Cerium oxide nanoparticles (CeNPs) have been shown to exhibit antioxidant capabilities, but their efficiency in scavenging reactive oxygen species (ROS) and the underlying mechanisms are not yet well understood. In this study, cerium dioxide nanoparticles (CeNPs) and nanorods (CeNRs) were found to exhibit much stronger scavenging activity than ·OH generation in phosphate buffered saline (PBS) and surrogate lung fluid (SLF). The larger surface area and higher defect density of CeNRs may lead to higher ·OH scavenging activity than for CeNPs. These insights are important to understand the redox activity of cerium nanomaterials and provide clues to the role of CeNPs in biological and environmental processes. Cerium dioxide nanoparticles and nanorods were found to exhibit much stronger scavenging activity than ·OH generation in quasi-physiological conditions. The SLF was used to mimic the key interface between human respiratory tract and inhaled air.In contrast to research about the antioxidant activity of CeNPs, inhalable CeNPs have been detected in ambient air and concerns have been raised about their potential adverse health effect.∅ 50 nm) and CeNPs (∅ 25 nm) have a heterogeneous size distribution with average diameters of <50 nm and <25 nm respectively. Moreover, samples of these commercial CeNPs contain predominantly cubic NPs. In contrast, the morphology of CeNRs  (2 g−1 (∅ 25 nm CeNPs) (2 g−1 (CeNRs) (∅ 50 nm) (∅ 25 nm) (3+ and Ce4+) on these NPs are quite similar. The six most prominent peaks of these spectra are attributable to Ce4+ ions.33 This indicates that Ce4+ was the dominant cerium species in all three samples. The peak fittings (dashed lines) in panels G, H and I are based on the method by Maslakov et al..33 The fitting based deconvolution of Ce 3d XPS spectra indicates that the concentration of surface Ce3+ in all these samples is <3%. Such a low abundance of surface Ce3+ is also supported by the absence of a shoulder peak of Ce 4f electrons at ∼1.1 eV in the XPS valence band spectrum of the CeNRs samples  , 39.2 ± m CeNPs) , and 106 (CeNRs) . Moreove∅ 50 nm) , CeNPs (∅ 25 nm) , and CeN∅ 25 nm)  indicatetert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide  and EPR spectra of aqueous mixtures of Fe2+, H2O2, SLF, and CeNPs (panel B). 35 The peak intensities of the spectra in 2+ + H2O2) > B (Fe2+ + H2O2 + CeNPs) > C (Fe2+ + H2O2 + SLF) > D (Fe2+ + H2O2 + CeNPs + SLF). This implies that the amount of ·OH decreases accordingly. Based on the spin-counting method,32 we quantified the concentration of BMPO–OH in these solutions. The results are shown in †∅ 50 nm) without (black circles) and with the addition of H2O2 (black triangles) under different CeNPs (∅ 50 nm) loading conditions. In the absence of H2O2, 0.1–30 mg mL−1 CeNPs (∅ 50 nm) can generate 0–0.8 and 0–0.5 μM ·OH in pH = 4.7 (∅ 50 nm) in acidic PBS is consistent with previous hypothesis that acid can catalyze the ·OH formation by CeNPs.36 In contrast to pure CeNPs (∅ 50 nm), mixtures of 0.1–30 mg mL−1 CeNPs (∅ 50 nm) with 10 mM of H2O2 can generate 0–5 (pH = 4.7) and 0–3 μM (pH = 7.4) ·OH, which also shows a positive correlation with the loading of CeNPs (∅ 50 nm) as shown in 37 H2O2 + Ce3+ → Ce4+ + ·OH + OH−.pH = 4.7  and 7.4 pH = 4.7  PBS, res∅ 50 nm) or CeNRs, Fe2+, and H2O2 in acidic and neutral PBS. 2+ and 10 mM H2O2 generated ∼53 and ∼17 μM ·OH in pH = 4.7 (−1 CeNPs (∅ 50 nm), concentration of ·OH decreased to 15 μM (pH = 4.7) and 11 μM (pH = 7.4), respectively (Tables S5 and S6−1 CeNRs. This implies that the scavenging activity of CeNPs (∅ 50 nm) is more pronounced under acidic conditions. The decrease of ·OH concentration may be induced by the following processes: first, CeNPs (∅ 50 nm) or CeNRs could scavenge ·OH directly  or CeNRs surfaces  or CeNRs.To evaluate the ·OH scavenging activity of CeNPs in aqueous solution, we measured the ·OH yield by mixtures of CeNPs  can be adsorbed on the surface of the CeNPs. Therefore, the surface adsorption of H2O2 by CeNPs cannot fully explain the reduction of ·OH concentration in et al.21 observed a H2O2 decomposition rate of ∼2.7 nmol min−1 through catalase-like reactions. Based on this value, we estimate that a concentration of 0.1 mg mL−1 of CeNPs would result in a H2O2 loss of <2% in our studies. Therefore, we suggest the direct scavenging process  and CeNRs loading. As the loading of CeNPs (∅ 50 nm) increased from 0.1 to 10 mg mL−1, the concentration of ·OH in SLF decreased by 38–85%. Within the same loading range, the CeNPs (∅ 25 nm) exhibited a similar efficiency. Whereas at higher loadings (1–5 mg mL−1), the ·OH scavenging potential of CeNPs (∅ 25 nm) was 9–55% higher than that of their 50 nm counterparts. In contrast to CeNPs, the CeNRs showed a much higher ·OH scavenging efficiency. Even with a loading as low as 0.1 mg mL−1, the CeNRs could reduce 88% of the ·OH. For CeNRs loadings that exceeded 1 mg mL−1, no ·OH could be observed. The trend of the ·OH scavenging efficiency according to CeNRs > CeNPs (∅ 25 nm) >CeNPs (∅ 50 nm) is in the same order as the surface area of these NPs (3+ on fresh CeNPs and CeNRs surface (3+ may be formed upon interactions of NPs with water.13 The larger surface area of CeNRs may increase the density of Ce3+ per unit particle mass and subsequently their ·OH scavenging activity. Previous works showed that CeNRs are prone to expose their (110) facets to reactive species.34 These facets were described as reactive “hybrid structures” between the (111) and (100) surfaces of CeNPs. Furthermore, the distinct crystallographic surface structure of CeNRs may act as binding site for reactive species (·OH and H2O2) exerting peroxidase-like effects. Additionally, Fe2+-dependent reactive oxygen chemistry may contribute to the observed ·OH scavenging processes.39 Finally, it has been suggested that glutathione could interact with CeNPs and influence the redox couple of Ce3+/Ce4+.40To assess the antioxidant activity of CeNPs under quasi-physiological conditions, we explored the ·OH scavenging activity of CeNPs and CeNRs in SLF. hese NPs . Given t surface , we sugg41 which may eventually influence its properties including ·OH scavenging efficiency and SOD-like characteristics.42 Thus, characterizing CeNPs or their functionalized derivatives in more realistic environments will be beneficial and promising in follow-up studies.It is worthy to note that a real physiologic environment is more complicated than SLF. A large number of redox chemistry processes may alter the agglomeration and distribution of CeNPs and relevant materials,−1. In the same loading range, CeNRs showed 5–50-fold higher ·OH scavenging potential than CeNPs, which may be attributable to the higher surface area and defect density of CeNRs. Furthermore, we found the scavenging activity of CeNPs is pH-dependent, exhibiting higher scavenging efficiency under lower pH condition. The observed ·OH scavenging efficiency of CeNPs and CeNRs in SLF took into account the effect of antioxidants at concentrations close to the epithelial lung fluid, reflecting the redox activity of CeNPs and CeNRs under more realistic in vitro conditions than previous studies. These findings are of critical importance for a better understanding of the relative ROS scavenging efficiency of CeNPs comparing to conventional antioxidants. Moreover, these results are also important for making accurate dose–response curves predicting the toxicity or antioxidant characteristics of CeNPs or their functionalized derivatives in biological and environmental processes.In this study, we compared the ability of CeNPs and CeNRs in scavenging hydroxyl radicals (·OH) under physiologically relevant conditions. We found that CeNPs and CeNRs exert high ·OH scavenging activity in both PBS and SLF. In SLF, the ·OH scavenging potential of CeNPs increased 4-fold as the loading increases from 0.1 to 10 mg mLThere are no conflicts to declare.RA-009-C9RA00642G-s001


R-enantiomers (GAT1665 and GAT1667) displayed mixed allosteric agonist-PAM activity at CB1R while the S-enantiomers (GAT1664 and GAT1666) showed moderate activity. Furthermore, we observed that the R and S-enantiomers had distinct binding sites on CB1R, which led to their distinct behavior both in vitro and in vivo. The R-enantiomers (GAT1665 and GAT1667) produced ago-PAM effects in vitro, and PAM effects in the in vivo behavioral triad, indicating that the in vivo activity of these ligands may occur via PAM rather than agonist-based mechanisms. Overall, this study provides mechanistic insight into enantiospecific interaction of 2-phenylindole class of CB1R allosteric modulators, which have shown therapeutic potential in the treatment of pain, epilepsy, glaucoma, and Huntington’s disease.Positive allosteric modulation of the type 1 cannabinoid receptor (CB1R) has substantial potential to treat both neurological and immune disorders. To date, a few studies have evaluated the structure-activity relationship (SAR) for CB1R positive allosteric modulators (PAMs). In this study, we separated the enantiomers of the previously characterized two potent CB1R ago-PAMs GAT591 and GAT593 to determine their biochemical activity at CB1R. Separating the enantiomers showed that the  The endogenous cannabinoid system (ECS) is composed of endogenous ligands (e.g. anandamide [AEA] and 2-arachidonoylglycerol [2-AG]), anabolic and catabolic enzymes, and receptors  . The ECSCannabis represent a major limitation to its use as medicine. The intoxicating effects of Cannabis are thought to be due to ∆9-tetrahydrocannabinol (THC) binding to the orthosteric site of CB1R -GAT228 was an allosteric-agonist and the opposite enantiomer, (S)-GAT229, showed PAM activity at CB1R within the assays and cell line used  and Sigma-Aldrich , respectively. All other tested compounds were obtained from Dr. Ganesh Thakur, Northeastern University. All compounds were initially dissolved in dimethylsulfoxide (DMSO) and diluted in a 10% DMSO solution in phosphate-buffered saline (PBS). Compounds were added directly to cell culture at the time and concentrations indicated at a final concentration of 0.1% DMSO.2 in DMEM/F-12 containing 10% fetal bovine serum (FBS) and 5 U × 104 U penicillin/streptomycin . In addition, 800 μg/ml geneticin was used for CHO-K1 hCB1R HitHunter cAMP cells and 800 μg/ml geneticin and 300 μg/ml hygromycin B was used for CHO-K1 hCB1R PathHunter βarrestin2 cells.HitHunter (cAMP) and PathHunter (βarrestin2) Chinese hamster ovary (CHO)-K1 cells stably-expressing human CB1R (hCB1R) from DiscoveRx  were maintained at 37°C, 5% CO2. Opti-MEM media was removed and replaced with cell assay buffer (DiscoveRx) and then cells were simultaneously treated with 10 µM FSK and experimental compounds (0.10 nM–10 µM) for 90 min cAMP antibody solution and working detection solutions were added according to the manufacturer’s protocol (DiscoveRx), and cells were incubated for 60 min at room temperature. cAMP solution A was added according to the manufacturer’s protocol (DiscoveRx) and cells were incubated for an additional 60 min at room temperature before chemiluminescence was measured on a Cytation5 plate reader . Data are presented as percent maximal CP55,940-dependent inhibition of cAMP accumulation.cAMP inhibition was performed in the presence of 10 µM forskolin (FSK) using the DiscoveRx HitHunter assay in hCB1R CHO-K1 cells. Cells  were incubated overnight in Opti-MEM (Invitrogen) containing 1% FBS at 37°C and 5% CO2. Cells were then simultaneously treated with experimental compounds (0.10 nM–10 µM) for 90 min. Detection solution was added to cells according to the manufacturer’s protocol (DiscoveRx), and cells were incubated for 60 min at room temperature. Chemiluminescence was measured on a Cytation5 plate reader . Data are presented as percent maximal CP55,940-dependent stimulation.βarrestin2 recruitment was determined using the hCB1R CHO-K1 cell PathHunter assay (DiscoveRx). Cells  were incubated overnight in Opti-MEM (Invitrogen) containing 1% FBS at 37°C and 5% CO3H]CP55,940 in Tris buffer , 1% bovine serum albumin (BSA), pH 7.4) with a total assay volume of 200 µl. The assay began with the addition of transfected hCB1R CHO-K1 cell membranes (25 µg protein per well). The assays were left to equilibrate at room temperature for 2 h before vacuum filtration using a Millipore Sigma 12-well sampling manifold and filter paper that had been soaked in wash buffer. Each reaction well was washed 3 times with a 2 ml aliquot of Tris-binding buffer. The filters were removed then submerged in 5 ml of scintillation fluid . Radioactivity was quantified by liquid scintillation spectrometry. Specific binding was defined as the difference between binding that occurred in the presence and in the absence of 1 µM unlabeled CP55,940. Data are presented as percent [3H]CP55,940 bound.Previous work from our group has shown that CB1R PAMs enhance orthosteric agonist binding  . RadioliThis assay was conducted as described in previous reports . To summad libitum access to food and water, and environmental enrichment. Compounds administered intraperitoneally (i.p.) were prepared in vehicle [ethanol and cremophor in saline (1:1:8)]. Catalepsy was assessed in the bar holding assay 5 min after drug administration. Mice were placed so that their forepaws clasped a 0.7 cm ring clamp positioned 4.5 cm above the surface of the testing space  between 4 and 7 months of age were used for these studies. Animals were group housed at the Laboratory Animal Services Unit (LASU) at the University of Saskatchewan  with a standard 12:12 light-dark cycle, ng space . The lenng space .A conformational analysis was performed on GAT1664, GAT1665, GAT1666, and GAT1667 using Spartan’18 V1.4.5 . A confovia exclusion spheres placed on each residue. The Generalized Born/Surface Area (GB/SA) continuum solvation model for water was used with the dielectric set to 80 outside of the implicit membrane region and 2 within. A truncated Newton conjugate gradient minimization was performed using the OPLS3e force field for one iteration up to a maximum of 1,000 steps and with a 0.1 kcal/mol gradient endpoint. Constraints of 1 kcal/mol were placed on the C-alpha atoms of residues R3.50, Y5.58, L6.33, and Y7.53 to prevent the intracellular opening present in the R* structures from closing during the minimization.The CB1R model has been described in detail in To identify potential binding site(s) for GAT1664, GAT1665, GAT1666 and GAT1667 at CB1R, we used the Forced-Biased Metropolis Monte Carlo simulated annealing program (MMC) as the first step . The MMCOf particular interest in MMC calculations are fragments that persist at particular sites on the protein throughout the annealing schedule, since these fragments clearly have high affinity for those sites. Receptor regions at which all molecular fragments of a studied ligand collect are identified from MMC output. This set of sites is then refined to include only those sites at which the order of the fragments reflects the structure of the entire molecule.GAT1664 and GAT1665 were broken into fragments that included a 6-fluoro-indole ring, a 2-fluoro-phenyl ring and a 3-fluoro-phenyl ring. GAT1666 and GAT1667 were broken into fragments that included 7-fluoro-indole ring, 2-fluoro-phenyl ring and 3-fluoro-phenyl ring. All fragments were prepared with partial charges using the Amber 2002 force field, a point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum MMC . Six MMCDocking: Once the two sites were confirmed, the structures of GAT1664-GAT1667 were docked in the intracellular TMH1/2/4 site with H2.41, F4.46, and W4.50 used as direct interaction sites. GAT1664 and GAT1666 were docked in the TM2/3/EC1 site with Y2.59 and D2.63 as direct interaction sites. In addition, after extensive previously published molecular dynamics calculations on unfluorinated versions of the GAT compounds (GAT1600-3), R (148) was modeled to interact directly with the carbonyl oxygens of the last turn of TMH1 and not the nitro group within the compounds  continuum solvation model for water was used with the dielectric set to 80 outside of the implicit membrane region and 2 within. A truncated Newton conjugate gradient minimization was performed using the OPLS3e force field for 1 iteration, up to a maximum of 1,000 steps and with a 0.1 kcal/mol gradient endpoint. Constraints of 1 kcal/mol placed on the c-alpha atoms of residues R3.50, Y5.58, L6.33, and Y7.53 were set to prevent the intracellular opening from closing during the minimization. The resulting docks were refined with the Induced Fit protocol . The Glide box size was set to 12 Å3 centered on the ligand and the SP docking algorithm employed. Residues within 5 Å of the docked ligand were included in the Prime refinement stage, except in the case of the TM2/3 PAM site where S2.60 and K3.28 were excluded based on mutation data . As the MMGBSA binding energies are approximate free energies of binding, a more negative value indicated stronger binding. For this computation, the VSGB solvation model was employed while the chosen force field OPLS4 was used.Each of the GAT1664-GAT1667 ligand-receptor complexes were evaluated 50 and Emax in 3H]CP55,940 bound  displayed greater PAM potency and efficacy than the S-enantiomers (GAT1664 and GAT1666), (ex vivo model systems (All enantiomers displayed G protein-mediated agonist activity at hCB1R as shown by their ability to inhibit cAMP production in the absence of an orthosteric agonist . The abiAT1666), . InitialAT1666), . ImportaAT1666), , includi systems . These o systems .R-enantiomers) recruit βarrestin2.For consistency with cAMP inhibition experiments and previous studies , compounThe operational model described by 3H]CP55,940 binding, consistent with their proposed PAM activity, with the R-enantiomers GAT1665 and to a greater extent GAT1667 increasing [3H]CP55,940 binding (50 = 28 [15–52] nM). Enantiomers were also tested for their ability to promote G protein coupling in the GTPγS assay (R-enantiomers GAT1665 and GAT1667 being more potent than the S-enantiomers and GAT1667 in particular displaying the greatest potency (EC50 = 8.0 [3.4–17] nM)  . When 1 –17] nM) . Therefoin vivo response to be modulated by our compounds of interest. It was found that GAT1665 and GAT1667 displayed significant cataleptic, hypothermic, and anti-nociceptive effects at 3 and 10 mg/kg i.p. compared to vehicle  compared to vehicle . Both GA vehicle . Therefoed alone .2.41 (4.46 (4.03Å) while the other fluorobenzyl has a T-shaped π-π interaction with each ring on W2414.50   . Upon bikcal/mol . GAT1666kcal/mol . This caate CB1R , 7 (Hursate CB1R .2 group on GAT1665 has a H-bond between 1 of the oxygens shown in red and a hydrogen atom on Y1722.59 (O–H distance 2.17 Å) (2.63 (H–O distance 2.29 Å)  (2.59 (4.05 Å) (2 rotates in order to H-bond with Y1722.59 (R-enantiomers (GAT1665 and GAT1667) undergo a smaller conformational cost than the S-enantiomers (GAT1664 and GAT1666) upon binding to their respective sites on hCB1R (R-enantiomers (GAT1665 and GAT1667) bind to the ago-PAM site  significantly weaker than the S-enantiomers (GAT1664 and GAT1666) bind to the PAM site . Of note, binding of enantiomers to either site was not absolute but was more energetically favourable for the S-enantiomers to bind near the intracellular surface and the R-enantiomers to bind near the first extracellular loop.GAT1665 and GAT1667 displayed the greatest modelled affinity to a distinct allosteric site on hCB1R relative to GAT1664 and GAT1666 , 8. The  2.17 Å) . Another 2.29 Å) . A catio 3.66 Å) . One of (4.05 Å) . Upon biY1722.59 . The conon hCB1R . The R-e3H]CP55,940 at hCB1R. Enhancing the binding of an orthosteric ligand is a characteristic of a PAM and these observations are consistent with all compounds operating in part via positive allostery and/or non-competitive allosteric agonism  acted as ago-PAM’s while GAT1664 and GAT1666 (S-enantiomers) showed little to no activity as either an agonist or PAM. In the G protein coupling assay, the R-enantiomers GAT1665 and GAT1667 also showed higher potency and efficacy than S-enantiomers as agonists and PAMs. As for the in vivo studies, no compounds tested produced catalepsy, hypothermia at or antinociceptive effects at 0.1, 1, 3 or 10 mg/kg i.p. compared to vehicle when tested alone, but GAT1665 and GAT1667 did potentiate the effects of THC as ago-PAMs. These data are congruent with previous observations that CB1R ago-PAMs are inactive or minimally active in vivo in wild-type, otherwise healthy animals under non-pathological and acute conditions , with GAT1665 and GAT1667 displaying the greatest potency and efficacy as CB1R PAMs in the presence of 100 nM CP55,940. In the βarrestin2 assay, GAT1665 and GAT1667 (nditions . In plannditions . In addiing site . GAT1664ing site . Howevering site . Moreoveing site . TherefoThis is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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Cells were divided into three groups: Control without irradiation; 4 h after irradiation; and 24 h after irradiation. In control, gene expression levels of OAT1 in all cells was below the limit of measurement. After irradiation, gene expression levels of OATP1B1/1B3/1B7 showed changes in each cell line. Gene expression levels of MRP1/2 tended to increase after irradiation. Gene expression levels of OATP1B1/1B3/1B7 were much lower than those of MRP1/2. Accumulation of 3H-MTX tended to decrease over time after irradiation. Irradiation of cancer cells thus alters gene expression levels of both solute carrier transporters (OATP1B1/1B3/1B7) and ABC transporters (MRP1/2) and decreases accumulation of 3H-MTX in cancer cells over time due to elevated expression of MRP1/2.Chemoradiotherapy is frequently used to treat cancer. Stereotactic body radiotherapy (SBRT) is a single high-dose radiotherapy used to treat a variety of cancers. The anticancer drug methotrexate (MTX) shows affinity for solute carrier (SLC) and ATP-binding cassette (ABC) transporters. This study investigated relationships between accumulation of methotrexate and gene expression levels of solute carrier and ATP-binding cassette transporters in cancer cells after a single and high-dose X-ray irradiation. Cancer cell lines were selected from lung and cervical cancer cell line that are commonly used for stereotactic body radiotherapy and effective with methotrexate. We examined expression levels of organic anion-transporting polypeptide (OATP)1B1, OATP1B3, OATP1B7, and organic anion transporter (OAT)1 as solute carrier transporters and multidrug resistance-associated protein (MRP)1 and MRP2 as ATP-binding cassette transporters, using real-time polymerase chain reaction and accumulation of  Chemoradiotherapy combines chemotherapy with anticancer drugs and radiotherapy to treat cancer. Chemoradiotherapy can be divided into three categories according to the timing of anticancer drug administration: Neoadjuvant; concurrent; and adjuvant . The accIn radiotherapy, stereotactic radiotherapy involves the delivery of higher doses (7–18 Gy or more) than the usual single-beam dose (1.8–2 Gy) and is used to treat various cancers . StereotMethotrexate (MTX) is a folate antagonist used as an anticancer drug . This ag2.The human-derived lung adenocarcinoma cancer cell lines NCI-H441  and PC-14  were used. The HeLa human-derived cervical cancer line (RIKEN Cell Bank) was also used. H441, PC-14 and HeLa cell lines were cultivated using RPMI-1640 , Dulbecco’s Modified Eagle’s Medium  and Eagle’s minimum essential medium  mixed with 10% fetal bovine serum and 1% sodium pyruvate at 37°C under conditions of 5% COAfter achieving 70–80% confluence in a 10-cm diameter plate, each cell line was irradiated with a single 10-Gy X-ray  using X-ray irradiation equipment . Cells were divided into three groups: Control without irradiation; 4 h after irradiation; and 24 h after irradiation.An RNeasy Plus Mini Kit  was used to extract RNA from the cancer cells used in this study. The quality of the extracted RNA was evaluated using the RNA integrity number (RIN) as an indicator of quality. The RIN is expressed as a number from 1 to 10, with a higher number reflecting higher quality of RNA. An Affinity Script QPCR cDNA Synthesis kit  was used for synthesizing cDNA.OATP1B1/1B3/1B7 as the combination of OATP1B1, OATP1B3 and OATP1B7 for SLC transporters (because the primer sequences of these transporters are quite similar) and MRP1 and MRP2 for ABC transporters were selected as the targets of PCR. The gene ACBT for β-actin was used as the internal control gene, as a housekeeping gene that is constantly expressed in all cells. Also, ACBT was used to correct for differences in the amounts of initial RNA and cDNA due to differences in sample organization and purification methods. Primer design was outsourced to Eurofins Genomics . Preparation of cloned plasmids used for the creation of standard curves was outsourced to GenScript . Primer sequences and concentrations of the genes used are shown in Real-time PCR was performed using an AriaMx 5P system (Agilent Technologies). 5 cells/well in 12-well plastic plates. At about 24 h after seeding, cells were irradiated and divided into three groups: 4 h after irradiation; 24 h after irradiation; and control without irradiation. Each group was pre-incubated for 5 min in phosphate-buffered saline (PBS), then incubated with 3H-MTX (10 kBq/well) for 5, 10, 30, or 60 min. After incubation, cells were washed twice with 600 µL of PBS and lysed by 500 µL of .1 M NaOH. Three hundred microliters of cell lysate were mixed with 5 mL of ULTIMA GOLD  and the radioactivity of the mixture was measured using a liquid scintillation counter . The results are shown as the percent injected dose/number of living cells measured by an automatic cell counter .Each cell was seeded at 1.0 ×10All cell lines showed RIN >9, indicating high-quality RNA. Measured expression levels of drug transporter genes in each cell line in the three groups by conducting Real-time PCR are shown in For ABC transporters, expression levels of MRP1/2, as the combination of MRP1 and MRP2, were higher than levels of OATP1B1/1B3/1B7 and OAT1 in control samples of all cancer cells. In control samples, MRP1/2 showed higher expression in HeLa than in H441 or PC-14. In addition, H441 and PC-14 showed higher gene expression levels of MRP1 than MRP2, while HeLa displayed higher gene expression levels of MRP2 than MRP1. After irradiation, gene expression levels of MRP1/2 tended to increase over time in all cancer cell lines.3H-MTX in H441, PC-14 and HeLa cells in the control, 4 h after irradiation and 24 h after irradiation groups at 5, 10, 30, and 60 min after 3H-MTX injection. Accumulation of 3H-MTX was decreased at 4 and 24 h after irradiation in H441 cells and at 24 h after irradiation in PC-14 cells. In HeLa cells, accumulation of 3H-MTX was significantly decreased compared to control from 10 min after 3H-MTX injection in the 4 h after irradiation group and from 30 min after 3H-MTX injection in the 24 h after irradiation group.3H-MTX in cancer cells was impacted by the effects of gene expression levels for SLC and ABC transporters after X-ray irradiation. Since the degree to which gene expression levels of SLC and ABC transporters are changed under the influence of irradiation was unknown, we examined these gene expressions after irradiation by conducting Real-time PCR  and ABC transporters (MRP1/2). In particular, changes in MRP1/2 were much greater than those in OATP1B1/1B3/1B7. Irradiation decreased accumulation of


The formed NiO was deposited on the surface of Sr2FeTaO6 to form a closely interfacial contact, leading to the formation of NiO/Sr2FeTaO6 heterojunction. The resulted samples were fully characterized by XRD, TEM, XPS, and UV-Vis DRS to gain their microstructure, crystal phase, atomic states and optical absorption properties. Introducing narrow-bandgap semiconductor of black NiO in NiO/Sr2FeTaO6 heterojunctions exhibits two major advantages. On the one hand, coupling with black NiO can significantly increase the light harvesting capacity of Sr2FeTaO6. On the other hand, the formed NiO/Sr2FeTaO6 heterojunctions benefited the separation and transfer of photogenerated charge carriers, which was confirmed by photo-electrochemical measurement, PL and TR-PL spectra. The activity of as-prepared samples was evaluated by photocatalytic hydrogen (H2) evolution (PHE) under visible light irradiation. The resulted NiO/SFT composites showed the improved PHE efficiency than that of NiO and Sr2FeTaO6, owing to the synergistic effects of synergistic effects of heterojunction formation for the efficient charge carrier transfer/separation and increased light harvesting capacity. However, the excess amount of NiO loaded in NiO/SFT composites will restrain the light harvesting of Sr2FeTaO6 component and decrease, leading to the decreased PHE activity. Our work provided an insight on the construction of high-efficiency heterojunction photocatalysts for PHE reaction.Series of black NiO/Sr The activity of as-prepared samples was evaluated by PHE reaction under visible light irradiation. The introduction of black NiO in NiO/SFT composites exhibited two advantages of increased light harvesting capacity and enhanced charge carrier separation and transfer, leading to the high PHE efficiency. A possible PHE mechanism was proposed based on the experimental results.In light of the above consideration, this work employed a facile hydrothermal process to synthesize black NiO/Sr2FeTaO6, Sodium oleate (300 mg) was firstly dissolved in a mixture of ethanol (52.5 mL) and oleic acid (7.5 mL) under constant stirring. Then ferric acetylacetonate (1.059 g), strontium acetate (1.234 g) and tantalum pentachloride (1.074 g) were separately added into above solution with a sealed condition. After stirring for 30 min, 15 mL sodium hydroxide (.5 M) was rapidly added and then a faint yellow emulsion appeared. After reaction for 60 min, the obtained faint yellow solution was transferred into a 100 mL Teflon-lined autoclave and heated at 200°C for 12 h. After cooling to room temperature, the obtained precipitates were gathered by centrifuged , washed with ethanol and deionized water several times, and subsequently dried under vacuum at 60°C to gain Sr2FeTaO6 powder.To prepare SrThe black NiO nanoparticles were prepared by a two-step approach containing hydrothermal method and subsequent calcination treatment. Nickel nitrate (5 mmol) and urea (15 mmol) were dissolved in mixed solution of 50 mL deionized water and 25 mL ethanol. After ultrasonic treatment for 30 min, aforementioned solution was transferred into a 100 mL Teflon-lined autoclave and heated at 120°C for 12 h. The obtained NiO precursor was washed with deionized water and ethanol several times, and then dried in a vacuum oven at 60°C. Finally, NiO precursor was directly calcined at 500°C for 2 h in air to gain black NiO nanoparticles.x mmol Ni(NO3)2·6H2O , (10-x) mmol Sr2FeTaO6 powders, and 15 mmol urea were added into the mixture of deionized water (50 mL) and ethanol (25 mL) under the continuous ultrasonic treatment for 30 min. The remained process for synthesizing xNiO/(10-x) SFT composites was similar to that of NiO. For comparison, black NiO nanoparticles were simply mixed with Sr2FeTaO6 to prepare the mixed sample of 3NiO/7SFT (MIXT), showing the same composition with 3NiO/7SFT.λ = 1.5408Å). The X-ray photoelectron spectroscopy (XPS) measurement was performed in a VG Scientifific ESCALAB250-XPS photoelectron spectrometer with an Al Kα X-ray (hν = 1486.6 eV) source. UV–Vis diffuse reflection spectroscopy (DRS) was obtained on a HITACHI U-3310 spectrophotometer to investigate the optical properties of the samples. Photoluminescence (PL) spectra were recorded on JASCO FP 6500 fluorescence spectrometer with using a He-Cd laser source in the excitation wavelength. Time-resolved photoluminescence (TR-PL) decay spectra of as-prepared samples were measured by a F900 fluorescence spectrometer with an excitation wavelength of 310 nm.The morphology, phase structure and composition of the samples were obtained by various characterization techniques. High-resolution transmission electron microscope (HRTEM) was operated on JEOL JEM-200CX to measure the microstructure and lattice fringes of photocatalysts. X-ray diffraction pattern (XRD) was collected on a powder X-ray diffractometer  using Cu Kα radiation  was used as visible light source. In the H2 production experiment, 50.0 mg photocatalyst was dispersed into the aqueous solution containing triethanolamine  as the hole sacrificial reagent and H2PtCl6 solution (2 wt% Pt) as cocatalyst. Prior to PHE reaction, the reactor system was vacuumed by using a vacuum pump to remove air completely. The amount of generated H2 was determined by gas chromatography .The photocatalytic performance of as-synthesized samples was evaluated by measuring H2FeTaO6, HRTEM images were conducted and presented in 2FeTaO6 sample has the similar particle morphology but a larger particle size  in 2FeTaO6, leading to the formation of heterojunction. This formed NiO/Sr2FeTaO6 heterojunction benefits the charge carrier separation and transfer, thereby boosting PHE efficiency  composites exhibit the dual characteristic signals of both NiO and Sr2FeTaO6. With the increasing of NiO loading amount, the peak intensity of NiO is gradually increased. Thus, it indicates that NiO is successfully coupled with Sr2FeTaO6 to form NiO/Sr2FeTaO6 heterojunction.The X-ray diffraction (XRD) patterns of as-prepared samples were measured to determine their crystal structure and composition. As shown in 3/2 (Ni2+) and Ni 2p1/2 (Ni3+), respectively  . Figure ectively . Three s2FeTaO6 at 529.7 eV and 531.8 eV are attributed to lattice Ta-O-Fe and surface oxygen (Osur), respectively, while only one characteristic peak of Osur at 531.8 eV is visible in NiO (7/2) and 25.15 eV (Ta 4f5/2) in Sr2FeTaO6 SFT composites  show widened absorption regions and increased light absorption edge values from 577 nm for 1NiO/9SFT to 645 nm for 9NiO/1SFT, which are strongly dependent on NiO deposition amount. Additionally, the bandgap values of various samples are resulted from the UV–Vis DRS absorption plots  were examined to investigate the optical absorption properties of as-prepared samples . The ligon plots . Accordiectively . This re2FeTaO6, and 3NiO/7SFT with a typical semicircular arc. The diameter of the semicircular arc can indirectly reveal the migration and separation mechanism of photogenerated electrons and holes  and transient photocurrent response of as-prepared samples were performed to analyze charge carrier migration and separation rate . Figure nd holes . A lowernd holes . Special2FeTaO6, and 3NiO/7SFT were measured under visible light for eight cycles  and Sr2FeTaO6 (τ = .56 ns), 3NiO/7SFT (τ = 3.11 ns) sample has the increased average emission lifetime  spectroscopy was developed to investigate the recombination capacity of the photogenerated electrons and holes in as-prepared samples . The PL lifetime , indicat2 evolution performance of NiO, Sr2FeTaO6 and xNiO/(10-x)SFT composites  was evaluated under visible light using TEOA aqueous solution as sacrificed electron donor. As shown in 2 evolution of NiO and Sr2FeTaO6 exhibit the relatively low rates of 268.9 and 910.9 μmol h−1 g−1, owing to the rapid recombination of photogenerated electrons and holes. In the xNiO/(10-x)SFT  photocatalytic system, the hydrogen production rate is increased with the increasing of NiO loading amount, showing the optimal example of 3NiO/7SFT with the maximum H2 evolution efficiency of 2,944 μmol h−1 g−1. With the loading amount increasing of NiO, the overall hydrogen production rates are correspondingly decreased. It demonstrates that the synergistic effects of heterojunction formation and increased light harvesting capacity play a vital role in boosting photocatalytic activity. However, the excess loading amount of NiO will inhabit the light absorption of Sr2FeTaO6 component and decrease the overall light utilization efficiency, leading to the reduced photocatalytic activity. In order to evaluate photo-stability, the photocatalytic H2 evolution experiment of 3NiO/7SFT sample was carried out for five cycles under the same condition  curves , respectively, by the intercept of X axis . According to the n-type semiconductor theory, the flat band potential is .2 V higher than CB potential (ECB). Thus, the ECB of Sr2FeTaO6, 3NiO/7SFT and NiO are calculated to be −.42 V, −.51 V, and −.71 V (vs. RHE), respectively. Combining the bandgap values of Sr2FeTaO6 (2.08 eV) and NiO (1.40 eV), the CB and valance band (VB) potentials of Sr2FeTaO6 and NiO are calculated to be −.42 V/+1.66 V (vs. RHE) and −.71 V/+.69 V (vs. RHE), respectively.The conduction band (CB) positions of Sr) curves . The pos2FeTaO6 and NiO are excited to generate electrons and holes. The generated electrons in CB of NiO will migrate to CB of Sr2FeTaO6 and then to Pt nanoparticles for photocatalytic H2 evolution reaction, while holes will oppositely transfer from VB of Sr2FeTaO6 to VB of NiO across the intimate interface due to the potential difference. The accumulated holes will be consumed by the sacrificial regent. The enhanced photocatalytic activity for H2 evolution is ascribed to the synergistic effects of heterojunction formation and increased light harvesting capacity.A possible PHE mechanism was proposed as follows. Under visible light, both Sr2FeTaO6 nanoparticles, and then fabricated series of black NiO/Sr2FeTaO6 (NiO/SFT) composites by a two-step process containing hydrothermal method and calcination treatment. The formed NiO was deposited on the surface of Sr2FeTaO6 to form a closely interfacial contact, resulting in the heterojunction formation between NiO and Sr2FeTaO6. Based on photo-electrochemical measurement, PL and TR-PL spectra, the formed NiO/Sr2FeTaO6 heterojunctions are beneficial to the charge carrier separation and transfer. The resulted NiO/SFT composites showed the higher PHE activity than that of NiO and Sr2FeTaO6, and the optimal sample of 3NiO/7SFT has the highest PHE efficiency of 2,944 μmol h−1 g−1. The enhanced PHE performance was ascribed to the synergistic effects of synergistic effects of heterojunction formation for the efficient charge carrier transfer/separation and increased light harvesting capacity. Notably, the excess loading amount of NiO in NiO/SFT composites will result in the reduced PHE activity due the decreased light harvesting capacity toward Sr2FeTaO6 component. Our work provided an insight on the development of high-efficiency heterojunction photocatalysts for PHE reaction by introducing narrow-bandgap semiconductor.In this work, a simple hydrothermal method was firstly employed to prepare Sr


N6-methyladenosine (m6A) RNA methylation is involved in governing the mechanism of tumor progression. We aimed to excavate the biological role and mechanism of the m6A methyltransferase METTL3 in cholangiocarcinoma (CCA).METTL3 expression was determined by database and tissue microarray analyses. The role of METTL3 in CCA was explored by loss- and gain-of-function experiments. The m6A target of METTL3 was detected by RNA sequencing. The role of AKR1B10 in CCA was explored, and the association between METTL3 and AKR1B10 was confirmed by rescue experiments.METTL3 expression was upregulated in CCA tissue, and higher METTL3 expression was implicated in poor prognoses in CCA patients. Overexpression of METTL3 facilitated proliferation, migration, invasion, glucose uptake, and lactate production in CCA cells, whereas knockdown of METTL3 had the opposite effects. We further found that METTL3 deficiency inhibited CCA tumor growth in vivo. RNA sequencing and MeRIP-qPCR confirmed that METTL3 enhanced AKR1B10 expression and m6A modification levels. Furthermore, METTL3 directly binds with AKR1B10 at an m6A modification site. A CCA tissue microarray showed that AKR1B10 expression was upregulated in CCA tissue and that silencing AKR1B10 suppressed the malignant phenotype mentioned above in CCA. Notably, knockdown of AKR1B10 rescued the tumor-promoting effects induced by METTL3 overexpression.Elevated METTL3 expression promotes tumor growth and glycolysis in CCA through m6A modification of AKR1B10, indicating that METTL3 is a potential target for blocking glycolysis for application in CCA therapy.The online version contains supplementary material available at 10.1186/s12935-022-02809-2. Cholangiocarcinoma (CCA) describes a rare tumor originating from the bile duct epithelium that can involve the entire biliary tract . AccordiN6-methyladenosine (m6A) is the most common methylation modification on mRNA molecules in eukaryotes. As an increasing number of m6A-related enzymes are recognized, such as methyltransferase-like 3 (METTL3), fat mass and obesity-associated gene (FTO), AlkB homolog 5 RNA demethylase (ALKBH5), and YTH domain family 1 (YTHDF1), the powerful biological functions of m6A modification have been gradually revealed. Increasing evidence suggests that m6A modification may contribute to carcinogenesis through different regulatory mechanisms, including the regulation of mRNA stability , splicinCancer cells are highly dependent on aerobic glycolysis for energy supply, known as the Warburg effect . AerobicIn the present study, we intended to reveal the biological role of m6A modification of aldo–keto reductase family 1 member B10 (AKR1B10) mediated by METTL3 in CCA progression. We clarified the expression patterns of METTL3 and AKR1B10 in CCA based on the results of database analysis and CCA tissue microarray and revealed the functions of METTL3 and AKR1B10 in CCA through in vitro and in vivo experiments. The regulatory roles of METTL3 and AKR1B10 in CCA were clarified by functional rescue experiments. Our study proposed that METTL3 may be a target of potential inhibitors for blocking glycolysis for application in CCA therapy.We characterized the expression profile of CCA RNA-seq datasets downloaded from TCGA-Cholangio carcinoma (CHOL) dataset and then the differential expression of eight m6A methylation-related genes  between CCA and normal control samples were evaluated using R package and plotted into heatmap using R package.The GEPIA2 database contained of 36 tumor tissues of CHOL and 9 adjacent tissues samples. We analyzed the differential expression of METTL3 and AKR1B10 between the CCA and adjacent tissues.2O2 in methanol to block endogenous peroxidases and incubated with 10% normal horse serum in TTBS to block non-specific binding. Next, sections were incubated with anti-METTL3  or anti-AKR1B10  overnight at 4 °C and then incubated with horse anti-mouse biotinylated antibody. Finally, sections were with colored by chromogen of DAB and counterstained with hematoxylin. Pictures were captured and exported using NDP.view 2.0. IHC staining results of METTL3 were assigned 1–3 scores and AKR1B10 were assigned 0–3 scores based on staining intensity of positive cells and percentage of positive cells. The section with strong staining intensity and diffuse of positive cells was assigned 3 score; strong staining intensity and focal distribution of positive cells was assigned 2 score; weak- medium staining intensity of positive cells was assigned 1 score; no staining or non-specific staining was assigned 0 score. METTL3 scores of 1 and 2 were categorized as low expression group and 3 as high expression group. AKR1B10 scores of 0 and 1 were categorized as low expression group, scores of 2 and 3 as high expression group.A CCA tissue microarray (No. LVC1202) was generated from 60 cancer tissues and paired pericarcinomas that purchased from Boster Biological Technology co.ltd. IHC staining for METTL3 and AKR1B10 were performed using the above microarray tissue blocks of CCA. Briefly, paraffin-embedded tissues were made into 6 μm sections following deparaffinization and hydration. Sections were repaired by high-pressure following incubated with 0.33% H2.Human liver bile duct carcinoma cell RBE and HCC9810 were purchased from Procell (China) and were maintained in RPMI 1640 with L-Glutamine  containing 10% FBS  and 1% penicillin/ streptomycin  at 37 °C and 5% COTo construct METTL3 stably overexpressed stable RBE cell line, the full-length of METTL3 was inserted into the lentiviral vector pLenti-EF1a-EGFP-P2A-Puro-CMV-3 × FLAG-WRPE (OE-METTL3 group) and then harvested-lentiviruses were infected with RBE cells using polybrene . Blank lentiviral vector was served as negative control (Vector group).To transient knockdown expression of METTL3 in HCC981 cells and AKR1B10 in RBE cells, we used small interfering RNA (siRNA) method. The synthesized sequence of siRNA targeted METTL3 (siMETTL3) or AKR1B10 (siAKR1B10) by GenePharma  were shown in Additional file Besides, for stable knockdown of METTL3 expression used in animal study, lentiviruses vector pLKO.1 puro containing METTL3 shRNA (shMETTL3) and non-targeting scrambled shRNA (shNC) were purchased from GenePharma .According to the manufacturer’s instructions, 5 μL of siMETTL3, siAKR1B10, shMETTL3 and shNC were diluted in 45 μL OPTI-MEM and then mixed with 10 μL Lipofectamine 2000 reagent pre-diluted with 45 μL OPTI-MEM. The mixture was added into cells and cultured for 24 h before further efficiency verification experiments.−ΔΔCq method. The primers were listed in Additional file TRIzol (Invitrogen Life Technologies) was applied for isolating total RNA from RBE cells and HCCC-9810 cells. Quality qualified RNA reverse-transcription into cDNA was carried out using High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and then mRNA expression of METTL3, AKR1B10, and GAPDH were measured by QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) with FastStart Universal SYBR Green Master mix  according to the product’s protocol. Relative expressions of genes were normalized to GAPDH using 2Total protein concentration was measured by the BCA protein assay kit . Then, 20 μg proteins were resolved by 10% SDS-PAGE and transferred onto PVDF membranes following blocking for nonspecific binding with 5% nonfat milk at 25 °C for 2 h. The membranes were incubated with anti-METTL3 , anti-AKR1B10 , and anti-GAPDH  at 4 °C overnight. After that, membranes were incubated with Goat Anti-Mouse IgG H&L (HRP)  at 25 °C for 1 h. Immunore-activity was imaged by Bio-Rad ChemiDoc XRS system and quantified by Image J.5 cells/well were seeded in 96-well plates and cultured for 24 h. Then, at each indicated times , 10 μL CCK-8 solution  was added and was incubated for another 1 h at 37 °C. The optical density was read at 450 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific).One hundred microliter cells with a density of 1 × 10Cell apoptosis was detected by TUNLE assay using a One Step TUNEL apoptosis kit (red Tunnelyte™ CY3 fluorescence detection)  according to the product instruction. Briefly, adherent CCA cells were washed with PBS and then fixed in immunostaining fixative solution  followed by permeabilized in immunostaining strong permeable solution . After that, cells were incubated with TUNEL solution for 1 h and sealed with anti-fluorescence quenching sealing tablets. Lastly, cells were photographed on a fluorescence microscope.Cell migration and invasion were accessed by using a Transwell assay. The Transwell chamber was coated with 0.8 μm Matrigel  for cell invasion assay, otherwise for migration assay. CAA cells were seeded into the upper chamber containing serum-free medium and complete medium was added to the lower chamber as a chemoattractant. The cells were cultured 24 h at 37 °C. The migrated cells to the lower chamber was photographed and calculated in three randomly fields under an inverted light microscope. The invaded-cells arriving at the lower chamber were fixed in 10% formaldehyde for 15 min and stained with 0.1% crystal violet for 10 min, and finally photographed in three randomly fields.Relative glucose uptake required by tumor cells was measured by Glucose Uptake Fluorometric Assay Kit , and relative lactate production was measured by Lactic Acid Content Assay Kit  according to the technical bulletin provided by manufacturer.6 HCCC-9810 cells were subcutaneously injected into axillary of mice. After injection, mice were continued to be raised normally for 3 weeks. Tumor volume (length × width × width × 0.5) was measured every 3 days using caliper. Mice were euthanized using CO2 inhalation after the last measurement, tumor was collected and weighted.A total of twelve 4-week-old female BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Company and were randomly divided into two groups: ShMETTL3 group (n = 6) and Vector group (n = 6). Mice were single housed at room temperature (21–26 °C) on a nature light cycle for one week before experiments to adapt laboratory environment. All mice were provided free access to diet and water. HCCC-9810 cells with METTL3 knockdown were digested by trypsin and made into single cell suspension. Next, 2 × 10RNA sequencing was performed at Yingbio Technology  using an Illumina HiSeq 2500. For differentially expressed genes (DEGs) identification, the thresholds was Log2fold change (FC) > 1 or < − 1 and false discovery rate (FDR) < 0.05. Gene onology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were carried out for the DEGs using R package. Transcriptome sequencing was repeated in three replicates.Total RNA was extracted from RBE cells-overexpressed METTL3 or not, and then isolated mRNA was purified by Dynabeads mRNA Purification Kit  according to the manufacturer’s instructions. Next, purified mRNA was fragmented by RNA Fragmentation Reagent  before immunoprecipitation. After that, the anti-m6A antibodies or anti-METTL3 were conjugated to protein magnetic beads for immunoprecipitation, anti-immunoglobulin G (IgG) was served as negative control. Finally, RNA was eluted from RNA–protein immunocomplexes followed by RT-qPCR analysis.RBE cells overexpressed METTL3 or NC were seeded in a 12-well plate and cultured for 24 h. Then, 5 μg/mL actinomycin D were added into cells and cultured another 0 h, 3 h, 6 h, 9 h, and 12 h following cell collection. Once collection, the total RNA was extracted from these RBE cells used for RT-qPCR as described above.For m6A reporter assays, the wild-type of AKR1B10 sequence and the mutated at m6A motif 1 , at m6A motif 2 , and at m6A motif 3  were inserted into XhoI/NotI site of the psiCHECK-2 luciferase reporter vector. Then, these recombinant plasmids were transfected into RBE cells overexpressed METTL3 and NC using Lipofectamine 2000 reagent as described above.Data analysis was performed by GraphPad Prism 9.0 and data were presented as mean ± SD. Kolmogorov–Smirnov test was used for evaluating the data normality, and Levene test was used for evaluating homogeneity of the variance of data. One-way ANOVA with Tukey test for three groups and t test for two groups were utilized when the data was normality and homogeneous. The chi-square test was used to evaluate the correlation between molecular expression and clinical data. P value less than 0.05 was considered significant.http://timer.comp-genomics.org/) showed that METTL3 was upregulated in a variety of tumors, including CHOL  or empty vector controls (Vector) were transfected into RBE cells. The overexpression efficiency of METTL3 was confirmed at both the mRNA , there were indeed 5 potential m6A modification sites on AKR1B10 mRNA (H) to catalyze its target. Emerging studies have identified that AKR1B10 can reduce a large number of endogenous carbonyl compounds, including retinal, isoprenyl aldehydes, cytotoxic aldehydes, and decrease glucose reductase activity characteristics . As a muIn conclusion, our study revealed that METTL3 was highly expressed in CCA and that elevated METTL3 expression was associated with poor prognosis. METTL3 exerted an oncogenic role in CCA progression in vitro and in vivo, which was also the case for AKR1B10. Moreover, AKR1B10 was an m6A-related target of METTL3, and knockdown of AKR1B10 rescued the tumor-promoting effects induced by METTL3 overexpression. Therefore, METTL3 may function as a novel therapeutic target for CCA.Additional file 1: Table S1. The primers used in this study.Additional file 2: Figure S1.A METTL3 expression in different cancers in the TIMER2 online database. B Bubble plot of KEGG enrichment of glycolysis-related pathways involved in DEGs. C Bubble plot of KEGG enrichment of intersection DEGs between the RNA-seq and TCGA datasets. D The results of MeRIP-qPCR in the siMETTL3 and siNC groups for four candidate DEGs.


PSEN1) are the most common cause of early onset familial Alzheimer’s disease (FAD). The PSEN1 mutations can alter the activity of γ-secretase on the cleavage of APP. Previous studies have shown that PSEN1 mutations increase the expression and activity of BACE1 and that BACE1 expression and activity are elevated in the brains of PSEN1 mutant knock-in mice, compared with wild-type mice, as well as in the cerebral cortex of FAD patients carrying PSEN1 mutations, compared with sporadic AD patients and controls. Here, we used a Psen1 knockout cell line and a PS1 inhibitor to show that PS1 affects the expression of BACE1 in vitro. Furthermore, we used sucrose gradient fractionation combined with western blotting to analyze the distribution of BACE1, combined with a time-lapse technique to show that PS1 upregulates the distribution and trafficking of BACE1 in the endoplasmic reticulum, Golgi, and endosomes. More importantly, we found that the PSEN1 mutant S170F increases the distribution of BACE1 in the endoplasmic reticulum and changes the ratio of mature BACE1 in the trans-Golgi network. The effect of PSEN1 mutations on BACE1 may contribute to determining the phenotype of early onset FAD.Neuritic plaques are one of the major pathological hallmarks of Alzheimer’s disease. They are formed by the aggregation of extracellular amyloid-β protein (Aβ), which is derived from the sequential cleavage of amyloid-β precursor protein (APP) by β- and γ-secretase. BACE1 is the main β-secretase in the pathogenic process of Alzheimer’s disease, which is believed to be a rate-limiting step of Aβ production. Presenilin 1 (PS1) is the active center of the γ-secretase that participates in the APP hydrolysis process. Mutations in the PS1 gene ( In the nonamyloidogenic pathway, APP can be cleaved sequentially by α- and γ-secretase. However, in amyloidogenic processing, APP can be cleaved sequentially by β- and γ-secretase to generate Aβ fragments, of which Aβpeptides . β-Secrepeptides ,4. Studipeptides ,6. It ispeptides .PSEN1) are a major cause of familial Alzheimer’s disease. Currently, more than 226 mutations have been identified in PS1 [PSEN1 mutations will be reported over time. Remarkably, those early onset familial Alzheimer’s disease (FAD)-linked PSEN1 mutations lead to increased molar ratios of Aβ42 over Aβ40 in cell lines and in the brains of transgenic animals, as well as in the plasma of FAD patients with PSEN1 mutations, compared with controls [PSEN1 can alter the Aβ42/Aβ40 ratio in the 2EB2 cell line [PSEN1 mutations can increase the expression and activity of BACE1 [γ-Secretase comprises four core subunits: presenilin 1 (PS1), nicastrin (NCT), anterior pharynx defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), among which, PS1 is the catalytic component . Mutatiod in PS1 . More PScontrols ,11,12. Oell line . It has ell line ,15,16. Iof BACE1  and compof BACE1 . Howeverof BACE1 ,20. Prevof BACE1 ,22. TheyPSEN1 were chosen to transfect the MEF cell line to prove the effect of PS1 on BACE1. Mutation at D257A is a negative control that does not lead to elevated secreted Aβ levels [Based on the above observations, we hypothesized that PS1 also influences the intracellular trafficking of BACE1. To clarify the effect of PS1 on BACE1, a wild-type (WT) and presenilin 1-deficient mouse embryonic fibroblast cell line (MEF) was used in the present study. Two mutations (D257A and S170F) in β levels . S170F iβ levels ,25. We cPsen1 knockout cell line (PS1−/−). The PS1 protein levels are shown in p = 0.013, −/− cells after overexpression of the PS1 wild-type plasmid was evidence of increased amyloidogenic processing.To investigate the effect of PS1 on the expression levels of BACE1, we transfected the PS1 plasmid (vector pcDNA4 as a control) into the MEF −/− cells were treated with L-685,458 . Cells were collected for western blot analysis at 0, 60, 90, and 180 min after L-685,458 treatment . The markers of the three organelles, i.e., Calnexin (marker of ER), EEA1 (marker of early endosome), Syntaxin-6 (marker of Golgi), and BACE1, were detected by WB. To determine how PS1 upregulates the expression levels of endogenous BACE1, many studies have been performed. There is a report stating that PS1 mutations can compromise the maturation of BACE1 . Further−/− cell line according to the western blot of all fractions from sucrose density gradient centrifugation, showing the ratio of BACE1 expression in each layer to the BACE1 expression of whole layers. PS1 and BACE1 co-overexpression resulted in the retention of BACE1 in the ER, compared to BACE1 overexpression alone, suggesting that proBACE1 might prolong its dwelling time in the ER. The increase in the ratio of BACE1 location in the fourth layer fraction indicated that more mature BACE1 was produced from proBACE1 in the Golgi/TGN, which might eventually contribute to the higher BACE1 activity.The results of sucrose density gradient centrifugation showed that when PS1 was transfected, BACE1 was enriched in the layer 3 and layer 4 fractions A,B. The −/− cell line. The results showed that in MEF PS1−/− cells, only a small ratio of BACE1 (green) protein colocalized with calnexin (red). When PS1 was transiently transfected into the MEF PS1−/− cells, the colocalization ratio of BACE1 and calnexin significantly increased, indicating that BACE1 protein migrated to the ER  has a higher BACE1 affinity than the wild-type and can be cleaved by β-secretase during maturation (ER-TGN) , while mPSEN1 mutation is known to be a key hereditary factor of FAD. Mutations in PSEN1 can cause changes in γ-secretase activities. To date, increasing evidence has indicated multiple physiological roles of PS1, such as those in calcium homeostasis, neuronal development, neurite outgrowth, apoptosis, synaptic plasticity, and tumorigenesis [42/Aβ40 ratio [PSEN1 mutations (D257A and S170F) to confirm the effect on BACE1  on BACE1. Due to the poor condition of cells after PS1 mutant transfection, 4 h of continuous filming will cause a shift in focusing cells. Our present data preliminarily provide a novel alternative for the interaction of PS1 and BACE1. Secretase inhibitors targeting PS1 or BACE1, but that will not interfere with the other normal functions of both, are required to establish the therapeutic potential for AD.Much remains to be learned about the mechanisms by which PS1 regulates BACE1. More work is needed. Unfortunately, we did not obtain acceptable films of the trafficking effect of PSEN1 (PS1-S170F and PS1-D257A) were generated by overlap extension PCR on the plasmid pcDNA4.1/PS1-WT using the following primers: Wild-type PS1 human cDNAs (pPS1-WT) and pBACE1-N3-EGFP were obtained from Weihong Song lab . Mutations in PS1-WT: 5′-GGCGAATTCGTTCTAGATATAAAATTGATGGA-3′ and 5′-CCCAAGCTTAAATGACAGAGTTACCTGC-3′; PS1-S170F: 5′-GCAACAATAGAAATGATATAATAAGCCAGGC-3′ and 5′-GCCTGGCTTATTATATCATTTCTATTGTTGC-3′; PS1-D257A: 5′-CAGCCACTAAAGCATATACTGAAATCACAGCC-3′ and 5′-GGCTGTGATTTCAGTATATGCTTTAGTGGCTG-3′. Psen1 knockout embryonic fibroblast cell line (MEF PS1−/−) was obtained from the De Strooper laboratory . Psen1 knockout MEF cell lines were maintained in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum , 100 IU/mL penicillin, 100 μg/mL streptomycin, pyruvic acid sodium salt (1 mM), glutamine (2 mM), 1% NEAA, 0.1 mM β-mercaptoethanol, and ESGRO recombinant mouse leukemia inhibitory factor . For subcellular fractionation experiment and immunofluorescence experiments, Lipofectamine 2000  was used for transient transfection of pcDNA4.1 or pPS1-WT constructs into MEF PS1−/− cells, according to the manufacturer’s instructions. For the time-lapse experiment, pBACE1-N3-EGFP with or without PS1-WT was transiently transfected into the MEF cell line using electroporation. For the inhibition of γ-secretase activity, pPS1-WT was transfected into MEF PS1−/− cells. A total of 48 h later, the γ-secretase inhibitor L-685,458 was added to the cell culture medium, reaching a final working concentration of 10 μM. MEF PS1−/− cells were collected at 0, 60, 90, and 180 min after L-685,458 treatment.The −/− cells were grown on 100 mm diameter dishes at a density of 10 × 106 cells per dish. Three dishes were transfected with PS1-WT, and three other dishes were transfected with pcDNA4.1. Sucrose gradient fractionation of raft-like proteins was conducted as previously described [g for 2 min at 4 °C. The supernatant was collected by centrifugation at 8000× g for 20 min at 4 °C. The precipitate was resuspended in cell homogenization buffer and 100 mM sodium carbonate buffer and then sonicated in continuous mode for three 5 s bursts, with an output of 6 W. A total of 2 mL of 80% sucrose was added to each sample to obtain a final volume of 4 mL and a final concentration of 40% sucrose. The mixture was laid on 4 mL of 35% sucrose solution, followed by 4 mL of 5% sucrose solution, and then centrifuged at 180,000× g for 6 h using a swing out rotor . After centrifugation, twelve 1 mL fractions were harvested from the top of the tube, and the fractions were subjected to western blot analysis.MEF PS1escribed . Cells w−/− cells were grown on confocal dishes at a density of 7 × 104 cells per dish. After 24 h, the pcDNA4.1 plasmid, PS1 plasmid, or PS1 mutation plasmid was transfected. A total of 48 h later, the cells were fixed for 15 min in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS. Then, the cells were blocked with blocking solution  for 1 h and incubated with primary antibody  overnight at 4 °C in blocking solution. After washing with PBS, the cells were incubated with the fluorescent secondary antibodies: Alexa Fluor 488- or Alexa Fluor 647 for 1 h at room temperature in the dark. Hoechst33258  was used to stain the nucleus. Images were obtained by laser scanning confocal microscopy . The colocalization data were quantified by FUJI software (Image J), which generated a Pearson’s correlation coefficient value.MEF PS1−/− cells were grown on confocal dishes at a density of 7 × 104 cells per dish. After 24 h, PS1+ BACE1-N3-EGFP plasmids or pcDNA4.1+ BACE1-N3-EGFP plasmids were transfected. A total of 48 h later, the cells were washed twice with Hank’s solution containing calcium and magnesium , and then tracker  was added to the dish. After incubation at 37 °C for 30 min, the working solution was discarded, and the cells were washed with Hank’s solution containing calcium and magnesium three times, followed by the addition of 10% fetal bovine serum-free medium without phenol red for observation under the microscope. A 100× objective lens with immersion oil was chosen for time-lapse imaging. The excitation light was 488 nm and 555 nm, the cell thickness was approximately 14 μm, and the Z-stack setting was 0.5 μm × 28 Z-slices in total. The duration of imaging was 8 h, with a 10 min interval of image acquisition. Imaris software was used to count the colocalization of BACE1 and different organelles in the cell.MEF PS1g for 10 min at 4 °C, and the protein levels were determined by the Quick Start™ Bradford protein assay . Cell lysates were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) with 16% Tris-tricine or 10% Tris-glycine gels. The samples were then transferred to PVDF membranes . For protein detection, membranes were blocked for 1 h with 5% skim milk and incubated with the primary antibody  at 4 °C overnight. After incubation with species appropriate secondary antibodies conjugated to horseradish peroxidase, immunocomplexes were visualized by chemiluminescence using an ECL  system, and the intensities of the bands were quantified with Image Lab™ Software .Cells were lysed 48 h after transfection in RIPA Lysis Buffer  with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitor cocktail , and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride . The samples were sonicated for 3 min and centrifuged at 14,000× BACE1: 5′-TGGAGGGCTTTGGAGGGCTTCTACGTTGTCTT-3′ and 5′-CATCATGGAAGGTTTCTATGTCGTCTTC-3′;qRT-PCR was used to analyze the mRNA levels of target genes using SYBR Green . Total RNA was isolated by TRIzol  following the manufacturer’s instructions. cDNA was synthesized by FastKing -RT SuperMix  following the manufacturer’s instructions. The results of three independent experiments were normalized, relative to that of GAPDH, to correct for differences in template input by applying the 2−ΔΔCt algorithm. The sequences of the primer pairs were as follows: GAPDH: 5′-GACTTCAACAGCAACTCCCACTCTTCC-3′ and 5′-TGGGTGGTCCAGGGTTTCTTACTCCTT-3′.p < 0.05 was considered statistically significant.All statistical analyses were performed using GraphPad Prism 6 software . Quantifications were performed from data generated during three independent experiments. Values represent the mean ± standard error. Comparisons of more than two groups were carried out using one-way ANOVA, and Dunnett’s post-hoc test was used to analyze PS1 construct values and the control group . Statist


Escherichia coli cells. The protocol utilizes the common shaker flask growth method and the well-known pET system (which provides expression control via the T7 promotor) for large-scale recombinant protein expression. One liter expression typically yields 5 to 50 mg of highly deuterated protein. Our data demonstrate that the optimized procedure produces a comparable quantity of protein in deuterium (2H2O) oxide M9 medium compared with that in 1H2O M9 medium. The protocol will enable a broader utilization of deuterated proteins in a number of biophysical techniques.Highly deuterated protein samples expand the biophysics and biological tool kit by providing, among other qualities, contrast matching in neutron diffraction experiments and reduction of dipolar spin interactions from normally protonated proteins in magnetic resonance studies, impacting both electron paramagnetic resonance and NMR spectroscopy. In NMR applications, deuteration is often combined with other isotopic labeling patterns to expand the range of conventional NMR spectroscopy research in both solution and solid-state conditions. However, preparation of deuterated proteins is challenging. We present here a simple, effective, and user-friendly protocol to produce highly deuterated proteins in  Most researchers find it arduous and inconsistent. Bacterial cells (Escherichia coli) are the preferred host, as insect and mammalian cells are difficult to culture in a D2O medium (2O (A600 ∼ 0.8), pelleted by centrifugation, resuspended in 100% D2O medium, and induced immediately. This method often leads to complete cell death during induction, due to lack of proper adaptation. If cells do survive, the level of protein expression is very low. In method (2), the multistep media switch adaptation, cells were grown and transferred, stepwise, from LB to 50%, 75%, and 100% D2O media, which discards considerable isotopic media in the adaptation phase. This method also results in low protein expression due to multiple centrifugation and resuspension steps, rendering a generally unhealthy cell culture. The approximate yield of deuterated proteins from these methods is less than 25% of the yield from our current protocol reported here. When expression efficiency is low, the combined cost of D2O, 15N/13C sources, and/or specific 13C-labeled metabolic precursor compounds can become very high, thus limiting widespread application and realization of the full potential.Although highly desired, deuterated  the carryover of any rich medium  into the final culture; (2) multiple media transfers during cell training, which involves repetitive cell pelleting, resulting in long recovery time for bacterial cells and unhealthy growth; (3) extended growth period that requires lengthy in-person monitoring; (4) an unhealthy cell growth curve that often leads to low expression and even cell death.2H-M9; for abbreviations and media descriptions see Fig. 1A). The protocol keeps A600 at or slightly above 0.2 each time a culture is initiated or transferred or following a dilution. This approach ensures that the cells are the most healthy and viable to adapt into a higher D2O percentage environment. The protocol is described for production of 1 liter 2H-M9 expression medium. LB and 1H-M9 cultures do not require training (adaptation). A modified simple protocol for expression in LB and 1H-M9 is described in the Figure 1B.The key to successful cell growth in deuterated medium  or 2H-M9(15N); store at 4 °C.Prepare 1 liter of the desired labeling medium, 1.A600 can reach ∼0.4 to 0.5 after about 2-h growth at 37 °C.Inoculate 15 ml LB media (in a 250-ml flask) with about two dozen freshly transformed colonies. The general guideline is to use enough colonies so that 2.A600 ∼0.4 to 0.5, add 15 ml of 2H-M9 medium in the same flask to a total volume of 30 ml, which is now ∼50% in 2H2O. Continue growth at 37 °C.Allow the initial 15 ml LB to grow to 3.2H2O) reaches A600 ∼0.4 to 0.5 , add 30 ml of 2H-M9 medium to a total volume of 60 ml . Continue growth at 37 °C until A600 ∼0.4 to 0.5 (in approximately 1 h).When the 30 ml culture (∼50% in 4.g), discard the medium, and resuspend the cell pellet in 100 ml fresh 2H-M9 medium .Centrifuge the cells  was added to the medium 30 min prior to IPTG induction. For myr-Arf1, addition of 50 mg sodium myristate and 50 mg coenzyme A are needed for posttranslational myristoylation. Other supplementary elements, such as Zn2+, which is required for high expression of ASAP1 PZA, can also be added at this growth stage, 30 min prior to IPTG induction.For methyl labeling, appropriate metabolic precursors If needed, supply additives specific to the desired sample, such as precursors for custom isotope labels or prosthetic groups for posttranslational modifications or other supplementary elements.5.•A600 during cool down. Induction is initiated by adding IPTG to 0.2 mM, and growth is continued at room temperature overnight. These cells are harvested in the morning of day 4. Specifically in this comparative study, the general procedure is followed for ASAP1 PH, ASAP1 PZA, and myr-Arf1; however, for Ube2g2, gp78c, and MSPDH5, cultures are maintained at 37 °C for 30 min after supplying additives and then induced with 1 mM IPTG for 3 h. Cells are harvested at the end of day 3. This 37 °C induction modification is necessary based on our knowledge and extensive testing on these proteins. We find that a significant number of mammalian proteins overexpressed in E. coli cells exhibit some level of toxicity, which can lead to reduced or completely abolished expression when induced at room temperature overnight. We recommend researchers test both induction conditions for their proteins of interest.Generally, cultures are cooled down (while shaking) for approximately 30 min in a shaker adjusted to room temperature. Cell density will expand moderately to about 0.9 to 1.0 Induce protein expression, wherein the temperature and duration time will vary based on the nature of proteins to be overexpressed.1.A600 of the final culture and collect a 20-μl sample for SDS-PAGE analysis.Record 2.g) and either proceed with protein-specific purification or store the cell pellet at −80 °C.Harvest cells by centrifugation  protein (myr-Arf1)  , 35, 36,yr-Arf1) , a complyr-Arf1) , and theyr-Arf1) , 41. Eac1H-M9(15N), (3) 2H-M9(15N) (1H-M9(15N) and 2H-M9(15N), and expression is only moderately reduced from LB. Each protein was purified according to the published procedures, and mass spectral data were acquired to enable determination of labeling efficiency , thus supporting a wide variety of structural and biophysical studies. In addition, we have observed that the expression level and the percentage of deuteration are not affected by other supplementary additives (such as a-keto acid precursors) in the medium, provided the carbon source (12C62H7-glucose or 13C62H7-glucose) is ≥97% deuterated (data not shown).The comparative performance for the six proteins is demonstrated by parallel expression in three different media: (1) LB, (2) -M9(15N) . It is oficiency . In each1H-M9 (15N) medium. One of the keys to this protocol is proper attention to cell viability and avoidance of cellular and metabolic stress. Our approach deviates from conventional adaptation methods that encompass multiple steps of cell pelleting, media transfer, and resuspension. All these treatments incur additional stress and slow down, or endanger, cell growth. Unlike other published methods (2H-M9) are needed for each 1 liter production. We achieve such simplicity by starting a small, healthy LB culture and expanding (by simple dilution) the LB culture with 2H-M9 medium in a stepwise fashion, while maintaining the growth within the exponential phase through all media adjustments. Another advantage of the protocol is that there is no carryover of any initial LB/H2O media into the final expression culture, since we complete adaptation to 100% D2O at the end of day 2, which leads to the high percentage of deuteration (2O) medium and (2) adapting cells to a higher D2O medium by stepwise addition of 2H-M9 medium, thus eliminating multiple cell-pelleting steps.The described procedure  has been methods , this prteration . In addi2O, the high cell density growth (A600 = 6.0–10.0) may incur unforeseeable cellular and metabolic stress and may not be commonly applicable for a wide range of proteins. Our protocol emphasizes healthy cell growth and is mindful of minimizing the waste of D2O. Overall, only 45 ml of 2H-M9 medium is discarded during cell adaptation per liter of growth production.Another protein deuteration method , 44 has Interestingly, by observing the induced protein band intensity , we find15N-labeling in the presence of deuteration, we can perform the full variety of methyl-specific labeling (1H-13C methyl transverse relaxation optimized spectroscopy (A) and the 1H-15N transverse relaxation optimized spectroscopy (B) spectra of 2H, 15N, ILVproS13CH3-labeled ASAP1 PZA illustrate the high spectral quality, sensitivity, and resolution that are afforded by the labeling scheme when precursors for methyl labeling are included. ASAP1 PZA is a 44.5-kDa multidomain protein that binds to membrane-associated Arf1 , facilicted NMR , and DEEcted NMR ). For SActed NMR , includicted NMR  and membcted NMR , 15. SinThe protocol has proven general and enables the use of deuteration to enhance NMR and other biophysical methodologies. The protocol should facilitate and expand the application of perdeuteration in a range of biophysical experiments and enable a broad range of systems to be examined in exquisite detail. Such studies will lead to better functional understanding and mechanistic detail in many biological systems.E. coli BL21 star (DE3) cells according to the protocol presented in Each of the six proteins were expressed in Mass spectrometry data were acquired on an Agilent 6130 Quadrupole LC/MS System  equipped with an electrospray source, operated in positive-ion mode. Separation was performed on a 300SB-C3 Poroshell column . The analytes were eluted at a flow rate of 1 ml/min with a 5 to 100% organic gradient over 5 min and holding the organic phase A for 1 min. Mobile phase A contained 5% acetic acid in water and mobile phase B was acetonitrile. Data acquisition, data analysis, and deconvolution of mass spectra were performed using Open Lab Chem Station Edition software (version C.01.05). Samples of purified proteins were typically 5 μl of a 5 μM solution.Isotopically labeled compounds were obtained commercially from the following sources:2H2O, Product no. DLM-4), a-keto acids .Cambridge Isotope Laboratories: deuterium oxide (15N-ammonium chloride (product no. 299251), 13C62H7-glucose (product no. 552151), 12C62H7-glucose (product no. 552003).Millipore Sigma/Isotech: δ1LVproS, TLAM- Iδ1MT, and other specialty labeling kits; for residue type and stereospecific 13CH3-labeling, see http://www.nmr-bio.com.NMR-Bio: TLAM-I1.et al. , the nitrogen source is natural abundance 14NH4Cl, and the carbon source is natural abundance 12C62H7 glucose (97% 2H).4.1H-M9(15N): M9 minimal medium wherein the solvent is 1H2O, the nitrogen source is 15NH4Cl (>98% 15N), and the carbon source is natural abundance 12C61H7-glucose.5.2H-M9(15N): M9 minimal medium wherein the solvent is 2H2O (99.9% 2H), the nitrogen source is 15NH4Cl (>98% 15N), and the carbon source is natural abundance 12C and 2H-labeled 12C62H7-glucose (97% 2H).6.2H-M9: M9 minimal medium wherein the solvent is 2H2O (99.9% 2H), the nitrogen source is 15NH4Cl (>98% 15N), and the carbon source is 13C62H7-glucose (99% 13C and 97% 2H).7.2H-M9: M9 minimal medium wherein the solvent is 2H2O (≥99% 2H), the nitrogen source is 15NH4Cl (>98% 15N), the general carbon source is 12C62H7-glucose (97% 2H), and specific metabolic precursors to label the methyl groups of Met, Ala, Val, Leu, Ile, and/or Thr are provided , and numerous other online variants exist for computing mass from primary sequence.All data are contained within the article. Copies of the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The software, Protein Sequence Analysis, is available from Ira Palmer, NIAMS, NIH (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Colorectal cancer (CRC) is one of the most common malignant cancers in human, and its incidence increases gradually every year. Metastasis is an important factor leading to tumor development. The epithelial–mesenchymal transition (EMT) has been proved to be closely related to tumor metastasis, yet its related mechanism in CRC remains to be explored.We obtained the differentially expressed gene C5aR1 with SETDB1 stable overexpression and knockdown cells by RNA-seq. Cell proliferation was tested by CCK8 and colony formation assay. Migration and invasion of CRC cells were determined by the wound healing and transwell invasion assay. The potential pathway of C5aR1 in CRC was preliminarily studied by western blotting.Sequencing results showed that C5aR1 was the most differentially expressed gene. By changing the expression of C5aR1 in CRC cells, this study found that C5aR1 promoted the proliferation, colony formation, migration and invasion of CRC cells in vitro. C5aR1 accelerated the EMT process and the expression of C5aR1 altered the molecular expression of key proteins in the Wnt/β-catenin pathway.C5aR1 promotes the development of CRC and accelerates the EMT process. Furthermore, C5aR1 may involve in the regulation of Wnt/β-catenin pathway in CRC. According to the data of 2020, CRC was still the most important factor in malignant tumors and related mortality worldwide . AlthougSETDB1 is a histone methyltransferase that specifically acts on lysine nine of histone H3 and is involved in histone methylation, transcription inhibition, and euchromatin gene silencing during cell transformation . In receC5aR1 is a membrane-bound G protein-coupled Receptor. Activation of C5aR1 can modulate the differentiation and function of multiple target cells and participate in multiple immune responses . C5aR1 iIn this study, the SETDB1 stable cell lines and control cell lines were used to analyze the transcriptome data by transcriptome sequence technology. By analyzing sequencing data, the most obvious differential gene, C5aR1, was screened out after the expression changes of SETDB1 in CRC cells. The results showed that C5aR1 promoted proliferation, colony formation, migration and invasion of CRC cells in vitro. We also showed that C5aR1 accelerated the EMT process, and may involve in Wnt/β-catenin pathway. Therefore, we demonstrated that C5aR1 promoted the development of CRC and might be a newly target and prognostic evaluation of CRC.2 |fold change|≥ 1.5 and p value < 0.05.Transcriptome sequencing technology was supported by Biomarker Technology Corporation . The qualified total RNA was extracted for mRNA purification and reverse transcription after fragmentation to construct the cDNA library. The threshold set for significant differences was loghttps://www.proteinatlas.org) is a database based on protein, transcriptome and system biology data, which can map tissues, cells and organs. Survival analysis of C5aR1 gene in CRC was conducted with data from HPA database.The Human Protein Atlas  and the SETDB1 knockdown cell line constructed with pLKO.Puro (target sequence: ATCCCTCCCATCCCATATTTG) were cultured in RPMI1640 medium containing 10% fetal bovine serum , 1% Penicillin–Streptomycin liquid and 1 μg/mL puromycin in an incubator at 37 °C and 5% CO2.HCT116, SW480, SW620, LoVo and RKO cells were all self-stored in the Department of Pathology, Affiliated Hospital of Southwest Medical University. The above five cell lines were cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% cyanin-streptomycin solution in an incubator at 37 °C and 5% COThe siRNA of the C5aR1 and negative control were designed by RiboBio . We transfected CRC cells with siRNA for 48 h using Lipofectamine 2000 . The C5aR1 full-length expression plasmid and its control plasmid were designed by Miaolingbio . All efficiency were analyzed by both RT-qPCR and western blotting.2. 100 μl prepared solution (CCK8 reagent l0 μl added to 90 μl medium) was added into each well and incubated for 2 h. The absorbance (OD) of each well at 450 nm was measured by SpectraMax ABS microplate reader. The growth curve was drawn according to the OD value.CCK8 assay was performed with CCK8 kit . 2500–3000 cells were seeded into 96-well culture plate and incubated for 0, 48, 72, 96 h, respectively, in the incubator at 37 ℃ in 5% CO2 till visible cell clones were observed. The clones were fixed with 4% methanol and stained with 1% crystal violet staining solution for 20 min. The number of formed clones was counted using a microscope.Eight hundred cells were seeded into 6-well culture plate and incubated for 2 weeks in the incubator at 37 ℃ in 5% CO5 cells were seeded into six-well culture plate. Cell scratches were performed using a 10 μl pipet tip when the cell density is about 95% (ensure that each scratch width is the same) and then cell debris were completely washed off using RPMI1640 medium. Complete medium containing 3% fetal bovine serum was added into wells and cells were incubated at 37 ℃ in 5% CO2. Reduced distance of cell scratches were recorded by microscope camera at 0, 24, 48 h, respectively.Transfection after 48 h, about 5 × 105 cells in serum-free PRMI1640 medium) was added in the upper chambers of the wells, while the lower chambers were filled with 600 μl RPMI1640 medium containing 30% fetal bovine serum. After cultured for 48 h, the chambers were placed into methanol to fix for 30 min and then stained with 1% crystal violet staining solution for 5 min. After air dried, the cells in the upper surface of the membrane were removed with a cotton swab. Cells migrated to the lower surface of the membrane were recorded and counted using microscope camera.The transwell chamber  were added with 40 μl mixed Matrigel (Serum-free medium: Matrigel = 8:1). Transfection after 48 h, 200 μl cell suspension , and the reverse transcription was performed using Primescript RT reagent Kit with gDNA Eraser .using.−ΔΔCT).TB Green Premix Ex Taq™ II  on LightCycler 480 II PCR  in RT-qPCR tests. The primers were designed by Sangon Biotech . Primer sequences were as follows: β-actinF-5'CATGTACGTTGCTATCCAGGC3', R-5' CTCCTTAATGTCACGCACGAT3'; C5aR1F-5' TATGGGCACTATGATGACAAGGATACC3', R-5'AAGACGACTGCAAAGATGACCAAGG3'. The expression of mRNAs were calculated by the comparative CT  and PMSF . Western blot was performed with the specific antibody, C5aR1 , β-actin , E-cadherin , Vimentin , β-catenin , Histone H3 .t test was used to analyse the results of CCK8, colony formation, wound healing and transwell invasion assay. p < 0.05 was considered to be statistically significant and the significance is presented as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.Statistical analyses were performed in SPSS 26.0 software and GraphPad prism 8.0 software. Two independent samples There were 200 differentially expressed genes (DEGs) between the SETDB1 overexpression group and the control group, and 431 DEGs between the SETDB1 knockdown group and the control group, among which 38 DEGs appeared simultaneously in the knockdown group and the overexpression group .Gene function analysis with GO and KEGG analysis of sequencing data found that biological functions related to C5aR1 gene were significantly enriched in complement component C5a signaling pathway and positive regulation of macrophage migration . The expression of C5aR1 was high in HCT116 and SW620 and low in RKO and LoVo (Fig. To investigate the function of C5aR1 in CRC, HCT116 and SW620 cells were transfected with the siRNA of C5aR1 and its control. For transfection efficiency verification, biological efficiency verification experiments were performed using siRNA C5aR1-1 and siRNA C5aR1-3. The efficiency of knockdown was confirmed by western blot and RT-qPCR (Fig. RKO was transfected with C5aR1 overexpression plasmid and its control cell line was also established. The validity of overexpression was confirmed by Western blot and RT-qPCR Fig. D. The reC5aR1 was found to promote metastasis by inducing EMT in hepatocellular carcinoma . Here, tWestern blotting assay was used to determine the expression level of β-catenin, a key protein of Wnt/β-catenin signaling channel, in CRC cells after C5aR1 knockdown or overexpression. The nuclear protein Histone H3 was used as the internal reference Fig. B. The reC5aR1 is composed of 350 amino acids and its protein molecular weight is 42 kDa. Tyrosine sulfate was located at position 11 and 14 at the end of NH2 of the receptor, which facilitates its binding to C5a . C5aR1 iRecent studies had found that C5aR1 was up-regulated in breast cancer, colon cancer, hepatocellular carcinoma, lung cancer, gastric cancer, kidney cancer, cervical cancer , 18–21. +T cells and regulate the production of cytokines and chemokines, thus initiating the occurrence of cancer [However, studies on C5aR1 in CRC were rarely reported. Studies had reported that C5aR1 recruited bone-marrow suppressor cells into inflamed CRC tissue through signal transduction to damage CD8f cancer . Anotherf cancer . In thisMany molecular mechanisms contribute to human carcinogenesis promoted by C5aR1. Previous studies had reported that complement can participate in EMT of mouse renal injury and fibrosis models , 28. In The Wnt/β-catenin pathway is a related pathway of EMT. Previous studies had confirmed that the Wnt/β-catenin pathway was involved in mediating EMT and played an important role in breast cancer, lung cancer, gastric cancer, colorectal cancer and other cancer types –34. In tIn conclusion, we demonstrate that C5aR1 promotes the development of CRC and may be involved in the regulation of the Wnt/β-catenin pathway. C5aR1 may be identified as a potential therapeutic target for CRC.