gene abcb1 nm 000927 human tagged orf  (OriGene)

Journal: iScience

Article Title: Human gut Actinobacteria boost drug absorption by secreting P-glycoprotein ATPase inhibitors

doi: 10.1016/j.isci.2024.110122

Figure Lengend Snippet: Eggerthella lenta inhibits P-gp efflux (A) P-gp inhibition test of E. lenta DSM2243 cell pellet lysate and cell-free filter-sterilized supernatant (CFS) using rhodamine (Rh123) accumulation assay in T84 human enterocyte model ( n = 3–4/condition, ANOVA with Tukey’s post hoc test). Vehicle (DMSO), cyclosporin A (CsA), vinblastine, and verapamil were included as controls. (B) Dose-response test of E. lenta CFS on Rh123 accumulation (P dose = 8.6e-13 ANOVA, n = 4 wells/condition). (C) CFS Rh123 accumulation test on T84 ABCB1 −/− knockout cells compared to parental T84 cells (see also Figure S1 ). (D) CFS activity test in 3 other human enterocyte cell lines. Activity tests in T84 cells were also completed using accumulation of other P-gp substrates, such as (E) calcein-AM and (F) doxorubicin (DOX). (G) Synergistic effects of E. lenta CFS and DOX were tested using MTT viability cell assay. DMSO was used as vehicle control. Unless otherwise noted, E. lenta was cultured in BHI + . n = 4/condition and two-way ANOVA with Sidak’s multiple testing correction. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Escherichia coli strains containing pCMV-dR8.91 and pMD2.G lentiviral packaging plasmids were a kind gift from the Mukherjee lab. An E. coli strain carrying the transfer plasmid (pCas9) with cas9 gene, eGFP, neomycin, and ampicillin marker genes and a separate E. coli strain carrying the transfer plasmid (pABCB1) with sgRNA against human ABCB1 gene, mCherry, puromycin, and ampicillin maker genes were purchased from Genecopoeia.

Techniques: Inhibition, Knock-Out, Activity Assay, Control, Cell Culture

Journal: iScience

Article Title: Human gut Actinobacteria boost drug absorption by secreting P-glycoprotein ATPase inhibitors

doi: 10.1016/j.isci.2024.110122

Figure Lengend Snippet: E. lenta inhibits P-gp ATPase activity (A) Possible mechanisms of P-gp inhibition reported in the literature ( Table S1 ). Created with Biorender.com . (B) Quantitative reverse-transcription PCR measurement of ABCB1 transcription in multiple human enterocytes with varying basal expression when treated with E. lenta CFS. GAPDH and beta-actin primers were used as loading controls. Amplification cycles (Ct) were normalized to untreated conditions for each cell line ( n = 3 biological replicates/condition; two-way ANOVA with Sidak’s correction; see also Figure S2 ). (C) Representative western blots of untreated, BHI sterile media, or E. lenta CFS-treated human enterocytes using C219 P-gp antibody and GAPDH antibody and densitometry quantifications normalized to untreated control for each cell line ( n = 1–3 biological replicates/condition; Wilcoxon test). (D) Cell-free P-gp vesicles were incubated with 3 kDa filtrate of E. lenta CFS prepared in EDM media in the presence of ATP. At the end of the incubation period, the amount of remaining ATP was quantified. Sodium orthovanadate (Na 3 VO 4 ) and verapamil P-gp inhibitors were included as controls for an ATPase inhibitor and an activator, respectively. n = 4/condition; ANOVA with Tukey’s post hoc test.

Article Snippet: Escherichia coli strains containing pCMV-dR8.91 and pMD2.G lentiviral packaging plasmids were a kind gift from the Mukherjee lab. An E. coli strain carrying the transfer plasmid (pCas9) with cas9 gene, eGFP, neomycin, and ampicillin marker genes and a separate E. coli strain carrying the transfer plasmid (pABCB1) with sgRNA against human ABCB1 gene, mCherry, puromycin, and ampicillin maker genes were purchased from Genecopoeia.

Techniques: Activity Assay, Inhibition, Reverse Transcription, Expressing, Amplification, Western Blot, Sterility, Control, Incubation

Journal: Molecular Oncology

Article Title: Repositioning VU ‐0365114 as a novel microtubule‐destabilizing agent for treating cancer and overcoming drug resistance

doi: 10.1002/1878-0261.13536

Figure Lengend Snippet: The role of M5 muscarinic acetylcholine receptor (mAChR) in the in vitro anticancer activity of VU‐0365114. (A) Protein expression levels of mAChRs (CHRM1 ~ CHRM5) in various human normal cell types were obtained from the Human Proteome Map. (B) Protein expression levels of mAChRs (CHRM1 ~ 5) in various human cancer types were obtained from the Human Protein Atlas. (C) The correlation of CHRM5 mRNA and growth inhibition by VU‐0365114 in NCI‐60 cancer cell panel. CHRM5 mRNA levels in NCI‐60 were obtained from the CellMinerCDB database. Statistical significance was determined using the Pearson's correlation coefficient ( r ) and the corresponding P value. (D) Protein lysates from HeLa, HCT116, HT29, BxPC‐3, PANC‐1, HPAC, AsPC‐1, and HepG2/C3A cell lines were subjected to Western blotting to assess basal CHRM5 protein expression (Fig. A). The correlation between the IC 50 values of VU‐0365114 and CHRM5 protein expressions (normalized to HeLa) in these cell lines was calculated using Pearson's correlation coefficient ( r ). Data represent two independent experiments. (E, F) HCT116 cells were transfected with CHRM5 siRNA in E or plasmid in F for 48 h, and then exposed to VU‐0365114 for 72 h. Knockdown or overexpression of M5 mAChR in E or F was confirmed by Western blotting (left panel). Cell viability was examined by an MTT assay (right panel). The error bars are the mean ± SD ( n = 5). Statistical significance, compared to the transfection control group at each dose (*** P < 0.001), was determined using a two‐tailed paired Student's t ‐test. si‐NC, negative control siRNA.

Article Snippet: For the gene overexpression analysis, pCMV3 (#CV011), pCMV3‐CHRM5 (#HG20062‐UT), pCMV3‐ABCB1 (#HG12030‐UT), and pCMV3‐MPS1 (#HG11529‐UT) plasmids were purchased from SinoBiological (Beijing, China).

Techniques: In Vitro, Activity Assay, Expressing, Inhibition, Western Blot, Transfection, Plasmid Preparation, Over Expression, MTT Assay, Two Tailed Test, Negative Control

Journal: Molecular Oncology

Article Title: Repositioning VU ‐0365114 as a novel microtubule‐destabilizing agent for treating cancer and overcoming drug resistance

doi: 10.1002/1878-0261.13536

Figure Lengend Snippet: The effect of VU‐0365114 on drug resistance in colorectal cancer cells. (A) HCT116 and HCT116‐OxaR cells were treated with various concentrations of oxaliplatin, VU‐0365114, and colchicine for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD ( n = 4). Statistical significance, compared to parental HCT116 cells at each dose (* P < 0.05 and *** P < 0.001), was determined using a two‐tailed paired Student's t ‐test. (B) The P‐glycoprotein (P‐gp) expression in MES‐SA, MES‐SA/Dx5, HCT116, and HCT116‐OxaR cells were examined by Western blotting. Data represent two independent experiments. (C) HCT116 and HCT116‐OxaR cells were treated with verapamil (Vera; 10 μ m ) or VU‐0365114 (VU; 10 μ m ) for 2 h. Then, an MDR assay was performed. The error bars are the mean ± SD ( n = 4). Statistical significance, compared to untreated controls (** P < 0.01 and *** P < 0.001), was determined using a one‐way ANOVA with Tukey's post hoc test. Statistical significance, compared between HCT116 and HCT116‐OxaR cells ( # P < 0.05), was determined using a two‐tailed paired Student's t ‐test. (D) The P‐gp expression in pCMV3‐ and pCMV3‐ABCB1 (pABCB1)‐transfected HCT116, as well as HCT116 and HCT116‐OxaR cells were examined by Western blotting. Data represent two independent experiments. (E) pCMV3‐ and pABCB1‐transfected HCT116 cells were treated with verapamil (10 μ m ) or VU‐0365114 (10 μ m ) for 2 h. Then, an MDR assay was performed. The error bars are the mean ± SD ( n = 3). Statistical significance, compared to untreated controls (* P < 0.05), was determined using a one‐way ANOVA with Tukey's post hoc test. Statistical significance, compared between pCMV3‐ and pABCB1‐transfected HCT116 cells ( ## P < 0.01), was determined using a two‐tailed paired Student's t ‐test. (F) pCMV3‐ and pABCB1‐transfected HCT116 cells were treated with VU‐0365114 (10 μ m ), doxorubicin (Doxo; 0.25 μ m ), and colchicine (Col; 100 n m ) for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD ( n = 5). Statistical significance, compared to pCMV3‐transfected HCT116 cells at each treatment (** P < 0.01), was determined using a two‐tailed paired Student's t ‐test. (G) Parental and p53‐knockout HCT116 (HCT116‐p53‐KO) cells were treated with various concentrations of 5‐fluorouracil (5‐FU), VU‐0365114, and colchicine for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD ( n = 4). Statistical significance, compared to parental HCT116 cells at each dose (** P < 0.01 and *** P < 0.001), was determined using a two‐tailed paired Student's t ‐test. (H) HCT116 and HCT116‐p53‐KO cells were treated with the indicated concentrations of 5‐fluorouracil (5‐FU), VU‐0365114 (VU), and colchicine for 48 h. Then, protein expressions were examined by Western blotting. Data represent two independent experiments.

Article Snippet: For the gene overexpression analysis, pCMV3 (#CV011), pCMV3‐CHRM5 (#HG20062‐UT), pCMV3‐ABCB1 (#HG12030‐UT), and pCMV3‐MPS1 (#HG11529‐UT) plasmids were purchased from SinoBiological (Beijing, China).

Techniques: MTT Assay, Two Tailed Test, Expressing, Western Blot, Transfection, Knock-Out

Journal: Molecular Oncology

Article Title: Repositioning VU ‐0365114 as a novel microtubule‐destabilizing agent for treating cancer and overcoming drug resistance

doi: 10.1002/1878-0261.13536

Figure Lengend Snippet: The effect of VU‐0365114 on drug resistance in colorectal cancer cells. (A) HCT116 and HCT116‐OxaR cells were treated with various concentrations of oxaliplatin, VU‐0365114, and colchicine for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD ( n = 4). Statistical significance, compared to parental HCT116 cells at each dose (* P < 0.05 and *** P < 0.001), was determined using a two‐tailed paired Student's t ‐test. (B) The P‐glycoprotein (P‐gp) expression in MES‐SA, MES‐SA/Dx5, HCT116, and HCT116‐OxaR cells were examined by Western blotting. Data represent two independent experiments. (C) HCT116 and HCT116‐OxaR cells were treated with verapamil (Vera; 10 μ m ) or VU‐0365114 (VU; 10 μ m ) for 2 h. Then, an MDR assay was performed. The error bars are the mean ± SD ( n = 4). Statistical significance, compared to untreated controls (** P < 0.01 and *** P < 0.001), was determined using a one‐way ANOVA with Tukey's post hoc test. Statistical significance, compared between HCT116 and HCT116‐OxaR cells ( # P < 0.05), was determined using a two‐tailed paired Student's t ‐test. (D) The P‐gp expression in pCMV3‐ and pCMV3‐ABCB1 (pABCB1)‐transfected HCT116, as well as HCT116 and HCT116‐OxaR cells were examined by Western blotting. Data represent two independent experiments. (E) pCMV3‐ and pABCB1‐transfected HCT116 cells were treated with verapamil (10 μ m ) or VU‐0365114 (10 μ m ) for 2 h. Then, an MDR assay was performed. The error bars are the mean ± SD ( n = 3). Statistical significance, compared to untreated controls (* P < 0.05), was determined using a one‐way ANOVA with Tukey's post hoc test. Statistical significance, compared between pCMV3‐ and pABCB1‐transfected HCT116 cells ( ## P < 0.01), was determined using a two‐tailed paired Student's t ‐test. (F) pCMV3‐ and pABCB1‐transfected HCT116 cells were treated with VU‐0365114 (10 μ m ), doxorubicin (Doxo; 0.25 μ m ), and colchicine (Col; 100 n m ) for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD ( n = 5). Statistical significance, compared to pCMV3‐transfected HCT116 cells at each treatment (** P < 0.01), was determined using a two‐tailed paired Student's t ‐test. (G) Parental and p53‐knockout HCT116 (HCT116‐p53‐KO) cells were treated with various concentrations of 5‐fluorouracil (5‐FU), VU‐0365114, and colchicine for 72 h. Then, cell viability was examined by an MTT assay. The error bars are the mean ± SD ( n = 4). Statistical significance, compared to parental HCT116 cells at each dose (** P < 0.01 and *** P < 0.001), was determined using a two‐tailed paired Student's t ‐test. (H) HCT116 and HCT116‐p53‐KO cells were treated with the indicated concentrations of 5‐fluorouracil (5‐FU), VU‐0365114 (VU), and colchicine for 48 h. Then, protein expressions were examined by Western blotting. Data represent two independent experiments.

Article Snippet: For the gene overexpression analysis, pCMV3 (#CV011), pCMV3‐CHRM5 (#HG20062‐UT), pCMV3‐ABCB1 (#HG12030‐UT), and pCMV3‐MPS1 (#HG11529‐UT) plasmids were purchased from SinoBiological (Beijing, China).

Techniques: MTT Assay, Two Tailed Test, Expressing, Western Blot, Transfection, Knock-Out