MOT48 and TWI1. Further structural and biochemical studies of PIH interacting proteins, including specific dynein HC/IC/LCs, are required for understanding assembly complexes, steps and specificity of each PIH protein required for ciliary dynein assembly.The TWI1 orthologue, DNAAF6/PIH1D3, has also been postulated to interact with DNAAF4/DYX1C1 , ortholoydomonas . Thus, Tydomonas , 19, 23 allele, referred to as mot48-2 was isolated from a CLiP library strain (LMJ.RY0402.055540)[APHVIII) insertional cassette used for selection but caused a slow-swimming phenotype. The original LMJ.RY0402.055540 strain was backcrossed with the wild-type (CC-125) strain to separate this mutation, and two mot48-2 progeny were isolated. The identification of the mot48 background was confirmed by Sanger sequencing using the primer pair: Ida10-2 GF2 (5’-TGGCAGCACATTCATAAGCA-3’) and Ida10-2 GR2 (5’-CGCTGTACTAGAGCCCCTCA-3’). The twi1 strain was first obtained from the CLiP library (LMJ.RY0402.076787)[twi1 mutant progeny (twi1-1) were used for experiments. Cells were grown in the tris-acetic acid-phosphate (TAP) liquid/solid media as previously described [Chlamydomonas PIH mutants were obtained using the standard tetrad procedure [The 2.055540) having a2.076787) and backescribed . Double rocedure . The decrocedure . For prerocedure .mot48-2, twi1-1, and mot48-2; twi1-1 strains was performed by the electroporation method using each wild-type gene cloned in the modified pGend vector (pGend-MCS-3HA-AphVIII/pGend-MCS-mCherry-3HA-AphVIII/pGend-MCS-3HA-Hyg)[mot48-2; MOT48::HA expressed exogeneous MOT48 with a 3HA tag at the C-terminus. The rescued strain, mot48-2; MOT48::mCherry-HA expressed exogeneous MOT48 with the mCherry-3HA tag at the C-terminus. The pGend-MOT48-3HA-AphVIII vector was previously described [twi1-1 rescued strains, twi1-1; TWI1::HA and mot48-2; twi1-1; TWI1::HA expressed exogeneous TWI1 with a 3HA tag at the C-terminus. The primer pair used for wild-type TWI1 cloning was as follows, TWI1-pGend-F1 (5’-CACAACAAGCCCATATGGACATTGGGAGCTTCACTGCTGA-3’) and TWI1-pGend-R1 (5’-GGTATCGATCGAATTCGAATGGCTCTTCCCGAATGATGCG-3’). The NdeI/EcoRI sites used for cloning are underlined.Phenotypic rescue of the -3HA-Hyg), 48, 49.escribed  and alsoA semi-quantitative estimation of the amount of dyneins in the isolated axonemes , 50, 51 cw15), mot48-1, and mot48-2 strains with the aim of comparing the dynein levels between the mot48-1 and mot48-2 strains. For normalization and comparison between samples, the peptide numbers of ciliary dyneins observed in the CC-124 strain were assigned a ratio of 1.0, and the observed peptide numbers of Hydin [http://www.proteomesoftware.com/products/scaffold/) for the first-set of analyses were set as follows: Protein Threshold: 90%/Minimal Peptides Number: 2/ Peptide Threshold: 70%. The averages of two independent experiments are summarized in A first set of experiments was performed on dyneins from the wild-type  strains with the aim of comparing the dynein levels between the single and double PIH preassembly mutants. For normalization and comparison between samples, observed peptide numbers in the CC-125 strain and also the peptide numbers of Hydin were used. The second set of experiments was generously performed by Drs. Scott Shaffer and Xuni Li . Thresholds in the Scaffold 4 software for the second-set analyses were set as follows: Protein Threshold: 99%/Minimal Peptides Number: 2/ Peptide Threshold: 70%. The results are summarized in A second set of experiments was performed on dyneins from wild-type (CC-125), twi1-1; TWI1::HA, pf22 (CC-1382 with the twi1-2 background) and pf22A (CC-2493) strains with the aim to check the dynein rescue and effect of the twi1-2 mutation in dynein assembly in pf22 strains. Observed peptide numbers were normalized using peptide numbers of CC-125 and Hydin, and the results were combined/incorporated into pf23 (CC-1383 with the twi1-2 background) normalized with the wild-type (137c) peptide counts were reanalyzed/refined from our previous paper [A third set of experiments was performed on dyneins from wild-type (CC-125), us paper  with theChlamydomonas cDNA library (Chlamydomonas Resource Center) and cloned into the NdeI/BamHI site of the pET15b vector (Novagen) by the In-Fusion HD Cloning enzyme (TAKARA). The primer pair to amplify the TWI1 cDNA sequence was as follows: TWI1-CF1 (5’-CGCGCGGCAGCCATATGGACATTGGGAGCTTCACTG-3’) and TWI1-CR1 (5’-GTTAGCAGCCGGATCCGAATGGCTCTTCCCGAATGATGCG-3’)(The NdeI/BamHI sites are underlined). The purified TWI1 protein with a 6His tag at the N-terminus was used as antigen to immunize two rabbits. The antisera from rabbits were blot and Protein-A purified before use, as previously described [Chlamydomonas TWI1 cDNA sequence determined in this study was deposited in the DNA Data Bank of Japan (DDBJ) under the accession No. LC461993.The TWI1 cDNA sequence was determined using the escribed , 54. TheChlamydomonas was assessed on free-swimming cells in liquid culture using our in-lab video system and ImageJ software (https://www.google.com/search?client=firefox-b-d&q=imagej+software) [pf13 and pf13; twi1-1 strains was counted and averaged on three days. Student’s t-test was performed on Excel (Microsoft).SDS-PAGE and immunoblotting were performed following standard procedures , 56. Foroftware) . VelocitS1 Figmot48-2, mot48-2; MOT48::HA, mot48-2; MOT48::mCherry-HA, twi1-1, twi1-1; TWI1::HA, mot48-2; twi1-1, and mot48-2; twi1-1; TWI1::HA. For wild-type (CC-125), twi1-1, twi1-1; TWI1::HA, mot48-2; MOT48::HA, and mot48-2; MOT48::mCherry-HA, more than 40 cells were measured. For mot48-2, mot48-2; twi1-1, and mot48-2; twi1-1; TWI1::HA, it was difficult to find ideal cells for the velocity measurement, but more than 15 cells were measured. As discussed in the main text, the swimming phenotypes of the preassembly mutants slightly varied from day to day and culture to culture because of the apparent compensatory and overlapping nature of the dynein preassembly. In this figure, swimming velocities are shown for cells cultured for 3 days in the liquid TAP media in mini petri-dishes under constant light. Asterisks indicate p < 0.01 in the Student’s t-test.Swimming velocities of wild-type (CC-125), (TIF)Click here for additional data file.S2 Figpf13 and mot48-2 nucleo-flagellar apparatuses. DHC11 was shown to be localized at the proximal part of the wild-type axonemes [pf13 and mot48-2 axonemes compared to wild-type axonemes. The bright puncta are non-specific staining/autofluorescence. Bar: ~ 5 μm.Immunofluorescence localization of DHC9 (IDA c HC), DHC11 (minor dynein HC) and α-tubulin in wild-type (CC-124), axonemes . Both DH(TIF)Click here for additional data file.S3 FigA) Spectral counting comparison of dyneins from axonemes of pf23 (5–4), pf23 , pf22A (CC-2493), and pf22 . The spectral data of pf23 (5–4) are from the second set of experiments. The spectral data of pf22A (CC-2493) and pf22 (CC-1382) are from the third set of experiments. The spectral data of pf23 (CC-1383) are refined/reanalyzed from our previous study [pf23 (5–4), pf22A (CC-2493), and pf22 (CC-1382), and 137c for pf23 (CC-1383)[pf23 strain  showed more than a 50% reduction compared to the pf23 (5–4) strain. B) Ciliary dynein species for which the spectral numbers in the pf23 (5–4) or pf22A (CC-2493) strain (without the twi1-2 background) showed a more than 50% reduction compared to wild-type (CC-125) are summarized.us study . The spe(CC-1383)). Asteri(TIF)Click here for additional data file.S4 Fighttp://clustalw.ddbj.nig.ac.jp/) by the default settings, and the phylogenetic tree was drawn by the Neighbor-Joining method [https://www.megasoftware.net/). The bootstrap consensus tree inferred from 1000 replicates is shown, and the bootstrap numbers are shown in percentile [Chlamydomonas MOT48 falls into the PIH1D1 group. The accession numbers of proteins used to draw this tree were as follows: Human DNAAF2/KTU (NCBI: ACN30493.1); Mouse DNAAF2/KTU (NCBI: NP_081545.3); Zebrafish KTU (NCBI: NP_001028272.1); Chlamydomonas PF13 (NCBI: BAG69288.1); Human PIH1D1 (NCBI: NP_060386.1); Mouse PIH1D1 (NCBI: AAH68254.1); Zebrafish PIH1D1 (NCBI: NP_001153400.1); Human PIH1D2 (NCBI: AAH19238.1); Mouse PIH1D2 (NCBI: AAH39645.1); Zebrafish PIH1D2 (NCBI: NP_001008629.1); Chlamydomonas MOT48 (NCBI: BAI83444.1); Human DNAAF6/PIH1D3 (NCBI: NP_001162625.1); Mouse DNAAF6a/PIH1D3a (NCBI: NP_083338.1); Mouse DNAAF6b/PIH1D3b/Twister2 (NCBI: AAI19079.1)[Chlamydomonas TWI1 ; Human DNAAF4/DYX1C1 (NCBI: NP_570722.2); Mouse DNAAF4/DYX1C1 (NCBI: NP_080590.3); Zebrafish DNAAF4/DYX1C1 (NCBI: NP_991251.1); Chlamydomonas PF23 (NCBI: BBA27223.1); Yeast Nop17 (NCBI: GAX71541.1).The protein alignment was performed using the ClustalW software (v2.1); Zebrafi(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


TNF, IL1B, IL6, IL8, NFKB1, NFKB2 and RELB genes. In addition to these pathways, lung fibrosis, keratinization/cornification, rheumatoid arthritis, and negative regulation of interferon-gamma production pathways were also significantly upregulated. We observed that these pathologic features of SARS-CoV-2 are similar to those observed in patients with chronic obstructive pulmonary disease (COPD). Intriguingly, tiotropium, as predicted by MT–DTI, is currently used as a therapeutic intervention in COPD patients. Treatment with tiotropium has been shown to improve pulmonary function by alleviating airway inflammation. Accordingly, a literature search summarized that tiotropium reduced expressions of IL1B, IL6, IL8, RELA, NFKB1 and TNF in vitro or in vivo, and many of them have been known to be deregulated in COPD patients. These results suggest that COVID-19 is similar to an acute mode of COPD caused by the SARS-CoV-2 infection, and therefore tiotropium may be effective for COVID-19 patients.The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects almost everyone in the world in many ways. We previously predicted antivirals  and non-antiviral drugs (tiotropium and rapamycin) that may inhibit the replication complex of SARS-CoV-2 using our molecular transformer–drug target interaction (MT–DTI) deep-learning-based drug–target affinity prediction model. In this study, we dissected molecular pathways upregulated in SARS-CoV-2-infected normal human bronchial epithelial (NHBE) cells by analyzing an RNA-seq data set with various bioinformatics approaches, such as gene ontology, protein–protein interaction-based network and gene set enrichment analyses. The results indicated that the SARS-CoV-2 infection strongly activates TNF and NFκB-signaling pathways through significant upregulation of the  The coronavirus disease COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 9 outbrea. Indeed,IL-6), interleukin 1B (IL-1B) and tumor necrosis factor (TNF) ), C–X–C motif chemokine ligand 10 (CXCL10), C–X–C motif chemokine ligand 9 (CXCL9) and C–X–C motif chemokine ligand 8  were also observed in H5N1 avian influenza virus-infected patients with severe lung injury [SARS-CoV-2 manipulates host defense systems, leading to overexpression of proinflammatory cytokines such as interleukin 6 , requires the angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the host cells . The ACEChronic obstructive pulmonary disease is characterized by respiratory symptoms such as dyspnea, cough, sputum production and airflow limitation . For theGiven that tiotropium can apply to multiple respiratory diseases, we hypothesize that it may have some impact on COVID-19. One small piece of evidence for our hypothesis comes from our previous study that identified several antiviral and non-antiviral drugs for COVID-19 . BrieflyThe purpose of this study is to define the molecular pathology of COVID-19 and to find evidence of top-ranked repurposed-drug candidates identified by our MT–DTI deep-learning-based drug–target affinity prediction model. To this end, we reanalyzed a recently available transcriptome data set, which was performed in normal human bronchial epithelial (NHBE) cells infected with SARS-CoV-2 . We usedhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147507, GSE147507). A batch effect correction between the groups was conducted using RUVSeq [p-value cutoff of 0.05. DEGs showing less than 1.5-fold-change between conditions were further discarded.A previous study  profiledg RUVSeq . Differeg RUVSeq  with an https://www.networkanalyst.ca/) with the minimum-order or zero-order network models [A list of upregulated DEGs  was usedk models . For thihttps://www.gsea-msigdb.org/gsea/msigdb/index.jsp) were used for the analysis.Gene set enrichment analysis (GSEA) was conducted using the fast gene set enrichment analysis (FGSEA) method . Hallmarhttp://159.149.160.88/pscan/) [Transcription factor binding motifs in the promoter regions of upregulated genes were predicted using PSCAN (/pscan/) . The prohttp://pantherdb.org/) [https://biit.cs.ut.ee/gprofiler/gost) with default parameters [Upregulated DEGs were annotated and functionally classified using the PANTHER gene ontology (GO) analysis application (db.org/) . In addirameters . The parhttps://software.broadinstitute.org/morpheus/) was used to draw heatmaps showing expression levels of given genes. The expression levels of the genes were normalized using the min–max normalization method [Morpheus , interleukin 8  and interleukin 6 (IL-6). Particularly, the tumor necrosis factor (TNF), intercellular adhesion molecule 1 (ICAM1) and nuclear factor kappa B subunit 2 (NFKB2) genes, which belong to the TNF- and/or NFκB-signaling pathways, were significantly upregulated. To gain further biologic insights, taking into account protein–protein interactions (PPI), we constructed lung tissue-specific PPI networks using NetworkAnalyst [n = 171). The results showed that ADRB2, NFKB2, NFKBIA, TNFAIP3, RELB, TNF and ICAM1 were the most highly connected proteins that may regulate various signaling pathways through PPI (ARDB2) gene, which is abundantly expressed in bronchial smooth muscle cells and plays important roles in regulating various systems, including cardiac, pulmonary and vascular systems [p-value < 1.41e−20) and/or NFκB-signaling  pathways were significantly overrepresented in the upregulated DEGs , BCL3 transcription coactivator (BCL3), RELB proto-oncogene (RELB), NFKB inhibitor alpha (NFKBIA), suppressor of cytokine-signaling 3 (SOCS3), TNF alpha induced protein 3 (TNFAIP3), baculoviral IAP repeat containing 3 (BIRC3), and ICAM1TNF  genes, which were significantly upregulated by SARS-CoV-2 infection, exert a profound influence on NHBE cells through the TNF- and/or NFκB-signaling pathways.To gain molecular insights on how the SARS-CoV-2 infection interferes with molecular signaling pathways in host cells, we reanalyzed RNA-seq data sets (GSE147507) performed in SARS-CoV-2-infected normal human bronchial epithelial (NHBE) cells cultured in bronchial epithelial growth media supplemented with BEGM SingleQuots . After bkAnalyst  with theough PPI B. Intere systems , was oneted DEGs D. Overalp-value < 2.90e−21) and TNF  signaling pathways, were significantly upregulated  and ‘rheumatoid arthritis’ , were significantly associated with the upregulated DEGs. Most of these genes were shared among the pathways. For example, the IL8, C–X–C motif chemokine ligand 2 (CXCL2), CSF2, IL6, TNF and interleukin 1 beta (IL1B) genes were overlapped between the ‘lung fibrosis’ and ‘rheumatoid arthritis’ pathways, while the IL6, TNF, ICAM1, IL1B, and NFKB2 genes were common in the ‘vitamin B12 metabolism’ and ‘photodynamic therapy-induced NF-kB survival-signaling’ pathways  were analyzed. We used the following 7 different gene sets for the analysis: ‘hallmark gene sets’, ‘positional gene sets’, ‘curated gene sets’, ‘regulatory target gene sets’, ‘GO gene sets’, ‘oncogenic gene sets’ and ‘immunologic gene sets’. The analysis revealed that there were common and unique gene sets in response to the virus infection. For example, ‘IL6 JAK-STAT3-signaling’, ‘interferon alpha response’ and ‘interferon gamma response’ pathways were upregulated in IAV-infected A549, SARS-CoV-2-infected A549 and SARS-CoV-2-infected NHBE cells compared to corresponding mock-infected cells, whereas they were downregulated in IAV-infected A549 cells according to the Hallmark database (IVL), transglutaminase 1 (TGM1), small proline rich protein 1A (SPRR1A), keratin 75 (KRT75), growth arrest specific 6 (GAS6), MAF BZIP transcription factor F (MAFF), cytochrome P450 family 27 subfamily B member 1 (CYP27B1), keratin 4 (KRT4), inhibin subunit beta A (INHBA), peptidoglycan recognition protein 4 (PGLYRP4), small proline rich protein 2A (SPRR2A), keratin 6B (KRT6B), keratin 24 (KRT24), small proline rich protein 2D (SPRR2D), and small proline rich protein 2E (SPRR2E) genes were the most highly upregulated genes among these pathways  and keratin  and small proline rich protein 1B (SPRR1B) genes were also upregulated uniquely to the SARS-CoV-2-infected NHBE cells. To understand the molecular mechanism, potentially controlling these genes, we searched for an upstream transcription factor by scanning promoter sequences of the genes. Intriguingly, the FOSL1  binding motif was significantly overrepresented   B. Concorp-value < 3.63e−04) such as serpin family B member 4 (SERPINB4), serpin family B member 1 (SERPINB1), serpin family B member 2 (SERPINB2), peptidase inhibitor 3 (PI3), cystatin B (CSTB), complement C3 (C3), serpin family A member 3 (SERPINA3), and BIRC3 were newly identified , NFKBIA, NFKB2, NFKB inhibitor zeta (NFKBIZ), MAF bZIP transcription factor F (MAFF) and interferon regulatory factor 7 (IRF7) were significantly upregulated, whereas MAF bZIP transcription factor (MAF) and PPARG coactivator 1 alpha (PPARGC1A) were downregulated in SARS-CoV-2-infected NHBE cells  or TNF  and TNF in the lung [There is an urgent need for finding effective treatment for COVID-19 patients. We had ranked available drugs based on affinity prediction using our MT–DTI deep-learning-based model and proposed available antiviral drugs that may act on inhibiting the replication complex of SARS-CoV-2 in a previous study . However) or TNF . Consequthe lung . On the the lung ,44, whilthe lung  and asththe lung , there athe lung . TherefoCXCL1), CXCL2, CXCL3, CXCL5 and CXCL6, were significantly upregulated as previously reported [CXCL14 with anti-inflammatory activity [IL1A), IL1B, IL32, IL36G, IL6 and IL8, were also upregulated in SARS-CoV-2-infected NHBE cells along with SOCS3, which has been shown to function as an IL-6 inhibitor [CSF3, IL8, CSF2, IL6, TNF, MMP9, IL1B and PDGFB) were significantly upregulated in SARS-CoV-2-infected NHBE cells , which are enzymes responsible for crosslinking [In this study, we attempted to define the molecular pathogenesis of COVID-19 by reanalyzing a transcriptome data set of SARS-CoV-2-infected NHBE cells  using vareported , while Cactivity  was subsnhibitor ,59. Thisnhibitor . For exanhibitor  showed tBE cells B. In addBE cells . These cslinking  and alsoslinking , were sislinking ,12,13,14slinking ,67.IVL), which is a marker of squamous metaplasia that is associated with airway obstruction in COPD. The squamous metaplasia of epithelial cells was also regarded as one of the main pathologic lung changes observed in COVID-19 patients [IVL gene was significantly upregulated in SARS-CoV-2-infected NHBE cells (IL1B, IL1A, S100A8, RHCG, KRT6B and SPRR1A genes, which were highly upregulated in the SARS-CoV-2-infected NHBE cells, were reported to be upregulated during a process of the squamous metaplasia in the same study [IL6, IL8 and MMP1 genes were increased in fibroblasts derived from lung tissues of COPD patients and the levels of the same were correlated with COPD stages. These genes were also significantly upregulated in SARS-CoV-2-infected NHBE cells. Interestingly, a recent study based on the human protein interactome also identified a similarity between COVID-19 and COPD [The molecular pathogenesis of COVID-19 inferred in this study implied that SARS-CoV-2-infected NHBE cells shared some features with those observed in COPD patients. For example, Araya et al.  reportedpatients . InteresBE cells A. In addme study . Furtherme study  reportedand COPD . Therefoand COPD ,72. BaseTiotropium—or similar treatments that the patients are receiving—would have several beneficial effects against SARS-CoV-2’s pathogenicity according to our proposed molecular pathogenesis of COVID-19. Tiotropium bromide has a favorable safety profile  and inhiWe provide all the results  used in


Drosophila to mammalian cells. Our findings demonstrate that Rack1 plays dual roles during Hh signal transduction and provide Rack1 as a potential drug target for Hh-related diseases.Hedgehog (Hh) pathway plays multiple roles in many physiological processes and its dysregulation leads to congenital disorders and cancers. Hh regulates the cellular localization of Smoothened (Smo) and the stability of Cubitus interruptus (Ci) to fine-tune the signal outputs. However, the underlying mechanisms are still unclear. Here, we show that the scaffold protein Rack1 plays dual roles in Hh signaling. In the absence of Hh, Rack1 promotes Ci and Cos2 to form a Ci–Rack1–Cos2 complex, culminating in Slimb-mediated Ci proteolysis. In the presence of Hh, Rack1 dissociates from Ci–Rack1–Cos2 complex and forms a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. Furthermore, we find the regulation of Rack1 on Hh pathway is conserved from  Drosophila through EMS-induced screening [decapentaplegic (dpp), ptc, and rdx [The evolutionarily conserved Hedgehog (Hh) pathway is first discovered in creening . Followicreening , 3, and creening , 5. In tcreening . Under tcreening . In turncreening –10. In tcreening , 12. Hh  and rdx , 14. As  and rdx . Contrad and rdx , suggest and rdx , but the and rdx , suggestDrosophila wing discs regardless of whether Hh is present [Another key component of Hh pathway is Smo, a GPCR-like transmembrane protein. Its cell surface accumulation is an essential step for Hh pathway activation. However, the underlying mechanisms governing Smo localization are still elusive. Inactivation of ubiquitin-activating enzyme Uba1 leads to Smo cell surface accumulation in  present , indicat present –22. The  present , 23, 24.The protein Rack1 (receptor for activated C-kinase 1) was originally identified as a receptor for protein kinase C . It interack1 increased Ci and Hh target genes expression, while overexpression of rack1 showed opposite results. Through genetic and biochemical analyses, we demonstrated that Rack1 interacted with both Ci and Cos2 and enhanced Ci–Cos2 association, resulting in Slimb-mediated Ci proteolysis. Intriguingly, with Hh stimulation, Rack1 dissociated from Ci–Cos2 complex and formed a trimeric complex with Smo and Usp8, leading to Smo deubiquitination and cell surface accumulation. These results revealed that Rack1 plays dual roles in coordinating Hh pathway activity. Furthermore, the mammalian ortholog of Rack1 had a similar role in regulating Hh pathway. Taken together, our study demonstrated that the Rack1 plays dual roles in Hh pathway, and provided Rack1 as a prudent drug target for Hh-related cancers.To find novel components of Hh signaling, we carried out a genetic screen and identified Rack1 as a regulator of Hh pathway. Knockdown of Drosophila wing, hh expresses exclusively in the posterior (P) compartment cells, while the unique transcription factor ci only expresses in the anterior (A) cells. After autoproteolytic cleavage and modification, the mature Hh protein is secreted from the P cells and moves to A cells to form a concentration gradient [nub-gal4 driver. Through this screening, we found that knockdown of rack1 apparently increased the area of L3/L4 intervein  by ApG4 apparently upregulated dpp-lacZ level , which the glutamine on 6 is replaced by a stop codon (Q6stop). Since rack11.8 fly was homozygous lethal at first instar larval stage, we employed FLP/FRT-mediated mitotic recombination and examined the expression of Hh target genes [rack11.8 homozygous clones, which were marked by loss of GFP expression, showed increased Ptc protein level , which only recognizes the full-length Ci [rack1 upregulated Ci protein levels in both wing and eye discs  and two Src homologs (Src42A and Src64B). Compared with the control disc  by adding a myristoylation signal at its N-terminus. Overexpression of Myr-rack1 decreased Ci protein  failed to affect Ci , Rack1 quickened the degradation of Ci  cell lines . In normSmo Fig. . Furthernes Fig. . We alsoion Fig. , suggestusp8 blocked rack1 RNAi-induced Smo degradation , and cloned them into the pUAST-Myc, pUAST-HA or pUAST-Fg backbone vectors respectively. UAS-Rack1-Y229/247F was generated via PCR-based site-directed mutagenesis. Truncated constructs including Rack1-WD2-7, Rack1-WD3-7, Rack1-WD4-7, Rack1-WD5-7, and Smo-C were generated by inserting the corresponding coding sequences into the indicated backbone vectors. A nuclear localization signal (NLS) from SV40 (PPKKKRKV) was inserted at the C terminus to generate Rack1-NLS plasmid. A myristoylation signal from Src (MGSSKSKPKDPSQRRRSLE) was inserted at the N terminus to generate Myr-Rack1. The GFP coding sequence was fused to the N terminus of Rack1 to get Rack1-GFP.MS1096 (BDSC #8860), ApG4 (BDSC #3041), dpp-lacZ [kn-lacZ [ptc-lacZ (BDSC #10514), En-gal4 (BDSC #6356), HA-rdx [slimb [usp8 was described previously [nub-gal4 (BDSC #42699), rack1-RNAi , rack11.8 (BDSC #24152), mirr-G4 (BDSC #29650), ci-lacZ (BDSC #6303), GMR-gal4 (BDSC #8605), cos2-RNAi (BDSC #44472), UAS-cos2 (BDSC #67187), UAS-cos2-S572A (BDSC #55046), UAS-cos2-927-935A (BDSC #55047), PKC53E-RNAi (NIG #6622R-1), PKC98E-RNAi (NIG #GL00174), PKN-RNAi (NIG #2055R-1), PKCδ-RNAi (BDSC #28355), aPKC-RNAi, UAS-PKCi (BDSC #4589), Src42A-RNAi (NIG #HMS02755), Src64B-RNAi (NIG #HMC03327), and slimb-RNAi  were obtained from BDSC or NIG. The detailed information of ptc-lacZ, dpp-lacZ, kn-lacZ, and ci-lacZ reporters have been described in FlyBase database (available at http://flybase.bio.indiana.edu/). UAS-rack1, UAS-rack1-GFP, UAS-rack1-NLS, UAS-Myr-rack1, UAS-Fg-hRack1, and UAS-rack1-Y229/247F transgenic flies were generated by injection of indicated constructs into Drosophila embryo according to the method described previously [Some stocks used in this study are kindly from Dr. Qing Zhang lab, such as dpp-lacZ , kn-lacZ[kn-lacZ , ptc-lac, HA-rdx , and Fg-x [slimb . UAS-uspeviously . UAS-GFPeviously . All storack1 mutant clones were as follows: hs-flp; rack11.8 FRT40A/ubi-GFP FRT40A. For mutant clone generation, the 1st larvae were heat shock at 37 °C for 1.5 h and kept at 25 °C for 2–3 days. The 3rd larvae were subjected to dissection according to standard protocols. Loss of GFP expression marked the mutant clones.Clones of mutant cells were generated by FLP/FRT-mediated mitotic recombination as previously described . GenotypImmunostaining of discs were performed with previous protocols . In brieDrosophila Medium (Gibco) with 10% heat-inactivated FBS (Gibco) and 1% penicillin/streptomycin (Sangon Biotech). 293T cells were purchased from the ATCC and cultured in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% FBS and 1% penicillin/streptomycin. Before experiments, cells were tested to be out of mycoplasma contamination. Cells were transfected using PEI (Sigma) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were harvested for IP and immunoblotting (IB) analysis with standard protocols as described [rack1 in S2 cells, the double stranded RNA (dsRNA) was generated by MEGAscript High Yield Transcription Kit (Ambion) according to the manufacturer’s instructions. Rack1 dsRNA targets the full-length coding sequence. dsRNA targeting the gfp full-length coding sequence was used as a negative control. The dsRNA was transfected into S2 cells by ExFect 2000 (Vazyme) according to the manufacturer’s instructions. For OA treatment, transfected S2 cells were treated with 10 μM OA (CST) for 2 h before cell harvesting. 10% acrylamide phos-tag gel with 20 μM phos-tag (Wako) acrylamide was used to examine Rack1 phosphorylation according to the manufacturer’s instructions. The following antibodies were used for IB: mouse anti-HA ; mouse anti-Myc ; mouse anti-Fg ; mouse anti-Ub ; mouse anti-Actin ; mouse anti-hRack1 ; rabbit anti-hSmo ; rabbit anti-Gli3 ; rabbit anti-Kif7 ; goat anti-mouse HRP  and goat anti-rabbit HRP .S2 cells (gift from Dr. Erjun Ling lab) were maintained in Schneider’s escribed . Two-steescribed . To knocProtein pulse-chase experiments were performed as previously described . Briefly® Q RT SuperMix with gDNA wiper (Vazyme) according to the instructions. Real-time PCR was performed on BIO-RAD CFX96TM with ChamQ SYBR® Color qPCR Master Mix . 2−∆∆Ct method was used for relative quantification. The primer pairs used were as follows: rack1, 5′-GAGCACAACGACATCATC-3′ (forward) and 5′-CTCC-TCAACGGTCTTCTT-3′ (reverse); ci, 5′-AGCGAGTAATACTTCGGTTA-3′ (forward) and 5′-TGATTGGTAGATGTGTTGTTC-3′ (reverse); ptc, 5′-AGATCGG- GCAAATCCTATG-3′ (forward) and 5′-GTCAGCGATGGATCATTAAG-3′ (reverse); dpp, 5′-CCTGGTCAACAATATGAATCC-3′ (forward) and 5′-TCATCTCCTGGTA-GTTCTTC-3′ (reverse); kn, 5′-CTTCAATTCCACGTCAAGG-3′ (forward) and 5′-TATAGCCCTGTGTATTGCA-3′ (reverse); actin, 5′-GTACCCCATTGAGCACGG-TA-3′ (forward) and 5′-CGAACATGATCTGGGTCATC-3′ (reverse). Data are presented as means ± SD of values from three experiments.About 40 wing discs were lysed in TRIzol (Invitrogen) for RNA isolation according to previous described . One mict test, and P < 0.05 was considered statistically significant. Where exact P values are not shown, statistical significance is shown as with ns, no significance, *P < 0.05, **P < 0.01 and ***P < 0.001.The density of western blot band or immunostaining was measured by Image J software. Statistical analysis was performed with GraphPad Prism software. The data shown in the Figures were representative of three or more independent experiments and were analyzed by one way student’s FigureS1FigureS2FigureS3FigureS4Supplementary information


Caenorhabditis elegans naturally thrives in a soil environment where they feed on bacteria and are in constant association with a diverse range of microbes . C. elegans egg laying is delayed or reduced when animals are infected with Burkholderia pseudomallei, Burkholderia thailandensis, Staphylococcus aureus, and Serratia marcescens . These changes in egg laying may be a protective response to pathogenic bacteria. Mutants of TGF-β-like DBL-1 signaling pathway also display reduced brood size . While the peak of egg-laying activity seen in normal animals between days two and four is depressed in dbl-1 pathway mutants, the reproductive span of these dbl-1 pathway mutants is increased to up to 13 days . To determine if the egg-laying observed during infection is DBL-1 pathway-dependent, we tested the effect of the DBL-1 signaling pathway on egg laying when C. elegans were fed on representative Gram-negative (S. marcescens) and Gram-positive bacteria (Staphylococcus epidermidis).E. coli strain OP50 and 20°C incubation) .Similar to previously published reports, we found that loss of DBL-1 pathway signaling decreases brood size and increases reproductive span in normal laboratory conditions (ubation)  and B (Ldbl-1(nk3) animals both significantly decrease their brood size when grown on S. marcescens . dbl-1 mutant animals laid even fewer eggs than the wild-type animals on S. marcescens, suggesting that the reduced brood size phenotype is independently affected by both S. marcescens exposure and by loss of DBL-1 (p< 0.001). While the decrease in brood size of wild-type animals on S. epidermidis was not significant (p= 0.115), the decreased brood size of dbl-1(nk3) animals was significant on this pathogenic bacterial strain (p= 0.045). Indeed, the decreases in brood size upon infection with either S. marcescens or S. epidermidis in both wild-type and dbl-1(nk3) populations are similar (p= 0.57), suggesting the pathogenic bacteria affect brood size independent of DBL-1. Because dbl-1(nk3) populations display a further reduced brood size upon infection by either pathogen compared to the wild type, the negative effects of pathogen exposure and loss of DBL-1 signaling on brood size appear to be additive (p< 0.05).Here, we report three new results. First, brood size reductions caused by infection and by loss of DBL-1 signaling are independent . Wild-tyS. marcescens survived until all animals ceased laying eggs, all dbl-1(nk3) animals died on S. marcescens by Day 5. These results explain why the extended reproductive span normally seen in dbl-1(nk3) populations was not observed on S. marcescens. These results also support previous reports of decreased viability of dbl-1 mutant animals on another variety of S. marcescens, Db11 .Second, while the wild-type population on S. epidermidis affects egg-laying patterns similar to loss of dbl-1 function. Initially, both wild-type and dbl-1(nk3) strains on S. epidermidis have reduced eggs laid in the first four days compared to strains grown on the E. coli control. Loss of DBL-1 further reduced the number of eggs laid during each of these days, suggesting that this phenotype is independently affected by both S. epidermidis exposure and by loss of DBL-1 (p= 0.004). Then, both wild-type and dbl-1(nk3) strains on S. epidermidis have similar extended reproductive spans, extending to at least day 13 (one tenacious wild-type hermaphrodite laid embryos until day 15). This S. epidermidis-induced reproductive span extension appears to be independent of DBL-1 signaling, because the numbers of eggs laid by both wild-type and dbl-1(nk3) populations between days 5 and 16 were similar at these time points (p= 0.509).Third, Escherichia coli OP50. Ten L4 animals were manually transferred to individual plates seeded with S. marcescens or S. epidermidis. Plates were completely covered by bacteria to prevent animals from avoiding the bacteria. Adults were daily transferred to new plates and the number of eggs laid on each plate was counted every 24 hours until no more eggs were laid. Statistical analyses were performed using repeated measures ANOVA and Tukey’s post-hoc test.Animals were age-synchronized by hypochlorite treatment  and grown on plates seeded with TM Peptone, 8 mg cholesterol, 2 g sodium chloride, 20 g agar, in water to 1 L . The C. elegans strains used were N2 and NU3 dbl-1(nk3). The Gram-negative bacterial strains used were Escherichia coli OP50 (CGC) and Serratia marcescens . The Gram-positive bacterial strain used was Staphylococcus epidermidis (ATCC 49134). S. marcescens and S. epidermidis were provided by A. J. Hammett, TWU. All bacterial strains were grown for 9 hours in tryptic soy broth at 37°C before plating on EZ media plates.Strains were maintained on EZ media plates at 20°C (0.55 g Tris-Cl, 0.24 g Tris base, 3.1 g BD Bacto


Drosophila. Terminal neuroblasts (NBs) in A8-A10 segments of Drosophila larval CNS are subdivided into two groups based on expression of transcription factor Doublesex (Dsx). While the sex specific fate of Dsx-positive NBs is well investigated, the fate of Dsx-negative NBs is not known so far. Our studies with Dsx-negative NBs suggests that these cells, like their abdominal counterparts (in A3-A7 segments) use Hox, Grainyhead (Grh) and Notch to undergo cell death during larval development. This cell death also happens by transcriptionally activating RHG family of apoptotic genes through a common apoptotic enhancer in early to mid L3 stages. However, unlike abdominal NBs (in A3-A7 segments) which use increasing levels of resident Hox factor Abdominal-A (Abd-A) as an apoptosis trigger, Dsx-negative NBs (in A8-A10 segments) keep the levels of resident Hox factor Abd-B constant. These cells instead utilize increasing levels of the temporal transcription factor Grh and a rise in Notch activity to gain apoptotic competence. Biochemical and in vivo analysis suggest that Abdominal-A and Grh binding motifs in the common apoptotic enhancer also function as Abdominal-B and Grh binding motifs and maintains the enhancer activity in A8-A10 NBs. Finally, the deletion of this enhancer by the CRISPR-Cas9 method blocks the apoptosis of Dsx-negative NBs. These results highlight the fact that Hox dependent NB apoptosis in abdominal and terminal regions utilizes common molecular players , but seems to have evolved different molecular strategies to pattern CNS.Neural circuitry for mating and reproduction resides within the terminal segments of central nervous system (CNS) which express Hox paralogous group 9–13 (in vertebrates) or Abdominal-B (Abd-B) in  Drosophila larval central nervous system highlights that region-specific Hox-dependent cell death of neural stem cells in abdominal and terminal regions utilizes common molecular players , but seems to have evolved different molecular strategies to pattern the developing central nervous system.Two major characteristic features of bilaterian organisms are the head to tail axis and a complex central nervous system. The Hox family of transcription factors, which are expressed segmentally along the head to tail axis, plays a critical role in determining both of these features. One of the ways by which Hox factors do this is by mediating differential programmed cell death of the neural stem cells along the head to tail axis of the developing central nervous system, thereby regulating the numerical diversity of the neurons generated along this axis. Our study with a subpopulation of neural stem cells in the most terminal region of the  CNS). This precision critically relies on generation of right cellular diversity (and numbers) in a region specific manner across the anterior-posterior (AP) axis of developing CNS , leading to a common end result of NB apoptosis.Existing literature implicates the role of Notch and Grh in determining the competence of the NBs , 46–49. F and resisted in males by DsxM-which is known to block NB apoptosis -GAL4, UAS-mCD8-GFP, hsflp1, w ; yw; tub-GAL80-LL9 FRT80B ; yw; FRT82B tub-GAL80-LL3 (BDSC 5135); Notch-RNAi (BDSC 28981), Dl-RNAi (BDSC 34322); Abd-B-RNAi (VDRC 12024), grh-RNAi (VDRC 101428) abd-A-RNAi (VDRC 106155), exd-RNAi (VDRC 7802) and hth-RNAi (NIG-17117-R4 and R2). F3B3-lacZ; 717-lacZ, Grhmutant-lacZ; Su(H)mutant-lacZ; Hox-Exd-Grhmutant-lacZ (AT-Grhmutant-LacZ) -GAL4, UASmCD8::GFP, hsflp1, w; FRT82B tub-GAL80-LL3 were crossed to males of FRT-Abd-BM1 /TM6Tb.FRT-exd1 MARCM clones: virgin females of the genotype FRT-exd1/FM7 were crossed to males of hsflp, FRT19A, tubGAL80; tub-GAL4, UASmCD8GFP/SM6 CyO-GFP.FRT-hthP2 MARCM clones: virgin females of genotype elav[C155]-GAL4, UASmCD8::GFP, hsflp1, w; FRT82B tub-GAL80-LL3 were crossed to males of FRT-hthP2/TM6Tb.All the eggs collected for a duration of 4 hours at 25°C were subjected to periodic heat-shock of 1 hour at 37°C after every 12 hour starting from the time of larval hatching to mid third instar larval stage. The dissections were performed at the late third instar larval stage.EMSAs were performed as described previously . For EMSThe following constructs were used for the affinity pulldown assay: N-terminal-GST-tagged fusion proteins were used for Abd-B-N (aa residues 313 to 493) and His tagged Grh (551–1333 aa). Bacterial cultures of GST tagged Abd-B-N and His tagged Grh were induced at OD600 with 0.5 mM IPTG for 2 h and 3 h at 18°C. The affinity pulldown experiments were performed as described in a previous report [S1 Fig(A-H) The temperature shift (TS) protocols used in different experiments are shown in different panels. The downward facing arrow indicates the time of dissection of the larvae. L1 and L2 stages were divided into three 8hr intervals which defined early, mid and late stages. L3 stage was divided into three 16 hr intervals to define early, mid and late L3 stages.(TIF)Click here for additional data file.S2 Figabd-A-RNAi is induced from early L1 stage. Yellow arrowheads indicate Dsx-negative NBs in panels “A-E”. Scale bars are 10μm. All images are single confocal sections except for panel “F” which is a partial z-project of confocal stacks. Graph shows mean±s.d. Significance  is from two-tailed Student's unpaired t-test.(A-C) Show the expression of apoptotic markers Dcp-1 and Casp-3 in A8-A10 NBs of female VNCs in mid L2 (A) and mid L3 stage (B-C). Dsx-positive NBs in female VNC die at mid L2 stage, therefore Dcp-1/Casp3 positive NBs in CNS at mid L3 stage are Dsx-negative NBs. (D-E) Show that expression of p35 (D) and knockdown of Abd-B (E) results in a block of Dsx-negative NB apoptosis in late L3 stage female VNCs. (F) Shows that knockdown of Abd-A by RNAi from early L1 stage TS,  results (TIF)Click here for additional data file.S3 Fig(A-E) Show Notch-GFP marked NBs and associated lineages expressing Notch ligand Delta in female VNCs at early L2 (A), late L2 (B), early L3 (C), mid L3 (D) and late L3 stages (E). (F) Shows Delta staining in NBs and associated lineages in female VNC at late L3 stage wherein expression of p35 blocks the death of A8-A10 NBs TS, . (G) Sho(TIF)Click here for additional data file.S4 Figgrh-GAL4 driven expression of UAS-nls-GFP closely correlate with Grh protein expression in abdominal and terminal NBs in early L2 (A), late L2 (B), early L3 (C) and mid L3 stages (D). (E-G) Shows that compared to wild type, grimA6C-reaper17 homozygous double mutants and MM3/M22 transheterozygotes show a block of Dsx-negative NB apoptosis in late L3 female VNCs. (H-J) Shows the expression of 717-lacZ in A8-A10 NBs in female VNCs in mid L2 (H), early L3 (I) and mid L3 (J) stages. (K) Graph showing the quantitation of lacZ intensity in A8-A10 NBs across different stages. Panels “E-G” are single confocal sections; rest all panels are partial z-projects. Yellow arrowheads indicate A8-A10 NBs. White arrowheads in panels “A-D” show abdominal NBs and in panels “F and G” indicate Dsx-positive NBs. Scale bars are 10μm. Graph shows mean±s.d. Significance  is from two-tailed Student's unpaired t-test.(A-D) Show that (TIF)Click here for additional data file.S5 Fig(A-I) EMSA autoradiogram for Abd-B, Grh and Abd-B-Grh binding on oligonucleotides for maintenance motifs-25 (A), 27 (E), 28 (D), 30 (B), 31 (C), 32 (G), 33 (F) and 34 (H) of 717bp enhancer are shown. Wild type and Hox mutant oligonucleotide sequences used are shown at the bottom of each gel. Hox and Grh binding sequences are colour coded in red and blue respectively. Mutations are shown in small case. Only motif-33 (F) shows Abd-B-Grh-DNA complex formation. (I) Shows a schematic of 717bp enhancer showing the relative position of various binding motifs. Proteins added to a specific lane are shown at the top of each lane. Lane with free probes are indicated by downward facing black arrows. EMSA indicate that Abd-A binding sites are also capable of binding Abd-B. Blue rectangles indicate a constant concentration of 200ng for Grh protein. An increasing concentration of 50ng and 100ng of Hox are indicated by red right triangles. Red arrow heads on the gels indicate Hox-DNA complex; while blue arrow heads indicate the Grh-DNA complex. Black arrowhead in panel “F” indicates Grh-Abd-B-DNA complex.(TIF)Click here for additional data file.S6 FigF3B3-lacZ, 717-lacZ and its mutant versions Grhmutant-lacZ, ATmutant-lacZ, AT-Grhmutant-lacZ and Su(H)mutant-lacZ (used in in vitro binding by Su(H) (indicated by black arrowhead). (C) EMSA autoradiogram showing that Su(H) does not bind on motif-30 which has Hox-Exd-Grh binding sites, lane-1 and 4 show free probes. Right triangle on top of EMSA indicates an increasing concentration of 150 and 300 ng of Su(H) protein. Sequence of oligonucleotides is shown at the bottom of EMSA. (D) Western blot showing in-vitro pulldown assay for bacterially expressed GST-tagged Abd-B  is able to pull down His-tagged Su(H) (lane-8 vs 9). (E) Western blot showing in-vitro pulldown assay for bacterially expressed GST-tagged Su(H)  is able to pull down His-tagged Grh (lane-11 vs 12). Coomassie Blue depicts almost equal loading of the GST-tagged protein samples in D and E.(A) Shows schematic of 1Kb (TIF)Click here for additional data file.S7 FigP-value) is from two-tailed Student's paired t-test.(A-D) Show expression of Grh and E(spl)mγ-GFP across early L2 (A), late L2 (B), early L3 (C) and mid L3 stages (D) in abdominal and terminal NBs of female VNCs. (E-G) Show graphs depicting quantitation of E(spl)mγ-GFP (E), Grh (F), and control Dpn staining (G) intensities across different stages. Graphs indicate that expression of Grh and E(spl)mγ-GFP is mostly constant across different stages in abdominal NBs. Slight but significant difference in E(spl)mγ-GFP expression is seen from early L3 to mid L3 stage. Partial z-projects are shown for A-D to show both abdominal and terminal NBs (indicated by white and yellow arrowheads respectively). Scale bars are 10μm. Graph shows mean±s.d. Significance ((TIF)Click here for additional data file.S8 FigP-value) is from two-tailed Student's unpaired t-test. Show that compared to A8-A10 NBs in control VNCs, knockdown of Grh (A-B) and Abd-B (D-E) does not affect E(spl)mγ-GFP expression in mid L3 stage VNCs.  Graphs showing quantitation of E(spl)mγ-GFP fluorescence in A8-A10 NBs of control VNCs versus Grh knockdown (C) and Abd-B knockdown (F). (G-I) Show that compared to p35 expressing A8-A10 NBs, knockdown for Grh (H) or Notch (I) TS,  does not(TIF)Click here for additional data file.S9 FigP-value) is from two-tailed Student's unpaired t-test.(A-B) Show that compared to control male VNC overexpressing GFP (A), overexpression of Abd-B in Dsx-positive NBs does not result in their apoptosis, indicating that Dsx-positive NBs in males are refractory to apoptosis induced by Abd-B. (C-E) Show that compared to controls (C) overexpression of Grh (D) or NICD (E) from early L1 stage TS,  does not(TIF)Click here for additional data file.S10 FigF (shown in red) cooperate to activate apoptotic enhancer to cause female specific apoptosis. This happens on a specific set of Abd-B-Dsx binding motifs (shown by green boxes) on the enhancer (shown by black line). While Dsx-negative NBs undergo apoptosis in mid L3 stage relying on presence of Abd-B, Grh (shown by dark green filled circle) and Notch (shown in dark blue triangle) using a completely different set of binding motifs (shown as light blue boxes). These motifs are required for maintenance of the apoptotic enhancer in Dsx-negative NBs. Abdominal NBs in A3-A7 segments also rely on the same motifs (shown as light blue boxes) for maintaining the activity of the enhancer in abdominal NBs.Dsx-positive NBs die at mid L2 stage, wherein Abd-B (shown in purple) and Dsx(TIF)Click here for additional data file.S1 Data(DOCX)Click here for additional data file.


Forkhead transcription factors (TFs) often dimerize outside theirextensive family, whereas bHLH transcription factors typically dimerize withE12/E47. Based on structural similarities, we predicted that a member of theformer, Forkhead Box P1 (FOXP1), might heterodimerize with a member of thelatter, MYOD1 (MyoD). Data shown here support this hypothesis and furtherdemonstrate the specificity of this forkhead/myogenic interaction among othermyogenic regulatory factors. We found that FOXP1-MyoD heterodimerizationcompromises the ability of MyoD to bind to E-boxes and to transactivate E box-containing promoters. We observed that FOXP1 is required for the full ability ofMyoD to convert fibroblasts into myotubules. We provide a model in which FOXP1displaces ID and E12/E47 to repress MyoD during the proliferative phase ofmyoblast differentiation. These data identify FOXP1 as a hitherto unsuspectedtranscriptional repressor of MyoD. We suggest that isolation of paired E-box andforkhead sites within 1 turn helical spacings provides potential for cooperativeinteractions among heretofore distinct classes of transcription factors. The five p1 members of the ~100 forkhead (Fkh) transcription factor(TF) family function primarily as transcriptional repressors by employing a highlyconserved Forkhead (Fkh) domain to bind DNA with high specificity followinghomodimerization . One sucAnother family of dimerizing TFs critical for heart development and othercellular systems are characterized by their basic helix-loop-helix (bHLH) DNAbinding domains. These include four highly conserved myogenic regulatory factors(MRFs) ,14. MuscMyoD1 transcription is largely limited to embryonic somiticprecursors and differentiated myogenic cells -conjugated anti-rabbit or anti-mouse)were purchased from Abcam. Filamentous actin (F- actin) was stained withFITC-conjugated phalloidin . Nuclei were labeledwith 4’-6-diamidino-2-phenylindol (DAPI).For immunoprecipitation, we employed protein-A immobilized on SepharoseCL-4B (Cat.No. P3391) from Sigma Chemical Company .Lysates from transfected or untransfected C2C12 and 10T1/2 cells wereprepared under non-dissociating conditions for SDS–polyacrylamide gelelectrophoresis (SDS-PAGE) fractionation and immunoblotting. Our Westernblotting procedure, described previously , was per32]ATP (6000 Ci/mmol). The probe was separated fromunincorporated [γP32]ATP on a Sephadex G-50 spin column. Thetypical EMSA mixture (20 pl) contained 12.5 mM Tris (pH 7.9), 50 mM KCI, 5 mMMgCl2 7.5% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 pg of poly(dI-dC),different concentrations of the bacterial-synthesized or in vitro translatedproteins, and 10-20 fmol of 32P-labeled probe. In most cases, 1 pl ofwhole-cell extract or nuclear extract (10 mg/ml) was added to the reaction. Whenheterodimeric complexes were formed, the proteins were added before the probeand left to incubate at 37°C for 10 min.Probes were labeled with polynucleotide kinase andUTP (800Ci/mmol). Reactions were terminated by the addition of RNase T1 (BoehringerMannheim) at 300C for 60 min, 0.2% SDS and proteinase K (660 ug/ml). After 10min at 37°C, reaction mixtures were extracted with phenol/chloroform 1:1(vol/vol), and transcripts were precipitated with ethanol. Reactions were thenvisualized on SDS-PAGE.Transcription reaction mixtures for crude nuclear extracts are detailedin Bengal et al. . NuclearSupplementary Figures and TableSupplementary Figure Legends and Methods