Journal: EBioMedicine

Article Title: Variants in oxidative stress-related genes affect the chemosensitivity through Nrf2-mediated signaling pathway in biliary tract cancer

doi: 10.1016/j.ebiom.2019.08.037

Figure Lengend Snippet: GPX4, CAT, and GSR regulate Nrf2 protein expression through ROS. (a and b) Representative FACS profile of ROS levels, which was measured by H2DCFDA staining, in GBC-SD (a) and QBC-939 (b) when GPX4, CAT, or GSR was overexpressed or knockdown. (c and d) Analysis of correlation between the protein level of Nrf2 and the mRNA level of GPX4 (c, left) or GSR (c, right) was performed in 36 GBC tissues, and the mRNA level of CAT (d, left) or GSR (d, right) was performed in 52 CC tissues. Spearman's correlation was used. (e and f) Immunoblot and qPCR analysis of Nrf2, and it target NQO1 from GBC-SD transfected with scrambled siRNA (si-Con) or siRNA (si- GPX4 and si- GSR ) and empty vector, GPX4 , or GSR (e), and from QBC-939 transfected with si-Con or siRNA (si- CAT and si- GSR ) and empty vector or CAT or GSR (F). n = 3; Bar, SEM. (g and i) qPCR analysis of NFE2L2, NQO1 and ABCG2 expression in GBC-SD (g: si-Con, si- GPX4 and si- GSR ), and in QBC-939 (i: si-Con, si- CAT and si- GSR ) cells cultured in standard media supplemented with or without 10 mM NAC. n = 3; Bar, SEM. (h and j) Immunoblots of Nrf2, NQO1 and ABCG2 protein in GBC-SD (h: si-Con, si- GPX4 and si- GSR ), and in QBC-939 (j: si-Con, si- CAT and si- GSR ) cells incubated with or without 10 mM NAC. * P < .05, ** P < .01, *** P < .001, Student's t- test.

Article Snippet: The following StableTM siRNA oligo (Genepharma, Shanghai, China) were used: nontargeting scrambled siRNA, human GPX4 siRNA, human CAT siRNA, human GSR siRNA, and human ABCG2 siRNA.

Techniques: Expressing, Staining, Knockdown, Western Blot, Transfection, Plasmid Preparation, Cell Culture, Incubation

Journal: EBioMedicine

Article Title: Variants in oxidative stress-related genes affect the chemosensitivity through Nrf2-mediated signaling pathway in biliary tract cancer

doi: 10.1016/j.ebiom.2019.08.037

Figure Lengend Snippet: GPX4, CAT, and GSR regulate chemosensitivity through Nrf2-mediated ABCG2 expression. (a and b) qPCR analysis of ABCG2 and NQO1 mRNA expression in paired GBC-SD (a) and QBC-939 (b) cells transfected with or without siRNA of GPX4, GSR, CAT . n = 3; Bar, SEM. (c and d) Immunoblots of ABCG2 and NQO1 in paired GBC-SD (c) and QBC-939 (d) cells that expressed GPX4, GSR , or CAT targeting siRNAs or a scrambled siRNA. (e and f) ABCG2-promoter luciferase assay in paired GBC-SD (a) and QBC-939 (b) cells that were transfected with or without siRNA of GPX4, GSR, CAT . n = 3; Bar, SEM. (g) The canonical sequence of the Nrf2-binding site (top, red), a potential Nrf2-binding site at -431 bp to -420 bp in the proximal promoter region of the human ABCG2 gene (middle, red), and introduced point mutations (bottom, green) used to inactivate the potential ABCG2-binding site are shown. (h and i) Determination of luciferase activity using vector only, wild type or mutant ABCG2 promoter in different pairs of GBC-SD (h) and QBC-939 (i) cells. (j) ChIP analysis of paired GBC-SD (sh-Con and sh- NFE2L2 ) cells immunoprecipitated by anti-Nrf2 or IgG antibody followed by qPCR using 2 primer sets for the Nrf2-binding site in the ABCG2 promoter or ABCG2 exon 1, respectively. Data represent the percent of input. n = 3; Bar, SEM. (k) Doxorubicin efflux of paired GBC-SD (left) and QBC-939 (right) cells were detected by FACS. * P < .05, ** P < .01, *** P < .001, Student's t- test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following StableTM siRNA oligo (Genepharma, Shanghai, China) were used: nontargeting scrambled siRNA, human GPX4 siRNA, human CAT siRNA, human GSR siRNA, and human ABCG2 siRNA.

Techniques: Expressing, Transfection, Western Blot, Luciferase, Sequencing, Binding Assay, Activity Assay, Plasmid Preparation, Mutagenesis, Immunoprecipitation

Journal: EBioMedicine

Article Title: Variants in oxidative stress-related genes affect the chemosensitivity through Nrf2-mediated signaling pathway in biliary tract cancer

doi: 10.1016/j.ebiom.2019.08.037

Figure Lengend Snippet: Nrf2 is the key element for growth and chemoresistance of BTC xenografts. (a and c) GBC-SD or QBC-939 cells stably expressing NFE2L2 -targeting shRNA (sh- NFE2L2 ) or scrambled shRNA (sh-Con) were injected into the flank of athymic nude mice s.c. ( n = 6 mice for each group) to create tumor xenografts. After tumors were about 5 mm in diameter, the first paired mice group was treated with saline, the second was treated with cisplatin (6 mg/kg), and the third was combined radical surgery and cisplatin (6 mg/kg) treatment. The chemotherapy was given once every nine days for 1 month. Tumor growth was determined by measurement of tumor volume, tumor weight, and the frequency of tumor formation. Tumor growth curves (left), tumor weigh (middle), and tumor-free percentages (right) at the indicated times were plotted. n = 6; Bar, SEM. (b and d) H&E, TUNEL, and IHC analysis of Ki-67, Nrf2, and ABCG2 expressions in tumor (b: GBC-SD; d: QBC-939) xenografts of the second paired mice groups. Representative images from 6 separate samples are shown. Original magnification, × 400; scale bars: 50 μm. * P < .05, ** P < .01, *** P < .001, Student's t- test.

Article Snippet: The following StableTM siRNA oligo (Genepharma, Shanghai, China) were used: nontargeting scrambled siRNA, human GPX4 siRNA, human CAT siRNA, human GSR siRNA, and human ABCG2 siRNA.

Techniques: Stable Transfection, Expressing, shRNA, Injection, Saline, TUNEL Assay

Journal: The Journal of Biological Chemistry

Article Title: p53-mediated regulation of LINE1 retrotransposon-derived R-loops

doi: 10.1016/j.jbc.2025.108200

Figure Lengend Snippet: Histone modification dynamics in L1 5′UTR-eGFP reporter cells upon HDACi exposure and withdrawal. A – D , chromatin immunoprecipitation followed by qPCR analyses was conducted on the 5′UTR region of A375 WT and p53 −/− L1 5′UTR-eGFP stable cells. Primers targeting the p53-binding region were utilized, with immunoprecipitation carried out using antibodies against histone modifications H3K9me3, H3K27me3, H3K9ac, and H3K4me3. The results illustrate levels of H3K9me3 ( A ) and H3K27me3 ( B ), representative of repressive histone marks, normalized to input values, as well as levels of activation marks H3K9ac ( C ) and H3K4me3 ( D ). Reporter cells were treated with HDACi (NaB drug) for 24 h, followed by withdrawal for 3 and 6 days. Samples for analyses were collected at first day, fourth day, and seventh day (for drug withdrawal), as depicted (refer to Fig. 5 ). It is noteworthy that alterations in H3K9me3, H3K27me3, H3K9ac, and H3K4me3 enrichment are specific to the p53 status (refer to Fig. S5 , A and B ). The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05, p ∗∗<0.01, and ns = non-significant. E , qPCR was performed to assess the knockdown of SETDB1 and G9A from the siRNA transfected WT and p53 −/− A375 L1 5′UTR-eGFP cell lines. The dark gray bar plot refers to the untransfected cells and the light gray being nontargeting control (NTC). The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05, and ns = non-significant. F and G , Western blotting was performed to assess the reduction in the expression levels of SETDB1 and G9A in the siRNA transfected samples of WT and p53 −/− A375 L1 5′UTR-eGFP cells. Please refer ( Fig. S5 , C and D ) for their quantification using Fiji software. H , qPCR to measure the transcript levels of L1 ORF1 in SETDB1 and G9A knocked down A375 WT and p53 −/− cell lines. The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05 and ns = nonsignificant. I , ChIP-qPCR to quantify the deposition of H3K9me3 marks at L1 5′UTR in SETDB1 knocked down cells of A375 WT and p53 −/− L1 5′UTR-eGFP cells. The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05 and ns = nonsignificant. J , ChIP-qPCR to quantify the deposition of H3K9me3 marks at L1 5′UTR in G9A knocked down cells of A375 WT and p53 −/− L1 5′UTR-eGFP cells.The individual data points indicate three biological replicates. The data represent mean ± SD across three independent biological replicates. Statistical significance was evaluated using the Mann–Whitney U test, with significance levels represented as follows: p ∗<0.05 and ns = nonsignificant; LINE1/L1, long interspersed nuclear element 1; HDACi, histone deacetylase inhibitor; NaB, sodium butyrate; ChIP, chromatin immunoprecipitation; qPCR, quantitative polymerase chain reaction.

Article Snippet: The siRNA sequences utilized for SETDB1 ( ) and G9A ( ) as described by along with the nontargeting siRNA control sequence (GenScript) are mentioned in .

Techniques: Modification, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Activation Assay, MANN-WHITNEY, Knockdown, Transfection, Control, Western Blot, Expressing, Software, ChIP-qPCR, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction