Lupinus luteus L.), a species that shows cleistogamy, the anthers split before the flowers open, but the course and regulation of this process are unknown. The specific control of anther development takes place via hormonal pathways, the wide action of which ensures reproductive success. In our previous research concerning flower and early pod development in yellow lupine, we showed that the lowest transcript level of LlDELLA1, a main repressor of gibberellin (GA) signalling, occurs approximately at the time of anther opening; therefore, the main purpose of this study was to precisely investigate the gibberellic acid (GA3)-dependent regulation of the anther dehiscence in this species.Anther dehiscence resulting in the release of pollen grains is tightly regulated in a spatiotemporal manner by various factors. In yellow lupine , and identified several genes widely associated with this process. The expression profile of genes varied over time, with the most intense mRNA accumulation in the phases prior to or at the time of anther opening. The transcriptional activity also revealed that these genes are highly coexpressed and regulated in a GA-dependent manner. The cellular and tissue localization of GA3 in the control of the extensive anther dehiscence process in yellow lupine. Understanding the processes underlying pollen release at the hormonal and molecular levels is a significant aspect of controlling fertility in this economically important legume crop species and is of increasing interest to breeders.The presented results show a clear contribution of GAThe online version contains supplementary material available at 10.1186/s12870-021-03085-4. Arabidopsis thaliana and rice (Oryza sativa) . The nucleotides are marked with lowercase letters, and amino acids are marked with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TGA codons, respectively. The ORF is shown in pink. Fig. S1b. Maximum likelihood phylogenetic tree of 15 cinnamyl alcohol dehydrogenase (CAD) proteins (BlastP) with the highest degree of similarity to LlCAD. Alignment and phylogenetic reconstructions were performed using the Environment for Tree Exploration3 (ETE3) v3.1.1 program as implemented in GenomeNet. The ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Fig. S1c. Multiple alignment  of 15 cinnamyl alcohol dehydrogenase (CAD) amino acid sequences (BlastP) that are closely related to LlCAD. The alcohol dehydrogenease GroES-like domain is shaded medium grey, and the zinc-binding dehydrogenase domain is shaded dark grey. The Zn-1 and Zn-2 binding motifs are shaded with violet. Three green amino acids, C, H, and C, are marked with green dots. Yellow letters define four cysteine (C) residues. Conserved glycine (G) residues (GxGGxG) are indicated with red letters. They represent NADPH cosubstrate-binding motif, which is highlighted in green. The conserved residues  are highlighted in blue. The serine (S) 212 is labelled with a red background. Most of the alignment information was identified according to the results described by . The nucleotides are marked with lowercase letters, and amino acids with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TAA codons, respectively. The ORF is shown in pink. Fig. S2b. Maximum likelihood phylogenetic tree of 12 CesA8 proteins  with the highest degree of similarity to LlCesA8/LlIRX1. Alignment and phylogenetic reconstructions were performed using the ETE3 v3.1.1 program as implemented in GenomeNet. The ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Fig. S2c. Multiple alignment  of 12 CesA8/IRX1 amino acid sequences (BlastP) that are closely related to LlCesA8/LlIRX8. Proteins contain N-terminus; globular/soluble central domain (CD) and the C-terminus. Short N-terminus (N) is prior to the Zinc-binding domain (Zn) (medium gray). This Zn domain contains strictly conserved CxxC motif (green box and red letters) beginning amino acids: CxxCx12FxACxxCxxPxCxxCxExxxxxDxxxCxxC, where x is any amino acid. Within the N-terminus is also a region rich in acidic amino acids designated as a hypervariable region . Following the N-terminal domains are two transmembrane domains . The CD contains variable region 2  composed mostly of the Class Specific Region (CSR) which on either side is flanked by conserved regions: CR1 [with the Plant Conserved Region (P-CR) in the middle highlighted in light grey)] and CR2. The four recognized catalytic motifs are marked as blue boxes, with the D, DxD, and D residues in bold red and the QxxRW residues in bold orange. The numerous basic residues of Arg (R) and Lys (K) (green leterrs) and acidic residues of Asp (D) and Glu (E) (red letters) of the VR2/CSR, as well as the conserved Cys  are conserved across species in an isoform-specific manner . The nucleotides are marked with lowercase letters, and amino acids with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TGA codons, respectively. The ORF is shown in pink. Fig. S3b. Maximum likelihood phylogenetic tree of 14 COBRA-like 4 (COBL4) / irregular xylem 6 (IRX6) proteins (BlastP) with the highest degree of similarity to LlCOBL4/LlIRX6. Alignment and phylogenetic reconstructions were performed using the ETE3 v3.1.1 program as implemented in GenomeNet. The ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Lang - Lupinus angustifolius (XP_019452983); Cc - Cajanus cajan (XP_020240142); Ap - Abrus precatorius (XP_027353541); Ss - Spatholobus suberectus (TKY65050); Mp - Mucuna pruriens (RDX75309); Ad - Arachis duranensis (XP_015946272); Ai - Arachis ipaensis (XP_016182359); Vr - Vigna radiata (XP_014522888); Gs - Glycine soja (XP_028215469); Ah - Arachis hypogaea (XP_025624246); Mt - Medicago truncatula (XP_013451095); Gm - Glycine max (XP_003554956); Pa - Prosopis alba (XP_028764818); At - Arabidopsis thaliana (NP_197067). Fig. S3c. Multiple alignment  of 14 COBL4/IRX6 amino acid sequences (BlastP) that are closely related to LlCOBL4/LlIRX6. Conserved domain characteristic to COBRA superfamily is marked in green background. The N-terminal predicted signal peptide is marked yellow, with cleavage site marked by black arrow. The underlined sequences with red letters show the putative cellulose-binding sites. The light blue sequences denote the Cys-rich (CCVS) motif characteristic for all COBL proteins, while CCVS amino acids are marked in dark blue background and white letters. Two conserved consensus N-glycosylation sites are indicated by a white letters on violet backgrounds. Locus corresponds to the predicted cleavage ω sites in the C terminus is indicated in middium grey background and black arrow. Fig. S3d. Comparison of COBL4/IRX6 proteins (BlastP) derived from different plant species using the DiAlign program (Genomatix). For each pairwise alignment, the similarity (relative to the maximum similarity) and the number of identical amino acids (in % of shorter sequence) are given. Maximum values are underlined. The similarity value of 1.000 denotes only the two most similar sequences; it does not necessarily mean that these sequences are identical. Lang - Lupinus angustifolius (XP_019452983); Cc - Cajanus cajan (XP_020240142); Ap - Abrus precatorius (XP_027353541); Ss - Spatholobus suberectus (TKY65050); Mp - Mucuna pruriens (RDX75309); Ad - Arachis duranensis (XP_015946272); Ai - Arachis ipaensis (XP_016182359); Vr - Vigna radiata (XP_014522888); Gs - Glycine soja (XP_028215469); Ah - Arachis hypogaea (XP_025624246); Mt - Medicago truncatula (XP_013451095); Gm - Glycine max (XP_003554956); Pa - Prosopis alba (XP_028764818); At - Arabidopsis thaliana (NP_197067). Fig. S4a. LlGAUT12  cDNA  identified in yellow lupine (Lupinus luteus L.) and its deduced amino acid sequence . The nucleotides are marked with lowercase letters, and amino acids with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TAG codons, respectively. The ORF is shown in pink. Fig. S4b. Maximum likelihood phylogenetic tree of 15 galacturonosyltransferase 12 (GAUT12) / irregular xylem 8 (IRX8) proteins (BlastP) with the highest degree of similarity to LlGAUT12/LlIRX8. Alignment and phylogenetic reconstructions were performed using the ETE3 v3.1.1 program as implemented in GenomeNet. The ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Lalb - Lupinus albus (KAE9588601); Lang - Lupinus angustifolius (XP_019437125); Gm - Glycine max (XP_003543290); Ap - Abrus precatorius (XP_027341076); Pp - Prunus persica (XP_007213904); Pm - Prunus mume (XP_008244342); Ca - Cicer arietinum (XP_004487615); Ss - Spatholobus suberectus (TKY48985); Cc - Cajanus cajan (XP_020235268); Ah - Arachis hypogaea (XP_025614714); Ad - Arachis duranensis (XP_015936483); Ai - Arachis ipaensis (XP_016170753); Md - Malus domestica (XP_008349951); Dz - Durio zibethinus (XP_022716962); At - Arabidopsis thaliana (NP_200280). Fig. S4c. Multiple alignment  of 15 galacturonosyl transferase 12 (GAUT12) / irregular xylem 8 (IRX8) amino acid sequences (BlastP) that are closely related to LlGAUT12/LlIRX8. N-terminal cytoplasmic domain and the transmembrane domain  were predicted using the protein homology/analogy recognition engine V 2.0 (Phyre2) web portal. The specific glycosyl transferase family 8 (GT8) domain  and the catalytic DxD motif (blue) were predicted using the CDD (NCBI). Fig. S4d. Comparison of GAUT12/IRX8 proteins (BlastP) derived from different plant species using the DiAlign program (Genomatix). For each pairwise alignment, the similarity (relative to the maximum similarity) and the number of identical amino acids (in % of shorter sequence) are given. Maximum values are underlined. The similarity value of 1.000 denotes only the two most similar sequences; it does not necessarily mean that these sequences are identical. Lalb - Lupinus albus (KAE9588601); Lang - Lupinus angustifolius (XP_019437125); Gm - Glycine max (XP_003543290); Ap - Abrus precatorius (XP_027341076); Pp - Prunus persica (XP_007213904); Pm - Prunus mume (XP_008244342); Ca - Cicer arietinum (XP_004487615); Ss - Spatholobus suberectus (TKY48985); Cc - Cajanus cajan (XP_020235268); Ah - Arachis hypogaea (XP_025614714); Ad - Arachis duranensis (XP_015936483); Ai - Arachis ipaensis (XP_016170753); Md - Malus domestica (XP_008349951); Dz - Durio zibethinus (XP_022716962); At - Arabidopsis thaliana (NP_200280). Fig. S5a. LlPG/LlQRT2 cDNA  identified in yellow lupine (Lupinus luteus L.) and its deduced amino acid sequence . The nucleotides are marked with lowercase letters, and amino acids with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TGA codons. The ORF is shown in pink. Fig. S5b. Maximum likelihood phylogenetic tree of 19 polygalacturonases (PGs) (BlastP) with the highest degree of similarity to LlPG/LlQRT2. Alignment and phylogenetic reconstructions were performed using the ETE3 v3.1.1 program as implemented in GenomeNet. ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Lang - Lupinus angustifolius ; Lalb - Lupinus albus ; Gm - Glycine max ; Gs - Glycine soja ; Ca - Cicer arietinum ; Ap - Abrus precatorius ; Va - Vigna angularis ; Ad - Arachis duranensis ; Ah - Arachis hypogaea ; Ai - Arachis ipaensis ; Mt - Medicago truncatula ; Cc - Cajanus cajan ; Cm - Cucurbita maxima ; Rc - Ricinus communis ; Gh - Gossypium hirsutum ; At – Arabidopsis thaliana ; A. thaliana ; A. thaliana ; Sl - Solanum lycopersicum (XP_004252072 PG QRT2). Fig. S5c. Multiple sequence alignment  of different polygalacturonases  showing the highest similarity to LlPG/LlQRT2. Four typical conserved domains of PGs, referred to as domains I, II, III and IV (RIKT) were indicatated in yellow, green, blue and red, respectively. A tyrosine (Y) is marked in pink. 12 cysteine (C) residues are marked in red letters. Three aspartic acids in NTD and DD structures  are marked with gray arrows. The histidine residue (H) in domain III is marked with purple arrow. Fig. S5d. Comparison of PGs derived from different plant species using the DiAlign program (Genomatix). For each pairwise alignment, the similarity (relative to the maximum similarity) and the number of identical amino acids (in % of shorter sequence) are given. Maximum values are underlined. The similarity value of 1.000 denotes only the two most similar sequences; it does not necessarily mean that these sequences are identical. Lang - Lupinus angustifolius (XP_019425177); Lalb - Lupinus albus (KAE9590458); Gm - Glycine max (XP_003545985); Gs - Glycine soja (KHM99207); Ca - Cicer arietinum (XP_004499889); Ap - Abrus precatorius (XP_027343054); Va - Vigna angularis (XP_017424613); Ad - Arachis duranensis (XP_015952899); Ah - Arachis hypogaea (XP_025642914); Ai - Arachis ipaensis (XP_020965356); Mt - Medicago truncatula (XP_003595746); Cc - Cajanus cajan (XP_020214988); Cm - Cucurbita maxima (XP_022982564); Rc - Ricinus communis (XP_002517823); Gh - Gossypium hirsutum (XP_016741024); At - Arabidopsis thaliana ; A. thaliana ; A. thaliana ; Sl - Solanum lycopersicum (XP_004252072). Fig. S6a. LlPCS1L cDNA  identified in yellow lupine (Lupinus luteus L.) and its deduced amino acid sequence . The nucleotides are marked with lowercase letters, and amino acids with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TGA codons, resceptively. The ORF is shown in pink. Fig. S6b. Maximum likelihood phylogenetic tree of 15 PCS1 proteins (BlastP) with the highest degree of similarity to LlPCS1. Alignment and phylogenetic reconstructions were performed using the ETE3 v3.1.1 program as implemented in GenomeNet. The ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Lang - Lupinus angustifolius (XP_019412745); Vr - Vigna radiata (XP_014497350); Gm - Glycine max (XP_003530215); Mt - Medicago truncatula (XP_013448893); Ap - Abrus precatorius (XP_027349738); Cc - Cajanus cajan (XP_020206919); Vu - Vigna unguiculata (XP_027927273); Ca - Cicer arietinum (XP_004514393); Tp - Trifolium pratense (PNX74271); Ql - Quercus lobata (XP_030925127); Pa - Prosopis alba (XP_028786489); St - Senna tora (KAF7825648); Qs - Quercus suber (XP_023913890); Tw - Tripterygium wilfordii (KAF5750924); At - Arabidopsis thaliana (OAO90380). Fig. S6c. Multiple alignment  of 15 PROMOTION OF CELL SURVIVAL1 (PCS1) amino acid sequences (BlastP) that are closely related to LlPCS1. Two motif in both N-terminal (DTGS) and C-terminal (DS/LGT) ends characteristic for pepsin like aspartic proteases are marked in yellow and light grey, respectivelly. Additionally, two catalytic residues (D) (white letters on red background) have been indicated. Active site flap ATLS and SSSS are marked green and light blue, respectively. Pepsin A like plant domain is shaded dark blue with white letters. TAXi_N domain  and TAXi_C domain (violet) are additionally underlined. Lang - Lupinus angustifolius (XP_019412745); Vr - Vigna radiata (XP_014497350); Gm - Glycine max (XP_003530215); Mt - Medicago truncatula (XP_013448893); Ap - Abrus precatorius (XP_027349738); Cc - Cajanus cajan (XP_020206919); Vu - Vigna unguiculata (XP_027927273); Ca - Cicer arietinum (XP_004514393); Tp - Trifolium pratense (PNX74271); Ql - Quercus lobata (XP_030925127); Pa - Prosopis alba (XP_028786489); St - Senna tora (KAF7825648); Qs - Quercus suber (XP_023913890); Tw - Tripterygium wilfordii (KAF5750924); At - Arabidopsis thaliana (OAO90380). Fig. S6d. Comparison of PCS1 proteins derived from different plant species using the DiAlign program (Genomatix). For each pairwise alignment, the similarity (relative to the maximum similarity) and the number of identical amino acids (in % of shorter sequence) are given. Maximum values are underlined. The similarity value of 1.000 marks only the two most similar sequences; it does not necessarily mean that these sequences are identical. Lang - Lupinus angustifolius (XP_019412745); Vr - Vigna radiata (XP_014497350); Gm - Glycine max (XP_003530215); Mt - Medicago truncatula (XP_013448893); Ap - Abrus precatorius (XP_027349738); Cc - Cajanus cajan (XP_020206919); Vu - Vigna unguiculata (XP_027927273); Ca - Cicer arietinum (XP_004514393); Tp - Trifolium pratense (PNX74271); Ql - Quercus lobata (XP_030925127); Pa - Prosopis alba (XP_028786489); St - Senna tora (KAF7825648); Qs - Quercus suber (XP_023913890); Tw - Tripterygium wilfordii (KAF5750924); At - Arabidopsis thaliana (OAO90380). Fig. S7a. LlGA3ox cDNA  identified in yellow lupine (Lupinus luteus L.) and its deduced amino acid sequence . The nucleotides are marked with lowercase letters, and amino acids with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TGA codons, resceptively. The ORF is shown in pink. Fig. S7b. Maximum likelihood phylogenetic tree of 10 GA3ox proteins (BlastP) with the highest degree of similarity to LlGA3ox. Alignment and phylogenetic reconstructions were performed using the ETE3 v3.1.1 program as implemented in GenomeNet. The ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Lang - Lupinus angustifolius (XP_019459798); Lalb - Lupinus albus (KAE9605408); Va - Vigna angularis (XP_017408358); Ss - Spatholobus suberectus (TKY63915); Gs - Glycine soja (KHN29357); Vv - Vitis vinifera (XP_019072492); St - Solanum tuberosum (XP_006339568); Ns - Nicotiana sylvestris (XP_009779750); Na - Nicotiana attenuata (XP_019262262); At -Arabidopsis thaliana (NP_178149). Fig. S7c. Multiple alignment  of 10 GIBBERELLIN 3-OXIDASE (GA3ox) amino acid sequences (BlastP) that are closely related to LlGA3ox. Gibberellin 3-β-dioxygenase domain (black background) and domain characteristic for 2-oxoglutarate (2OG) and Fe (II)-dependent oxygenase (Oxy) superfamily (blue background) are indicated. The putative His-x-Asp-(x)n-His (HxD … H) and Arg-x-Ser (RxS) motif locations are highlighted in red and green, respectively. Domains and specific, highly conserved motifs were delineated from the CDD (NCBI) and . The nucleotides are marked with lowercase letters, and amino acids with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TGA codons, resceptively. The ORF is shown in pink. Fig. S8b. Maximum likelihood phylogenetic tree of 15 GIBBERELLIN 2-OXIDASES  with the highest degree of similarity to LlGA2ox1. Alignment and phylogenetic reconstructions were performed using the ETE3 v3.1.1 as implemented in GenomeNet. The ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Lang - Lupinus angustifolius (XP_019424643); Lalb - Lupinus albus (KAE9590556); Mp - Mucuna pruriens (RDX87676); Ss - Spatholobus suberectus (TKY49301); Ap - Abrus precatorius (XP_027342450); Va - Vigna angularis (NP_001316752); Cc - Cajanus cajan (XP_020204195); Vu - Vigna unguiculata (XP_027926310); Gm - Glycine max (XP_003543155); Ps - Pisum sativum (AAD45425); Gs - Glycine soja (XP_028201454); Ca - Cicer arietinum (XP_004488652); Jr - Juglans regia (XP_018824979); Nt - Nicotiana tabacum (XP_016500243); At - Arabidopsis thaliana (Q8LEA2). Fig. S8c. Multiple alignment  of 15 GIBBERELLIN 2-OXIDASE (GA2ox) amino acid sequences (BlastP) that are closely related to LlGA2ox1. Gibberellin 2-β-dioxygenase domain (black background) and domain characteristic for 2-oxoglutarate (2OG)-Fe (II)-dependent oxygenase superfamily (blue background) are indicated. Both domains designated using the CDD (NCBI). Additionally, amino acid residues presumed to bind Fe2+ at the active site of protein are indicated with red (HxD … H) and green (RxS) letters. Fig. S8d. Comparison of GIBBERELLIN 2-OXIDASES (GA2oxs) derived from different plant species using the DiAlign program (Genomatix). For each pairwise alignment, the similarity (relative to the maximum similarity) and the number of identical amino acids (in % of shorter sequence) are given. Maximum values are underlined. The similarity value of 1.000 marks only the two most similar sequences; it does not necessarily mean that these sequences are identical. Lang - Lupinus angustifolius (XP_019424643); Lalb - Lupinus albus (KAE9590556); Mp - Mucuna pruriens (RDX87676); Ss - Spatholobus suberectus (TKY49301); Ap - Abrus precatorius (XP_027342450); Va - Vigna angularis (NP_001316752); Cc - Cajanus cajan (XP_020204195); Vu - Vigna unguiculata (XP_027926310); Gm - Glycine max (XP_003543155); Ps - Pisum sativum (AAD45425); Gs - Glycine soja (XP_028201454); Ca - Cicer arietinum (XP_004488652); Jr - Juglans regia (XP_018824979); Nt - Nicotiana tabacum (XP_016500243); At - Arabidopsis thaliana (Q8LEA2). Fig. S9a. LlGAMYB cDNA  identified in yellow lupine (Lupinus luteus L.) and its deduced amino acid sequence . The nucleotides are marked with lowercase letters, and amino acids with capital letters. The START and STOP codons (yellow background) are indicated. 5′ and 3′ UTR regions are marked with small italic letters before the ATG and after the TGA codons, resceptively. The ORF is shown in pink. 21 nucleotide sequence in blue background corresponds to target site for Ll-miR159. Fig. S9b. Maximum likelihood phylogenetic tree of 16 GAMYB proteins (BlastP) with the highest degree of similarity to LlGAMYB. Alignment and phylogenetic reconstructions were performed using the ETE3 v3.1.1 as implemented in GenomeNet. The ML tree was inferred using PhyML v20160115. Branch supports are the Chi2-based parametric values returned by the approximate likelihood ratio test. Lang - Lupinus angustifolius (XP_019449331); Lalb - Lupinus albus (KAE9617167); Ss - Spatholobus suberectus (TKY68413); Cc - Cajanus cajan (XP_020219565); Ap - Abrus precatorius (XP_027354365); Mp - Mucuna pruriens (RDX95167); Gm - Glycine max (AHB19229); Gs - Glycine soja (XP_028187659); Ah - Arachis hypogaea (XP_025615172); Ai - Arachis ipaensis (XP_016171706); Ad - Arachis duranensis (XP_015937001); At - Arabidopsis thaliana , , , , . Fig. S9c. Multiple alignment  of 16 MYB amino acid sequences (BlastP) that are closely related to LlGAMYB. The R2R3 domain near the 5′ terminus (black) and Box 1 (yellow), Box 2 (green), Box 3 (blue) domains distributed throughout the protein were designated. REB1 domain characteristic for Myb superfamily proteins (underlined), Myb_DNA-binding domain (pink) and SANT  domain (gray) have been designated using the CDD (NCBI). Lang - Lupinus angustifolius (XP_019449331); Lalb - Lupinus albus (KAE9617167); Ss - Spatholobus suberectus (TKY68413); Cc - Cajanus cajan (XP_020219565); Ap - Abrus precatorius (XP_027354365); Mp - Mucuna pruriens (RDX95167); Gm - Glycine max (AHB19229); Gs - Glycine soja (XP_028187659); Ah - Arachis hypogaea (XP_025615172); Ai - Arachis ipaensis (XP_016171706); Ad - Arachis duranensis (XP_015937001); At - Arabidopsis thaliana , , , , . Fig. S9d. Comparison of GAMYB proteins derived from different plant species using the DiAlign program (Genomatix). For each pairwise alignment, the similarity (relative to the maximum similarity) and the number of identical amino acids (in % of shorter sequence) are given. Maximum values are underlined. The similarity value of 1.000 marks only the two most similar sequences; it does not necessarily mean that these sequences are identical. Lang - Lupinus angustifolius (XP_019449331); Lalb - Lupinus albus (KAE9617167); Ss - Spatholobus suberectus (TKY68413); Cc - Cajanus cajan (XP_020219565); Ap - Abrus precatorius (XP_027354365); Mp - Mucuna pruriens (RDX95167); Gm - Glycine max (AHB19229); Gs - Glycine soja (XP_028187659); Ah - Arachis hypogaea (XP_025615172); Ai - Arachis ipaensis (XP_016171706); Ad - Arachis duranensis (XP_015937001); At - Arabidopsis thaliana , , , , . Fig. S10. The cDNA sequence of Ll-MIR159 identified in yellow lupine (GenBank accession number MW240683). The 21-nucleotide fragment that constitutes the mature miR159 is marked in yellow. Fig. S11. The domain structures of LlCAD , LlCesA8/LlIRX1 , LlCOBL4/IRX6 (COBRA-like4), LlGAUT12/LlIRX8 , LlPG/LlQRT2  and LlPCS1 (PROMOTION OF CELL SURVIVAL1) identified in yellow lupine (Lupinus luteus L.). (A-F) The predicted tertiary 3D models constructed by the ROBETTA protein modelling server visualized by the ChimeraX program. (A’-F′) Conserved domains, motifs and specific amino acids identified via different tools described in the Methods section. The colours used in the 3D protein models correspond to the colours presented in the protein diagrams. The specific functions of domains/motifs/amino acids are described in detail in Table Fig. S12. The domain structures of LlGA3ox (gibberellin 3-oxidase), LlGA2ox1 (gibberellin 2-oxidase1) and LlGAMYB identified in yellow lupine (Lupinus luteus L.). (A-C) The predicted tertiary 3D models constructed by the ROBETTA protein modelling server visualized by the ChimeraX program. (A’-C′) Conserved domains, motifs and specific amino acids identified via different tools described in the Method section. The colours used in the 3D protein models correspond to the colours presented in the protein diagrams. The specific functions of domains/motifs/amino acids are described in detail in Table Fig. S13. (A) Image of the electrophoretic separation of the PCR product carried out with degenerate primers to identify a cDNA fragment of LlGAMYB in yellow lupine on a 1.2% agarose gel in 0,5 × TBE buffer at 5 V/cm in the presence of the GeneRuler 100 bp DNA ladder marker ; (B) Simplified diagram of the pSC-A-amp/kan vector (Agilent Technologies); (C) Representative Petri dish containing S-Gal/LB agar blend with one shot Mach1-T1 E. coli. White bacterial colonies took the recombinant form of the pCRII-TOPO vector, while dark blue bacterial colonies took the vector without the insert. (D) Image of electrophoretic separation of plasmid DNA digested with the restriction enzyme EcoRI (Fermentas) to confirm the presence of the insert - the cDNA fragment of LlGAMYB. Separation was performed on a 1.2% agarose gel in 0.5 × TBE buffer at 5 V/cm and in the presence of a GeneRuler 100 bp DNA ladder marker (M). The arrow indicates the sample that was sent for sequencing . Fig. S14.  Electrophoretic separation images of the PCR products  and 3’RACE-PCR  for LlGAMyb in 1.2% agarose gels in 0.5 × TBE buffer at 5 V/cm in the presence of GeneRuler 100 bp DNA Ladder marker ;  Electrophoretic separation images of plasmid DNA containing inserts of various sizes that were digested with the restriction enzyme EcoRI (Fermentas) on 1.2% agarose gels in 0.5 × TBE buffer at 5 V/cm and in the presence of GeneRuler 100 bp DNA Ladder marker (M); Transformation results obtained with the reaction products carried out using the first pair of primers (C) and the second pair of primers (D); arrows indicate the individual samples that were sent for sequencing . Fig. S15. The negative control required for validation of the immunohistochemical reactions was carried out by omitting incubation with the primary antibody, and showed no labelling. The autofluorescence signal of the cell walls and pollen grains is visible. DAPI was used to stain cell nuclei. Scale bar = 50 μm. Tab. S1. (A) Sequences of degenerate primers designed based on the cDNA sequences of GAMYB genes derived from different plant species (BlastP); (B) Sequences of specific, degenerate and universal primers used in 3′ RACE (rapid amplification of cDNA ends)-PCR; GSOP – gene specific outer primer, OP – outer primer, GSIP – gene specific inner primer, IP – inner primer; (C) Sequences of specific primers, their melting temperature (Tm) and UPL (UNIVERSAL PROBE LIBRARY) probe sequences and numbers used in qPCR for all studied genes and LlACT. The length of the amplified product in PCR and the annealing temperature of the primers are also given. (PPTX 19524 kb)c manner . The C-ts XP_0273541; Ss -


Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19.IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus family member that most recently adapted to humans, is the etiologic agent of the coronavirus disease of 2019 (COVID-19). While the four coronaviruses endemic to humans  impact mainly the respiratory tract and usually cause mild symptoms, SARS-CoV-2, like the other emerging coronaviruses, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), can cause lethal systemic symptoms  have been used to model SARS-CoV-2 spread in cardiac tissue –24. Base–We assessed whether ACE2, the SARS-CoV-2 receptor, and the spike-activating proteases transmembrane protease serine 2 (TMPRSS2) and cathepsin B (CTSB) are expressed during the differentiation process of human embryonic stem cells (hESC) into cardiomyocytes. The ACE2 transcription level peaked at day 20, those of cathepsin B remained stable, and TMPRSS2 transcripts were never detectable . Thus, we characterized our hiPSC-CMs  at this differentiation stage. Super resolution immunofluorescence confocal microscopy documented cell surface expression of ACE2 and striated F-actin organization characteristic of contractile cardiomyocytes . In partTo further characterize day 20 differentiated cardiomyocytes, we analyzed their total cellular transcriptome by RNA sequencing (RNA-Seq). Cardiomyocyte differentiation markers were expressed in our hiPSC-CMs at levels similar to those documented in two other hiPSC-CM lines used for SARS-CoV-2 infection studies , 26 wereWe used for our studies SARS-CoV-2/UW-001/Human/2020/Wisconsin (UW-001). This virus was isolated from a mild case in February 2020 and passaged in VeroE6 cells expressing TMPRSS2 to promote maintenance of the multibasic furin cleavage site , 27. Dee6 infectious units/ml were produced (We inoculated two independent lines of spontaneously beating hiPSC-CMs at a 0.01 multiplicity of infection (MOI) and monitored virus titer in the supernatant by plaque assay. Two days after inoculation, about 10produced . StrikinWe then sought to document expression, processing, and localization of the viral proteins. Immunoblot analyses of viral spike (S), nucleocapsid (N), and membrane (M) proteins confirmed high expression levels and accurate processing (We then assessed by transmission electron microscopy (TEM) whether SARS-CoV-2 infection of hiPSC-CM recapitulated features characteristic of other coronavirus infections. TEM analyses revealed canonical double-membrane vesicles, endoplasmic reticulum-Golgi intermediate complex, and smooth-walled exocytic vesicles containing numerous 65- to 90-nm particles , inset. We assessed whether typical SARS-CoV-2 particles are present on the surface of hiPSC-CMs by scanning electron microscopy (SEM), which revealed numerous particles on the plasma membrane. We also monitored the cytopathic effects of SARS-CoV-2 infection of hiPSC-CMs by IF confocal microscopy. In To quantify SARS-CoV-2-mediated hiPSC-CM fusion, α-actinin and SARS-CoV-2 M protein colabeled cells were imaged by IF confocal microscopy, and the percentage of nuclei found within syncytia was determined. In infected cells about half of the nuclei were found within syncytia, while in mock-infected cells less than 1% of the nuclei were found within syncytia . Thus, mTo characterize the mechanism of cell fusion, we engineered a SARS-CoV-2 full-length recombinant spike protein fused to modified emerald green fluorescent protein at its carboxyl terminus (CoV-2 S-mEm) . FluoresWe then assessed whether CoV-2 S-mEm fuses cardiomyocytes. Despite overall transfection efficiency of <5%, CoV-2 S-mEM expressing hiPSC-CMs produced syncytia with nuclei frequently arranged in clusters or rosettes . Some sytrans-Golgi apparatus that contributes to SARS-CoV-2 spike activation, is expressed in hiPSC-CM  with high ACE2 receptor transcript levels, while the ACE2 receptor transcript levels were intermediate in the experiments of Sharma et al.  with hig. We alsoin vitro models of human airway epithelium infection , cell adhesion structures that connect adjacent cardiomyocytes and support synchronized contraction. Several viruses exploit cell adhesion structures to spread rapidly in tissue , 49, andFinally, our observation that SARS-CoV-2-induced cardiomyocyte fusion is recapitulated by recombinant spike protein expression alone facilitates the development of therapeutics for COVID-19 induced cardiac disease. In particular, the fusion inhibition activity of peptide-based decanoyl-RVKR-CMK derivatives can be precisely quantified , 54 afteObtained under the Mayo Clinic institutional review board (IRB) protocol, patient and control human fibroblast-derived iPSCs were maintained in mTESR1 basal medium with mTESR supplement on plates coated with Geltrex (in Dulbecco’s modified Eagle’s medium [DMEM]/F12 medium). Undifferentiated hiPSCs were transitioned and expanded in suspension/spinner culture in DMEM/F12 plus GlutaMAX, StemPro supplement, bovine serum albumin (BSA), and basic fibroblast growth factor (bFGF) with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor Y27632 combined with mTESR1 medium and then chemically differentiated by CHIR (Wnt [Wingless and Int-1] activator) IWP-4 (Wnt inhibitor) into cardiomyocytes (CMs) in RPMI 1640 plus B27 minus insulin supplement as beating aggregates. The detailed spinner culture cardiac differentiation protocol is available from J.W.S. upon request. Differentiated hiPSC-CMs were maintained in Gibco cardiomyocyte maintenance medium and attached to fibronectin-coated glass coverslips. Human H9 embryonic stem cells (WiCell) were chemically differentiated into CMs using an analogous protocol in monolayer culture. EdU (5-ethynyl-2′-deoxyuridine) labeling of growing hiPSC-CMs and detection were done as described by the manufacturer (Thermo-Fisher).SARS-CoV-2/UW-001/Human/2020/Wisconsin (UW-001) was isolated from a mild case in February 2020 and passaged in VeroE6 cells expressing TMPRSS2. The virus was used to infect hiPSC-CMs in monolayer at an MOI of 1.0 to 0.001 for 30 min at 37°C. Unbound virus was then washed off, and replaced with fresh medium. At the various time points, cells were fixed or extracted, and samples were collected. An MOI of 0.01 for 24 to 48 h proved optimal for observing early stages of SARS-CoV-2 infection in hiPSC-CMs. Beyond 72 h, even at a low starting MOI, cytopathic lysis overwhelmed hiPSC-CM cultures. Highly permissive SARS-CoV-2 infection was observed in 3 different, equivalently differentiated hiPSC-CMs from unrelated donors.5) for 30 min at 37°C. After initial exposure, the Vero/TMPRSS2 cells were washed three times to remove unbound virus, and the medium was replaced with 1.0% methylcellulose medium. After an incubation of 3 days at 37°C, the cells were fixed and stained with crystal violet solution, and the plaques were counted to determine PFU/ml.The titer of SARS-CoV-2 infectious virus produced by hiPSC-CM was determined by plaque-forming assay done in confluent Vero E6/TMPRSS2 cells. Then, 12-well tissue culture plates were infected with supernatant (undiluted and 10-fold dilutions from 10 to 10The hiPSC CMs were infected with SARS-CoV-2/UW001/Human/2020/Wisconsin (UW-001) at an MOI of 0.01. Cells were lysed in TRIzol and were kept at −80°C. Total RNA of the lysate was extracted using a Direct-zol RNA miniprep kit (R2050). Library preparation and sequencing were performed at the Mayo Clinic Genome Analysis Core (GAC).Briefly, cDNA libraries were prepared using 100 ng of total RNA according to the manufacturer’s instructions for the Illumina TruSeq stranded total RNA sample prep kit with Ribo-Zero Gold . The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip  and Qubit fluorometry . Libraries were sequenced at three samples per lane to generate approximately 119 to 137 million fragment reads per sample following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE cluster kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 instrument using a HiSeq 3000/4000 sequencing kit and HD 3.4.0.38 collection software. Base-calling was performed using Illumina’s RTA v2.7.7.https://usegalaxy.org). The aligned reads were counted using HTSeq-count v0.9.1  using the Burrows-Wheeler Aligner MEM algorithm (BWA-MEM) v0.7.17.1 (https://arxiv.org/abs/1303.3997). Alignment summary statistics were computed using SAMtools idxstats v2.0.3 (GSE150392) and GSE156754), except for SARS-CoV-2 genome (GenBank accession number MN985325.1). The raw RNA-seq data from this study are available at the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) under accession number GSE184715.The quality of the raw RNA-seq data was assessed using fastqp v0.20.1 , and quat v0.9.1 , and 0.5t v0.9.1  and log2in limma . For thes v2.0.3 . The samCoverslips were fixed with neutral buffered formalin for 15 min at room temperature, washed with phosphate-buffered saline (PBS)/0.05% Tween 20 and blocked in PBS/5% normal goat serum or 3% BSA/0.3% Triton X-100 at room temperature for 1 h. Coverslips were incubated in primary antibodies diluted in PBS/1% BSA/0.3% Triton X-100 overnight at 4°C, washed extensively, incubated with diluted secondary antibodies (1:400) at room temperature for 1 h, then DAPI stained for 10 min at room temperature. Coverslips were mounted on slides with Prolong Gold antifade mountant (Thermo Fisher) and stored at 4°C. Coverslips were imaged using a Zeiss LSM780 or Elyra PS.1 Super Resolution confocal microscope. Antibodies and reagents for immunocytochemistry included ACTC1 , phalloidin Alexa Fluor-568 conjugated (Invitrogen), SARS-CoV-2 spike MAb clone 1A9 (GeneTex), SARS-CoV-2 M rabbit polyclonal Ab (Argio Biolaboratories), SARS-CoV-2 nucleocapsid clone 1C7 (Bioss Antibodies), ACE2 goat polyclonal Ab (R&D Systems), and ATP2A2/SERCA2 rabbit polyclonal Ab .Cells were washed with PBS and placed in Trump’s universal electron microscopy (EM) fixative   , tissue 2.HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Vero-hSLAM   were maiYP_009724390) was synthesized by Genewiz in a pUC57-Amp plasmid (kindly provided by M. Barry). The S-protein coding sequence was cloned into a pCG mammalian expression plasmid  supplemented with cOmplete protease inhibitor cocktail , and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4 to 15% gradient) under reducing conditions. For hiPSC-CMs transfected with CoV-2 S (2 μg/well in 6-well plates), extracts were prepared in cell lysis buffer as described above  and separated by SDS-PAGE under reducing (β-mercaptoethanol) or nonreducing conditions. The S-proteins were visualized on an immunoblot using the anti-S specific monoclonal antibody 1A9 , which binds the S2 subunit of SARS CoV and SARS-CoV-2 S-proteins. An anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody was used to reveal the bands. MERS S-protein was detected using a monoclonal anti-FLAG M2-HRP-conjugated antibody  which bound to a C-terminal FLAG-tag. The expression of the mEmerald tag was verified using a polyclonal anti-green fluorescent protein GFP antibody . For hiPSC-CMs infected with SARS-CoV-2 , extracts were prepared in cell lysis buffer as described above , separated by SDS-PAGE and blotted with S, M, and N antibodies as described in “Immunohistochemistry,” above.Vero cells were transfected with spike protein expression constructs using the GeneJuice transfection reagent (Novagen). The indicated S-protein expression constructs (1 μg) were transfected into 2.5 × 105 Vero cells in 24-well plates were transfected with 0.5 μg of the indicated S-protein expression vector using the GeneJuice transfection reagent (Novagen) and syncytium formation monitored for 24 to 48 h posttransfection. Images were collected with a Eclipse TE300 microscope using NIS-Elements F 3.0 software . For recombinant spike glycoprotein-mediated fusion in hiPSC-CMs, subconfluent day-20 differentiated cells plated on fibronectin-coated glass coverslips in 6-well plates were transfected with 1 to 2 μg plasmid using Lipofectamine 3000. For CoV-2 S-mEm in hiPSC-CM experiments, syncytium formation became obvious within 6 h of transfection.For spike glycoprotein-mediated cell-to-cell fusion, 1.5 × 10Furin inhibitor I  dissolved in dimethyl sulfoxide (DMSO) was added to Vero or hiPSC-CM cell culture medium 2 h posttransfection. Cell-cell fusion was followed for 72 h for Vero cells and 5 days for hiPSC-CMs, with medium and inhibitor refreshed on day-3. Whole-cell extracts were separated on SDS-PAGE and immunoblotted for SARS-CoV-2 S as described above, or cells were fixed and stained with crystal violet.Quantification of percent nuclei in syncytia for mock- versus SARS-CoV-2-infected cells was done as follows. Following immunocytochemistry as previously described, mock-infected and SARS-CoV-2-infected hiPSC-CMs were imaged on a Zeiss LSM780 confocal microscope at ×20 magnification. DAPI-stained nuclei within alpha-actinin stained cell bodies were counted manually using Zen 3.2 Blue software. Then, 12 images from 3 biological replicates were counted for each condition, with an average of 44 nuclei per image. Syncytia were defined as cell bodies containing 3 or more nuclei. Percent nuclei within a syncytium denotes the percentage of total nuclei counted within a syncytium at 48 h postinfection.Quantification of percent nuclei in syncytia for SARS-CoV-2 spike transfected cells was done as follows. Human iPSC-CMs were plated on 35-mm round glass-bottom dishes and transfected with SARS-CoV-2 spike protein, with or without furin inhibitor treatment, or transfected with SARS-CoV-2 R682S mutant spike protein, as previously described. Bright-field microscopy images were taken at ×10 magnification from randomly chosen areas of each culture dish. Five images from three independent replicates were counted for each condition. Images were manually counted for number of nuclei, number of syncytia, and number of nuclei per cell using Olympus Dimension cellSens software. Percent nuclei within a syncytium denotes the percentage of total nuclei counted within a syncytium at 48 h posttransfection. Syncytia are defined by a cell containing three or more nuclei. Analysis of variance (ANOVA) statistical analysis was carried out using GraphPad Prism software.5 in a 6-well plate) were transfected with the indicated S-protein expression plasmids (2 μg using GeneJuice transfection reagent). Then, 36 h posttransfection, cells were washed in PBS and detached by incubating with Versene (Life Technologies) at 37°C for 10 min. The resuspended cells were washed twice with cold fluorescence-activated cell sorter (FACS) wash buffer  and then incubated with the anti-S-protein MAb 1A9  for 1 h on ice. Cells were washed three times with cold FACS wash buffer and incubated with an AF647-conjugated secondary antibody  for 1 h on ice. After three washes with FACS wash buffer, cells were fixed in 4% paraformaldehyde and analyzed with a FACSCalibur  cytometer and FlowJo software .To determine S-protein cell surface expression levels, HeLa cells (8 × 10https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi) under accession number GSE184715.The raw RNA-Seq data from this study are available at Gene Expression Omnibus (


Rationale: Macrophages are the frontline immune cells in response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Angiotensin-converting enzyme 2 (ACE2) serves as the binding receptor to SARS-CoV-2 Spike glycoprotein for fusion and internalization into the human host cells. However, the mechanisms underlying SARS-CoV-2-elicited macrophage inflammatory responses remain elusive. Neutralizing SARS-CoV-2 by human ACE2 (hACE2) decoys has been proposed as a therapeutic approach to ameliorate SARS-CoV-2-stimulated inflammation. This study aims to investigate whether an engineered decoy receptor can abrogate SARS-CoV-2-induced macrophage inflammation.Methods: hACE2 was biotinylated to the surface of nano-liposomes (d = 100 nm) to generate Liposome-human ACE2 complex (Lipo-hACE2). Lentivirus expressing Spike protein (D614G) was also created as a pseudo-SARS-CoV-2 (Lenti-Spike). Liposome-hACE2 was used as a decoy receptor or competitive inhibitor to inhibit SARS-CoV-2 or Lenti-Spike-induced macrophage inflammation in vitro and in vivo.Results: Both SARS-CoV-2 and Lenti-Spike stimulated strong inflammatory responses by inducing the expression of key cytokine and chemokines, including IL-1β, IL-6, TNFα, CCL-2, and CXCL-10, in murine and human macrophages in vitro, whereas Lipo-hACE2 decoy abolished these effects in macrophages. Furthermore, intravenous injection of Lenti-Spike led to increased macrophage and tissue inflammation in wild type mice, which was also abolished by Lipo-hACE2 treatment. Mechanistically, Spike protein stimulated macrophage inflammation by activating canonical NF-κB signaling. RNA sequencing analysis revealed that Lenti-Spike induced over 2,000 differentially expressed genes (DEGs) in murine macrophages, but deficiency of IκB kinase β (IKKβ), a key regulator for NF-κB activation, abrogated Lenti-Spike-elicited macrophage inflammatory responses.Conclusions: We demonstrated that the engineered Lipo-hACE2 acts as a molecular decoy to neutralize SARS-CoV-2 or Spike protein-induced inflammation in both murine and human macrophages, and activation of the canonical IKKβ/NF-κB signaling is essential for SARS-CoV-2-elicited macrophage inflammatory responses. The novel coronavirus disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuing to evolve globally as the pandemic persists It has been recognized that SARS-CoV-2 cellular entry is achieved by the homotrimeric Spike protein-mediated virus-receptor engagement through the receptor-binding domain (RBD), followed by virus-host membrane fusion Macrophages are the key frontline immune cells contributing to the pathogenesis of chronic diseases To this end, we sought to block the interaction of SARS-CoV-2 Spike protein and host hACE2 receptors and to also neutralize the impact of SARS-CoV-2 Spike protein on macrophage activation. In addition to using live SARS-CoV-2 virus, we also cloned and inserted the Spike sequence into the lentiviruses (Lenti-Spike) as pseudo-SARS-CoV-2, and we biotinylated the surface of nano-liposomes (d = 100 nm) with human hACE2 (Lipo-hACE2) as a decoy. We demonstrated that SARS-CoV-2 or Lenti-Spike-induced inflammatory responses were abrogated by Lipo-hACE2 in both murine and human macrophages. Furthermore, RNA sequence analysis revealed that Lenti-Spike infection resulted in over 2,300 differentially expressed genes (DEGs) that were enriched in key pathways including “NF-κB and TNF-α signaling pathways”, “cytokine-cytokine receptor interaction”, and “Toll-like receptor signaling pathways” in control macrophages. However, deficiency of IκB kinase β (IKKβ), a key regulator for NF-κB activation, abolished Spike protein-elicited macrophage inflammatory responses. Overall, our engineered Lipo-hACE2 inhibits SARS-CoV-2-induced macrophage inflammatory responses in both murine and human macrophages, and the canonical IKKβ/NF-κB signaling was implicated in SARS-CoV-2-mediated inflammatory responses independent of hACE2 in macrophages.Liposomes incorporating rhodamine were purchased from Encapsula Nano Sciences . The total lipid concentration in Immunosome-Biotinyl Cap was 22.45 mM. 1% mol of the lipid in liposomes contains a Biotinyl Cap group, exposing half of them to the surface of the liposomes, equal to 0.11 mM of reactive conjugable lipid. At UCLA Cardiovascular Engineering Laboratory, liposomes were reconstituted in 2mL of phosphate buffered saline (PBS) 1X at a concentration of 0.22 uM. Liposomes-Biotinyl cap were poured in a conical tube, vortexed gently, and mixed with Neutravidin Protein  while vortexing at the ratio of 1:10 fold-molar liposomes:neutravidin, respectively. The solution was incubated at room temperature for 1 h during shaking (110 rpm). Next, the solution was placed at 4 °C overnight. The following day the unbound neutravidin from the prep was removed by dialysis using the Float-A-Lyzer dialysis cassette with 300K MWCO (Spectrum Labs). Liposomes/Neutravidin solution was dialyzed in 1 liter of 1X PBS at pH 7.4 for 8 h while stirring at 4 °C. After 8 h, PBS was replaced with fresh PBS for a further 8 h dialysis. After this step, Liposomes-Biotinyl Cap/Neutravidin were separated from unbound Neutravidin. The following day the liposomes-Biotinyl Cap/Neutravidin solution was poured in a conical tube and vortexed gently. During vortexing Biotinylated human-ACE2 (Acrobiosystem) was added to the Liposomes-Biotinyl Cap/Neutravidin solution at the ratio of 1:2 fold-molar Liposomes-Biotinyl Cap/Neutravidin:Biotinylated human-ACE2, His, Avitag, respectively. The solution was incubated at room temperature for 1h during shaking (110 rpm). After 1 h incubation, the solution was placed at 4 °C overnight. The Following day, the unbound neutravidin from the prep was removed by dialysis using Float-A-Lyzer dialysis cassette with 300K MWCO.Liposomes/Neutravidin/Biotinylated-Spike hACE2 solution was dialyzed in 1 liter of 1X PBS at pH 7.4 while stirring at 4 °C. After 8 h the 1X PBS was replaced with fresh 1X PBS for a further 8 h dialysis. After this step, Liposomes/Neutravidin/Biotinylated-hACE2 preparation was separated from the unbound hACE2. Next, the purified immunoliposome solution was ready to be used. We did not observe the significant changes with the size of liposome (d = 100 nm) by using electron microscope (data not shown). Stock concentration of 0.22 uM Liposomes/hACE2 was diluted for the experiments.We validated the correct complex liposome-hACE2 by using a Human ACE2 ELISA Kit. The liposome-hACE2 complex was incubated in the ACE2 ELISA plate and the fluorescence from the rhodamine incorporated in the liposomes was measured at λEx 546 nm and λEm 568 nm. Next anti-hACE2 antibodies were added to the wells and the absorbance was measured at 450 nm OD according to manufacture. The double signal (fluorescence and absorbance) from the same wells confirmed the successful construction of the liposome-hACE2 complex.Expression of cloned DNA (625 ng) was mixed with packaging plasmids pVSV-G and pCMV delta R8.2 at the ratio of 8:1 by mass. Viafect transfection reagent (Promega) was incubated with OPTIMEM (Gibco)  for 20-30 min at room temperature, prior to adding to the DNA mix at 31 ml per well. HEK293T cells were plated to 70-90% confluence prior to transfection. After 16 h, the medium was replaced with fresh medium. After 48 h, the virus was harvested and filtered in 45 μM in a 15 mL tube. Lenti-X concentrator (Takara) was mixed with the medium containing the virus in the ratio of 1:3 media:Lenti-X, respectively. The mixture was left overnight at 4 °C, and 45 min centrifugation was performed at 1500 xg. The supernatant was removed by aspiration and the pellet was resuspended in 400 μL of 1X PBS (Gibco).5 pfu/mL of SARS-CoV2 was used for the experiments. Studies involving live SARS-CoV-2 virus were approved by the University of California, Los Angeles Institutional Biosafety Committee (IBC) and were performed in the UCLA Biosafety Level 3 (BSL3) high-containment facility.SARS-CoV-2, B.1.526, was acquired from the Biodefense and Emerging Infections (BEI) Resources of the National Institute of Allergy and Infectious Diseases (NIAID) via UCLA Virology Program. SARS-CoV-2 was passaged once in Vero-E6 cells (ATCC), and viral stocks were aliquoted and stored at -80 °C. Virus titer was determined by plaque assay using Vero E6 Cells. 2.5x10AlamarBlue (AB) assays were used to elucidate the inhibitory effect on Cytopathic effect (CPE) induction caused by SARS-CoV-2 infection. Briefly, Vero E6 cells (infected and non-infected) were incubated with Dulbecco's Modified Eagle Medium (DMEM) without FBS or supplements, and AB solution (5% [v/v] solution of AB dye). Following 3 h incubation, AB absorbance was quantified at 600 nm using a Fisher AccuSkan FC microplate reader. Twelve technical replicates per experiment at concentration of 25 nM was carried out (n = 5) for cytopathic effect evaluation of SARS-CoV-2 in the presence of free-hACE2 protein or Lipo-hACE2. The reading was performed in triplicate . The relative cell viability rate (%) was calculated by comparing infected and non-infected wells [(mean fluorescence of treated-SARS-CoV-2 infected wells/mean fluorescence of treated non-infected wells) × 100]. Each assay was carried out in triplicate. To score SARS-CoV-2 induced CPE, we performed microscopy to image the infected cells in the presence or absence of Lipo-hACE2 or free hACE2 treatment.ΔMye) mice on C57BL/6J background  were generated by crossing mice carrying loxP-flanked IKKβ alleles (IKKβF/F) with LysM-Cre transgenic mice, as previously described 6 pfu/mL) or control lentivirus followed by IV injection of 100 µL control liposome or Lipo-hACE2 (4 nM) 1 h post infection. On the day of euthanasia, mice were fasted for 6 h following the dark cycle (feeding cycle), and blood and tissues were collected as described previously C57BL/6J wild-type (WT) mice were purchased from The Jackson Laboratory . Myeloid-specific IKKβ knockout  or control lentivirus for 1 h followed by treatment with 25 nM Lipo-hACE2 or control liposome for another 23 h. In the UCLA BSL3 Laboratory, macrophages were infected with SARS-CoV-2 (2.5×105 pfu/mL) or inactivated SARS-CoV-2 (control virus) for 1 h followed by treatment with 25 nM Lipo-hACE2 or control liposome for 23 h.Peritoneal Macrophages were isolated from WT, IKKβSupplemental Table 1.Total RNA was isolated from mouse tissues or cells using TRIzol Reagent following the manufacture's protocol  as previously described F/F and IKKβΔMye mice as previously described Salmon tool EnsemblTPM) was used as the unit of mouse gene expression level. We then used the edgeR algorithm TMM algorithm implemented in the edgeR package to perform reads count normalization and effective library size estimation. Group-wise differential expression was estimated by the likelihood ratio test included in the edgeR package. The genes with a false discovery rate (FDR) < 1% and fold change (FC) >3 was deemed differentially expressed. We further performed gene ontology analysis upon the differentially expressed genes using the definition from Kyoto Encyclopedia of Genes and Genomes (KEGG) FAIME) algorithm Peritoneal macrophages were isolated from 8-week-old male IKKβImmunofluorescence staining was performed on sections of lung and heart aortic roots that were freshly embedded in OCT t test unless otherwise noted, with P < 0.05 to be statistically significant. One-way ANOVA with Dunnett's test was used to assess the difference among more than 2 groups. Two-way ANOVA was used when multiple comparisons were made, followed by a Bonferroni multiple comparisons test.All data were presented as the mean ± SEM except for the high-throughput sequencing. Individual pairwise comparisons were analyzed by two-sample, two-tailed Student's We engineered the liposome surface conjugated hACE2 (Lipo-hACE2) for targeting SARS-CoV-2 Spike protein. We validated that rhodamine encapsulated liposomes were biotinylated with hACE2 at an excitation wavelength of 546 nm and emission of 568 nm. Both the fluorescence emission and absorbance signals were measured at 450 nm OD to confirm the conjugation of liposomes with hACE2 Figure A.50 = 10.68 nM as compared with free hACE2 (IC50 = 38.19 nM).Next, Vero E6 cells were infected with SARS-CoV-2 B.1.526 at a multiplicity of infection (MOI) of 0.05, along with lipo-hACE2 or free-hACE2. Both free-hACE2 and Lipo-hACE2 inhibited SARS-CoV-2-induced cell death, whereas Lipo-hACE2 treatment significantly improved cell viability in SARS-CoV-2-infected cells to a greater extent than free-hACE2 proteins at the same concentrations  and control IKKβ-flox (IKKβF/F) mice, and infected control lentivirus or lentivirus expressing Spike protein  of < 1% and a fold change (FC) >3 as a cut-off threshold (Figure FAIME algorithm F/F mice (Figure ΔMye mice (Figure FAIME analysis, the DEGs associated with these pathways in macrophages of IKKβF/F mice were upregulated by Lenti-Spike treatment, whereas deficiency in IKKβ abolished the Spike-induced gene expression (Figure To elucidate IKKβ/NF-κB signaling as the molecular mechanism underlying Spike protein-mediated macrophage inflammation n Figure A 22. RNAd Figure . No DEGsd Figure B. KEGG ad Figure C. By usie Figure D. IKKβ de Figure D. Consisn Figure E.F/FLDLR-/- mice compared with the controls, but deficiency of IKKβ abolished Lenti-Spike-elevated geneset scores in the macrophages of IKKβΔMye LDLR-/- mice (F/F mice, and IKKβ deficiency abolished Spike protein-stimulate gene expression. Collectively, IKKβ/NF-κB signaling is implicated in the SARS-CoV-2 Spike-mediated macrophage inflammatory responses.In addition to KEGG analysis, we further performed gene ontology analysis using the definition from the GO -/- mice B. For ex-/- mice B. FurtheSARS-CoV-2 variants were reported to evade COVID-19 vaccines and to escape neutralization by antibodies in recovered COVID-19 patients SARS-CoV-2 is well-recognized to promote the dysregulated immune responses in patients with severe COVID-19 et al.Neutralizing SARS-COV-2 by hACE2 decoys has been proposed as a therapeutic target to ameliorate SARS-CoV-2-stimulated inflammation We further assessed the potential molecular mechanisms underpinning SARS-CoV-2-mediated macrophage inflammation. The severity of COVID-19 is associated with the SARS-CoV-2 load, and the activation of host inflammatory responses. For this reason, mitigating the inflammatory response is critical to develop anti-SARS-CoV-2 therapies Whilst SARS-CoV-2 infects primarily the respiratory system, it has a tropism for the cardiovascular system, leading to arrhythmias and myocarditis In summary, we established that engineered Lipo-hACE2 as a decoy efficiently abrogated SARS-CoV-2 or Spike protein-mediated macrophage inflammatory responses, and IKKβ/NF-κB signaling pathway is implicated in SARS-CoV-2-stimulated inflammation. We demonstrated that SARS-CoV-2 induced the expression of cytokines and chemokines in both murine and human macrophages, suggesting a non-hACE2 mechanism to activate IKKβ/NF-κB signaling. Taken together, engineering Lipo-hACE2 offers a therapeutic target to mitigate by myeloid-derived immune cells from priming cytokine storm amid severe SARS-CoV-2 infection.Supplementary figure and tables.Click here for additional data file.


Bag-1 protein is a crucial target in cancer to increase the survival and proliferation of cells. The Bag-1 expression is significantly upregulated in primary and metastatic cancer patients compared to normal breast tissue. Overexpression of Bag-1 decreases the efficiency of conventional chemotherapeutic drugs, whereas Bag-1 silencing enhances the apoptotic efficiency of therapeutics, mostly in hormone-positive breast cancer subtypes. In this study, we generated stable Bag-1 knockout (KO) MCF-7 breast cancer cells to monitor stress-mediated cellular alterations in comparison to wild type (wt) and Bag-1 overexpressing (Bag-1 OE) MCF-7 cells. Validation and characterization studies of Bag-1 KO cells showed different cellular morphology with hyperactive Akt signaling, which caused stress-mediated actin reorganization, focal adhesion decrease and led to mesenchymal characteristics in MCF-7 cells. A potent Akt inhibitor, MK-2206, suppressed mesenchymal transition in Bag-1 KO cells. Similar results were obtained following the recovery of Bag-1 isoforms  in Bag-1 KO cells. The findings of this study emphasized that Bag-1 is a mediator of actin-mediated cytoskeleton organization through regulating Akt activation. Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer-related death of women worldwide . AlthougBag-1 (Bcl-2 associated athanogene-1) protein regulates multifarious signaling pathways involving cell proliferation, survival, transcription, apoptosis, autophagy, and metastasis through its various direct or indirect molecular partners. Bag-1 is alternatively translated from one single mRNA transcript and consists of 3 major isoforms Bag-1S (33 kDa), Bag-1M 46 kDa), and Bag-1L 52 kDa)  (ab50887), FAK Antibody #3285, phospho-FAK (Tyr397) Antibody #3283, Paxillin #2542, CD133 (D2V8Q) XP® Rabbit mAb #64326, α-Actinin (D6F6) XP® Rabbit mAb #6487, Anti-Cofilin (ab54532), phospho-cofilin (ab12866), Vimentin (D21H3) XP® Rabbit mAb #5741, Vinculin Antibody #4650.30 μg total cell lysates and 20 μg total breast tissue lysates were boiled for 5 min with 5x Laemmli Sample Buffer at 95°C, fractionated on a 12% SDS-PAGE, and transferred to PVDF membranes . After blocking with 5% BSA in TBS-Tween 20 for 1h at room temperature, membranes were incubated with primary antibodies  overnight at 4°C, and anti-rabbit or anti-mouse IgG-horseradish peroxidase-conjugated secondary antibodies  overnight at 4°C. Following a gentle washing step with TBS-Tween 20, the membranes were treated with enhanced chemiluminescence detection reagent and analyzed using Chemidoc MP Imaging System . Densitometric analyses were performed using Image Lab Software . All proteins were quantified relative to the GAPDH internal loading control. The information about the primary antibodies used in this study was as the following: Bag-1 (3.10G3E2) Mouse mAb #3920, GAPDH (D16H11) XPTotal RNA of wt, Bag-1 KO, Bag-1 OE cells and S. M and L isoform transfected Bag-1 KO cells were extracted with PureZOL RNA Isolation Reagent (Bio-Rad) according to the manufacturer’s instructions. Total RNA samples were converted to cDNA by using iScript Reverse Transcription Supermix for RT (Bio-Rad). Quantitative real-time PCR was performed using SYBR Green Master Mix (Bio-Rad) and analyzed on a CFX Connect Real-Time System (Bio-Rad). Relative target mRNA expressions were normalized to the level of GAPDH mRNA as an internal control. All experiments were performed in three biological and two technical replicates.5 cells were seeded in the upper part of the Transwell chamber with 200μl serum-free media. 750 μl complete medium was added into the lower chamber in a 24-well plate. Cells were incubated at 37°C in a humidified 5% CO2 incubator for 24h. Cells on the upper surface were washed with 1xPBS, and were fixed with 3.7% formaldehyde. Permeabilization of cells was maintained by incubation with 100% methanol for 20 min. After washing with 1xPBS cells were stained with %0.5 crystal violet for 15 min. Following the last washing step, the non-migratory cells on the upper part of the chamber were scraped with a cotton swab. The number of cells that invaded to the lower part of the filter membrane were visualized at 100x magnification under a light microscope Olympus 1X71.Transwell chamber: 24-well, 8 μm pore membranes (Corning) were used to investigate the migration potential of wt, Bag-1 KO, and Bag-1 OE cells. 2.5x105 cells/well were seeded into a 6-well plate and maintained until their at least 80% confluency. Plates were straightly scratched using a 200 μl sterile pipette tip. The wound closure was measured in time dependent manner to quantify the migration rate of cells and imaged by light and fluorescence microscopy (Olympus IX70).The wound healing assay was performed to investigate the cell migration of wt, Control KO, Bag-1 KO and Bag-1 OE cells in vitro. Basically, 6x10https://www.graphpad.com/. MTT Cell viability assay  was performed as three replicates and statistically analyzed using Two-Way ANOVA Sidak’s multiple comparison tests. Trypan Blue Dye Exclusion Assay  analyzed by Two-Way ANOVA Dunnett’s multiple comparison test. Path Scan analysis  was performed by Two-Way ANOVA, Tukey’s multiple comparison tests. The densitometric calculations of three replicates of immunoblotting images were performed using Image Lab Software (Bio-Rad) and analyzed by Two-Way ANOVA, Tukey’s multiple comparison test. Differences were considered statistically significant when *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Error bars represent ± standard deviation values.All the statistical analyses of experiments were performed using GraphPad Prism version 8, Bag-1 KO MCF-7 cells were generated by co-transfection of Bag-1 KO plasmid (sc-417179) and Bag-1 HDR Plasmid (sc-417179-HDR). Three sgRNAs on Bag-1 KO plasmid target exon 2 (sgRNA A), exon 3 (sgRNA B) and exon 6 (sgRNA C) . TransfeTo investigate the potential biological effects of Bag-1 loss on MCF-7 breast cancer cells, we utilized cell sensitivity assays such as MTT and trypan-blue dye exclusion assay. We examined the relative cell viability of each condition at 24 h and 48 h in comparison to wt cells. Bag-1 KO cells showed less cell density with the lower cell viability ratio compared to wt cells (**** p<0.0001) in 24 h. The longer seeding of cells for 48 h increased the density of cells in wt, Control KO, Bag-1 KO and Bag-1 OE cells . We concConsidering the important roles of RTKs in breast cancer development, we investigated the effect of Bag-1 expression on the phosphorylation status of RTKs in MCF-7 cells by using Pathscan RTK Signaling Antibody Array kit . VariousConsidering the significant Akt phosphorylation of both Ser473 and Thr308 residues in Bag-1 KO cells, we assessed the expression profile of upstream and downstream members of Akt, EMT and cytoskeletal proteins through MK-2206, a potent Akt inhibitor . The celin vitro wound closure compared to wt and Control KO MCF-7 cells after 48 h growth of cells. Bag-1 OE cells showed significant wound closure in 48 h. Akt inhibition suppressed the migration of cells into wound area significantly in Bag-1 KO cells and other cell models . To examine the effect of different Bag-1 expression levels on actin cytoskeleton regulation, generated MCF-7 cell models were stained with FITC-Phalloidin to mark F-actin and were visualized by immunofluorescence imaging. Untransfected wt, isoform-specific Bag-1 transfected wt and Bag-1 OE cells showed increased F-actin accumulation in the cell, whereas knockout of Bag-1 caused cytoplasmic condensation with the decrease in F-actin stress fiber. Bag-1 S, M and, L isoform transfected Bag-1 KO cells showed slightly more phalloidin stain compared to untransfected Bag-1 KO cells. Nuclear formation of cells was visualized by DAPI staining S5E Fig.The multifunctional Bag-1 protein interacts with several molecular targets to regulate cell survival, proliferation and apoptosis. Several studies explained the anti-apoptotic potential and survival-targeted role of Bag-1 expression in cancer, whereas the effect of complete knockout of Bag-1 in breast cancer has not been revealed in depth yet. In the previous studies, complete loss or ablation of Bag-1 expression was found to be associated with the cell death of neuronal precursor cells and overexpression of Bag-1 was found to associated with the cell death of neural cells . In a rectn binds to its cytoplasmic domain. Then, the cytoplasmic domain is phosphorylated by casein kinase II which leads to its binding to β-catenin, α-catenin and α-actinin. During the maturation process, the pro-region is cleaved by furin protease, and N-cadherin-catenin complex transported to the plasma membrane to link with actin cytoskeleton which regulates the cell-cell adhesion [Bag-1 KO and Bag-1 OE MCF-7 cells showed a significant reduction in both total and phosphorylated PTEN levels independent from Akt inhibition . Active adhesion . Thus, wadhesion . Increasadhesion , 28. Theadhesion . In addiadhesion . Inhibitadhesion . Akt inhDue to the morphological changes of Bag-1 KO MCF-7 cells compared to wt, Control KO and Bag-1 KO cells, Bag-1 KO caused the redistribution of actin filaments . The F-aMultifunctional Bag-1 possesses a central role in the survival-related signaling axis to maintain normal cell physiology. The enhanced stress conditions due to homozygous deletion of Bag-1 gene hyperactivated Akt with reduced PTEN expression to maintain cell survival with dysregulated mesenchymal cellular characteristics. The findings in this study highlighted the functional role of Bag-1 on the PI3K/Akt signaling axis, which orchestrates cell cytoskeleton targets in MCF-7 breast cancer cells . Stress-S1 TableA. Schematic representation of target sgRNA sequences for specific Bag-1 gene loci. Different sgRNA sequences of Bag-1 KO plasmid (sc-417179) were obtained from Santa Cruz and the regions were found from comparing the human nucleotides sequences through BLAST and also checked at Bag-1 genomic sequence (NG_029018.1). B) Designed primers to validate Bag-1 deficiency in MCF-7 cells.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FigA) The genomic PCR analysis of exon 2, exon 3, and exon 6 in Bag-1 KO clones and wt cells following standard PCR reaction with designed Guide A, B, and C primers. B) Sanger sequencing results of forward and reverse reads targeting exon 2 of Bag-1 in Bag-1 KO cells by Guide A PCR product. C) Sanger sequencing results of forward and reverse reads targeting exon 3 of Bag-1 in Bag-1 KO cells by Guide B PCR product. D) Sanger sequencing results of forward and reverse reads targeting exon 6 of Bag-1 in Bag-1 KO cells by Guide C PCR product. E) Densitometry analysis of (PDF)Click here for additional data file.S2 FigA) The overexpression of Bag-1 was confirmed by the immunoblotting assay. Bag-1 OE cells were generated by the transfection of the TAP-tag Bag-1 expression vector into MCF-7 cells and the upper band profile showed the TAP-tag Bag-1L isoform. β-actin was used as the loading control. B) Morphological images of wt, Control KO, Bag-1 KO, and Bag-1 OE cells were obtained by light microscopy in a time-dependent manner. Magnification 40x, scale 10 μm. C) Colony-forming potentials of wt, Control KO, Bag-1 KO, and Bag-1 OE cells determined by Colony formation assay. The number of colonies represented the mean ± SD of three repetitive experiments and measured using Image J based on their densities and analyzed by Two-way ANOVA, Tukey’s multiple comparison test. Scale bar is 10 μm and magnification is 10x. .(PDF)Click here for additional data file.S3 Fighttps://www.graphpad.com/. B) Densitımetry analysis of immunoblotting results of A) The expression profiles of 39 different RTKs that were obtained by PathScan RTK assay. The signal intensity of these proteins was normalized to positive control and analyzed GraphPad Prism version 8, (PDF)Click here for additional data file.S4 FigA) The effect of 24 h treatment of 500 nM MK-2206 on cells was determined by MTT Cell Viability assay. The bar histograms represent the mean ± SD of three independent experiments with at least four replicates. . B) The effect of MK-2206 on colony formation potentials of wt, Control KO, Bag-1 KO, and Bag-1 OE cells was determined by colony formation assay. The number of colonies represented the mean ± SD of three repetitive experiments, measured by Image J based on their densities and were analyzed by Two-way ANOVA, Tukey’s multiple comparison test. (**** p< 0.0001). C) Cellular ROS generation of wt, Control KO, Bag-1 KO, and Bag-1 OE cells was measured by DCFH-DA staining and analyzed by BD Accuri C6 flow cytometer . D) Densitometry analysis of (PDF)Click here for additional data file.S5 FigA) The cell viability of Bag-1 KO cells and transfected with Bag-1S, M and L isoforms, respectively was determined by MTT Cell Viability assay. B-C) Densitometry analyses of immunoblotting results of Bag-1 KO cells and transfected with Bag-1S, M and L isoforms. **** p< 00001 by Two-way ANOVA, Tukey’s multiple comparison test). D) Time-dependent wound healing images of Bag-1 KO cells and transfected with Bag-1S, M and L isoforms. E) Visualization of the actin cytoskeleton in Bag-1 isoform plasmid transfected MCF-7 wt and Bag-1 KO cells through fluorescent-labeled Phalloidin incubation by immunofluorescence experiment described in “Material and methods”.(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Scientific Reports 10.1038/s41598-020-73747-0, published online 27 October 2020Correction to: This Article contains an error in the entry Neutrophil-to-lymphocyte ratio (relative count) Median (IQR) reported in Table 1.“Neutrophil-to-lymphocyte ratio (relative count) Median (IQR) ABBREVIATIONS MUST BE DEFINED”.should read:“Neutrophil-to-lymphocyte ratio (relative count) Median (IQR)”.Secondly, the caption of Table 1 contains an error.RT radiotherapy; CT chemiotherapy.”“Table 1. Characteristics of the 56 patients and their cancers. should read:RT radiotherapy; CT chemiotherapy; IQR interquartile range.”“Table 1. Characteristics of the 56 patients and their cancers.


This analysis assessed improvements in patients with radiographic axial spondyloarthritis (r-axSpA) treated with ixekizumab in the Assessment of Spondyloarthritis International Society (ASAS) treatment response domains and additional patient-reported outcomes at 1 year of treatment.COAST-V and COAST-W were 52-week, phase 3, randomized controlled trials evaluating the efficacy and safety of ixekizumab in biologic disease-modifying antirheumatic drug (bDMARD)-naïve and tumor necrosis factor inhibitor (TNFi)-experienced patients with radiographic spondyloarthritis, respectively. Patients were treated with 80-mg ixekizumab either every 2 weeks or every 4 weeks. Patient-reported outcomes included Patient Global Disease Activity, Spinal Pain, stiffness as measured by Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) Questions 5 and 6, function as measured by the Bath Ankylosing Spondylitis Functional Index, fatigue as measured by the Fatigue Numeric Rating Scale and BASDAI question 1, Spinal Pain at Night, and sleep quality as measured by the Jenkins Sleep Evaluation Questionnaire. Mixed-effects models for repeated measures were used to analyze changes from baseline in patient-reported outcomes from weeks 1 to 16, and descriptive statistics were reported from weeks 20 to 52. Analysis of covariance with Scheffé’s method was used for the ASAS response association analyses.p < 0.001), with up to 10.5-fold greater improvements observed in ASAS40 responses compared with ASAS20 non-responders. Function and fatigue demonstrated the highest values.This study assessed 341 bDMARD-naïve and 316 TNFi-experienced patients in the placebo-controlled blinded treatment dosing period (weeks 1–16) as well as 329 bDMARD-naïve and 281 TNFi-experienced patients in the dose double-blind extended treatment period (weeks 20–52). bDMARD-naïve or TNFi-experienced patients treated with ixekizumab every 2 weeks and every 4 weeks reported improvements in patient global disease activity, spinal pain, function, stiffness, fatigue, spinal pain at night, and sleep quality through week 52. Greater correlations with improvements in all response domains were seen when comparing ASAS40 responders to ASAS20 non-responders (Ixekizumab-treated bDMARD-naïve and TNFi-experienced patients with radiographic axial spondyloarthritis achieving ASAS40 reported sustained and consistent improvement in all ASAS response domains and other patient-reported outcomes though week 52, with spinal pain, function, and stiffness as major drivers of the response.NCT02696785 and NCT02696798, March 2, 2016.The online version contains supplementary material available at 10.1186/s41927-021-00205-3. Radiographic axial spondyloarthritis (r-axSpA) is a chronic inflammatory condition that affects the axial skeleton and is also referred to as ankylosing spondylitis (AS). Up to 0.5% of adults worldwide are reported to have the disease –3. R-axSIxekizumab is a high-affinity monoclonal antibody that selectively targets interleukin (IL)-17A, a proinflammatory cytokine. In the context of r-axSpA, ixekizumab has demonstrated efficacy in patients who are naïve to biologic disease modifying antirheumatic drugs (bDMARD) and in patients intolerant of or inadequate responders to tumor necrosis factor inhibitors (TNFi); ixekizumab treatment in these two patient populations were evaluated in the COAST-V and COAST-W trials, respectively , 7.The Assessment of Spondyloarthritis International Society (ASAS) response criteria , which include ASAS20, ASAS40, and ASAS partial remission, are commonly used to measure r-axSpA treatment efficacy in clinical research . The COACOAST-V NCT02696785) and COAST-W (NCT02696798) were phase 3, multicentre, randomised, double-blind, active (COAST-V only) and placebo-controlled, 52-week trials. The trial designs for COAST-V and COAST-W have been previously published [96785 andInclusion criteria for COAST-V and COAST-W have been previously described , 7, 9. EThe dosing period regimens for COAST-V and COAST-W have been published , 9, 10. The primary endpoint for both COAST-V and COAST-W was the proportion of patients achieving an ASAS40 response at week 16. Secondary efficacy endpoints included ASAS20 and ASAS partial remission, and changes from baseline in ASAS treatment response domains , in addition to the clinically impactful symptoms of fatigue, spinal pain at night, and sleep quality.Function was evaluated as the average score of Bath Ankylosing Spondylitis Functional Index (BASFI) responses . StiffneWeek 52 data were pooled across treatment groups to assess the relationship between improvements in individual PROs and the achievement of different levels of ASAS response. These pooled data were then stratified by ASAS response level: ASAS20 non-responders, ASAS20 but not ASAS40 responders, and ASAS40 responders.Efficacy and health outcome analyses for the placebo-controlled blinded treatment (weeks 0 to 16) and the dose double-blind extended treatment period (weeks 16 to 52) were conducted on all patients randomized to treatment groups in COAST-V and COAST-W. Comparisons between treatment groups, and least square mean changes from baseline for the ASAS treatment response domains and other PROs up to week 16 were analyzed using a mixed-effects model for repeated measures, as described previously . The mixPost hoc association analyses were performed using data from week 52 between ASAS response levels and changes from baseline in the four ASAS response domains  and the additional PROs fatigue, spinal pain at night, and sleep quality. From weeks 0 to 52, changes from baseline in these PROs were compared between the ASAS responder groups listed above using analysis of covariance (ANCOVA) models after adjusting for baseline values, age, and gender. Standard error and standard deviation were used as measures of dispersion for ANCOVA. Post hoc comparisons were conducted with Scheffé’s correction, which was based on multiple comparisons of outcome measures. Fold increases were determined by the following calculations: (ASAS40 responder/ASAS20 non-responder) -1 or (ASAS40 responder/ASAS20 but not ASAS40 responder) -1. Modified baseline observation carried forward (mBOCF) was used for imputation of missing data .All analyses were conducted using SAS 9.4. Residual plots were generated via SAS to confirm normality of residuals.This study assessed 341 bDMARD-naïve and 316 TNFi-experienced patients in the placebo-controlled blinded treatment dosing period (weeks 0 to 16) as well as 329 bDMARD-naïve and 281 TNFi-experienced patients in the dose double-blind extended treatment period (weeks 16 to 52). Baseline disease characteristics for the bDMARD-naïve and TNFi-experienced patients in the dose double-blind extended treatment period population are displayed in Table As previously reported, improvements were seen in the four ASAS treatment domains at week 16 in patients treated with ixekizumab compared with placebo . ImproveImprovements were reported at 16 weeks for these outcomes in ixekizumab-treated patients compared with placebo , and theChanges from baseline in individual ASAS domains  were compared at week 52 among three ASAS response groups in order to evaluate the relationship between individual PROs and the achievement of ASAS response for both bDMARD-naïve and TNFi-experienced ixekizumab (Q2W and Q4W)-treated patients Fig. .Fig. 3Ap < 0.0001) in PtGA, a 4.7-fold improvement  in spinal pain, a 5.0-fold improvement  in function, and a 3.5-fold improvement  in stiffness were observed. Greater improvements were also reported when TNFi-experienced patients who achieved ASAS40 were compared with ASAS20 non-responders. A 5.2-fold improvement  in PtGA, 4.6-fold improvement  in spinal pain, a 10.5-fold improvement  in function, and a 3.6-fold improvement  in stiffness was observed.Greater improvements were reported for bDMARD-naïve patients who achieved ASAS40 when compared with ASAS20 non-responders. A 7.3-fold improvement  were also reported for the PROs of PtGA, spinal pain, function, and stiffness, for ixekizumab treated patients through 52 weeks of treatment, although more moderate (0.8-fold to 1.6-fold) than when compared with ASAS20 non-responders Fig. .p < 0.0001) in fatigue NRS, a 5.1-fold improvement  in fatigue (BASDAI Q1), a 3.3-fold improvement  in spinal pain at night, and 2.3-fold improvement  in sleep quality were observed  demonstrated significant improvements from baseline in fatigue, spinal pain at night, and sleep quality for bDMARD-naïve patients who achieved ASAS40 when compared with ASAS20 non-responders. A 4.6-fold improvement  in fatigue NRS, a 5.6-fold improvement  in fatigue (BASDAI Q1), a 4.3-fold improvement in spinal pain at night , and a 0.9-fold improvement  was observed for sleep quality  in fatigue (NRS and BASDAI Q1), spinal pain at night, and sleep quality for bDMARD-naïve patients , and 2.3 to 5.6-fold improvements for other PROs . In this study, the major drivers responsible for achieving the ASAS40 response are PtGA (7.3-fold improvement), BASDAI fatigue (5.1-fold improvement), and function (5.0-fold improvement) for the bDMARD-naïve patients, and function (10.5-fold improvement) and BASDAI fatigue (5.6-fold improvement) for the TNFi-experienced patients. Patient-reported fatigue defined by BASDAI Q1 is a prevalent and major clinical feature of r-axSpA . TherefoA recent study investigated the effect of ixekizumab treatment in bDMARD-naïve and TNFi-experienced patients with r-axSpA on work productivity, including absenteeism, presenteeism, and overall work impairment through week 52 . PatientIn conclusion, this post hoc analysis demonstrated the sustained and consistent effectiveness of ixekizumab in r-axSpA patients who achieved ASAS40 compared with patients who achieved ASAS20 or ASAS20 non-responders providing greater improvements in clinically relevant PROs including PtGA, spinal pain, spinal pain at night, function, stiffness, fatigue, and sleep quality through 1 year of treatment. Striving to reach an ASAS40 level of improvement will translate to better clinical disease management and improvement in common symptoms and patient quality of life.Additional file 1: Table S1. Changes from baseline in patient reported outcomes at week 52. Baseline is defined as the last non-missing assessment recorded on or prior to the date of first study drug injection at week 0. Data shown as mean (SD) at baseline, and mBOCF (SD) at week 52. ASAS = Assessment of Spondyloarthritis International Society; BASDAI = Bath Ankylosing Spondylitis Disease Activity Index; BASFI = Bath Ankylosing Spondylitis Functional Index; bDMARD = biologic disease-modifying anti-rheumatic drugs; IXE = ixekizumab; JSEQ = Jenkins Sleep Evaluation Questionnaire; mBOCF = modified baseline observation carried forward; N = number of patients in the analysis population; n = number of patients in each treatment subgroup; NRS = numeric rating scale; PtGA = patient global disease activity; Q = question; Q2W = every 2 weeks; Q4W = every 4 weeks; SD = standard deviation; TNFi = tumor necrosis factor inhibitor. Figure S1. Association between ASAS response and improvements in ASAS PROs for ixekizumab Q2W-treated patients after 52 weeks. Q2W ixekizumab-treated patients. § p<0.001, achieved ASAS20 but not ASAS40 vs. ASAS20 not achieved; * p<0.0001, ASAS40 achieved vs. ASAS20 not achieved; † p<0.0001, ASAS40 achieved vs. achieved ASAS20 but not ASAS40. Results were compared using ANCOVA. Values are LSM improvements from baseline (SE). mBOCF was used for imputation of missing data. Fold difference = (ASAS40 responder/ASAS20 non-responder) -1, or (ASAS40 responder/ASAS20 but not ASAS40 responder) -1. ANCOVA = analysis of covariance; ASAS = Assessment of Spondyloarthritis International Society; BASDAI = Bath Ankylosing Spondylitis Disease Activity Index; BASFI = Bath Ankylosing Spondylitis Functional Index; bDMARD = biologic disease-modifying antirheumatic drugs; LSM = least squares mean; mBOCF = modified baseline observation carried forward; Nx = number of observations; PROs = patient-reported outcomes; PtGA = patient global disease activity; Q = question; Q2W = every 2 weeks; SE: standard error; TNFi = tumor necrosis factor inhibitor. Figure S2. Association between ASAS response and improvements in ASAS PROs for ixekizumab Q4W-treated patients after 52 weeks. Q4W ixekizumab-treated patients. § p<0.001, achieved ASAS20 but not ASAS40 vs. ASAS20 not achieved; * p<0.0001, ASAS40 achieved vs. ASAS20 not achieved; † p<0.0001, ASAS40 achieved vs. achieved ASAS20 but not ASAS40. Results were compared using ANCOVA. Values are LSM improvements from baseline (SE). mBOCF was used for imputation of missing data. Fold difference = (ASAS40 responder/ASAS20 non-responder) -1, or (ASAS40 responder/ASAS20 but not ASAS40 responder) -1. ANCOVA = analysis of covariance; ASAS = Assessment of Spondyloarthritis International Society; BASDAI = Bath Ankylosing Spondylitis Disease Activity Index; BASFI = Bath Ankylosing Spondylitis Functional Index; bDMARD = biologic disease-modifying antirheumatic drugs; LSM = least squares mean; mBOCF = modified baseline observation carried forward; Nx = number of observations; PROs = patient reported outcomes; PtGA = patient global disease activity; Q = question; Q4W = every 4 weeks; SE = standard error; TNFi = tumor necrosis factor inhibitor. Figure S3. Association between ASAS response and improvements in other PROs for ixekizumab Q2W-treated patients after 52 weeks.Q2W ixekizumab-treated patients. § p<0.001, achieved ASAS20 but not ASAS40 vs. ASAS20 not achieved; * p<0.0001, ASAS40 achieved vs. ASAS20 not achieved; † p<0.0001, ASAS40 achieved vs. achieved ASAS20 but not ASAS40. Results were compared using ANCOVA. Values are LSM improvements from baseline (SE). mBOCF was used for imputation of missing data. Fold difference = (ASAS40 responder/ASAS20 non-responder) -1, or (ASAS40 responder/ASAS20 but not ASAS40 responder) -1. ANCOVA = analysis of covariance; ASAS = Assessment of Spondyloarthritis International Society; BASDAI = Bath Ankylosing Spondylitis Disease Activity Index; bDMARD = biologic disease-modifying antirheumatic drugs; JSEQ = Jenkins Sleep Evaluation Questionnaire; LSM = least squares mean; mBOCF: modified baseline observation carried forward; NRS = numeric rating scale; Nx = number of observations; PROs = patient reported outcomes; Q = question; Q2W = every 2 weeks; SE = standard error; TNFi = tumor necrosis factor inhibitor. Figure S4. Association between ASAS response and improvements in other PROs for ixekizumab Q4W-treated patients after 52 weeks.Q4W ixekizumab-treated patients. § p<0.001, achieved ASAS20 but not ASAS40 vs. ASAS20 not achieved; * p<0.0001, ASAS40 achieved vs. ASAS20 not achieved; † p<0.0001, ASAS40 achieved vs. achieved ASAS20 but not ASAS40. Results were compared using ANCOVA. Values are LSM improvements from baseline (SE). mBOCF was used for imputation of missing data. Fold difference = ASAS40 responder/ASAS20 non-responder) -1, or (ASAS40 responder/ASAS20 but not ASAS40 responder) -1. ANCOVA = analysis of covariance; ASAS = Assessment of Spondyloarthritis International Society; BASDAI = Bath Ankylosing Spondylitis Disease Activity Index; bDMARD = biologic disease-modifying antirheumatic drugs; JSEQ = Jenkins Sleep Evaluation Questionnaire; LSM = least squares mean; mBOCF = modified baseline observation carried forward; NRS = numeric rating scale; Nx = number of observations; PROs = patient-reported outcomes; Q = question; Q4W = every 4 weeks; SE = standard error; TNFi = tumor necrosis factor inhibitor.


Correction to: BMC Health Serv Res 20, 726 (2020)https://doi.org/10.1186/s12913-020-05560-4Statistical analyses section under the heading Method.Following publication of the original article , the autThe sentence currently reads:segment 2 , segment 3 The sentence should read:segment 2 , segment 3 The original article  has been