Figure 4.
Knockout of TCF7L2 and JMJD6 depletes target gene expression, reduces cell viability, and resensitizes OTX-P/R cells to the lethal effects of BETi treatment. (A) SET-2 and SET-2-OTX P/R cells were transfected with negative control sgRNA or sgRNAs against TCF7L2 exon 3 or exon 6 and incubated for 5 days. Then, cells were cytospun onto glass slides and stained with anti-β-catenin, TCF7L2, or TBL1 antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Confocal microscopy analysis was performed (original magnification ×100). Representative images are shown for each condition. (B) Representative immunoblot analysis of SET-2 and SET-2-OTX P/R cells transfected with negative control sgRNA or sgRNAs against TCF7L2 and incubated for 5 days. The expression levels of β-actin in the cell lysates served as the loading control. KO, knockout. (C) SET-2 and SET-2-OTX P/R cells were transfected with negative control sgRNA or sgRNAs against TCF7L2. Following this, the percent cell viability was monitored over 8 days. **P < .01; ***P < .005 for cell viability values significantly less in SET-2-OTX P/R cells transfected with TCF7L2 sgRNA compared with SET-2 parental cells transfected with TCF7L2 sgRNA. (D) SET-2 and SET-2-OTX P/R cells were transfected with negative control sgRNA or sgRNA against JMJD6 and incubated for 5 days. Immunoblot analysis was conducted on the total cell lysates. The expression levels of β-actin in the cell lysates served as the loading control. (E) SET-2 and SET-2-OTX P/R cells were transfected with negative control sgRNA or sgRNAs against JMJD6. Following this, the percent cell viability was monitored over 10 days. Asterisks (***) indicate cell viability values significantly less (P < .005) in SET-2-OTX P/R cells transfected with JMJD6 sgRNA compared with SET-2 parental cells transfected with JMJD6 sgRNA. (F) SET-2-OTX P/R and HEL-OTX P/R cells were transfected with negative control sgRNA or TCF7L2 or JMJD6 sgRNAs and incubated for 3 days. Following this, cells were treated with the indicated concentrations of OTX015 for 48 hours. The percentage of annexin V–positive, apoptotic cells were determined by flow cytometry. (G) SET-2 cells were transfected with a vector for overexpression of TCF7L2 or JMJD6. The overexpression was confirmed by immunoblot analysis. The expression levels of β-actin in the cell lysates served as the loading control. (H) SET-2 cells were transfected with a vector for overexpression of TCF7L2 or JMJD6. Parental and TCF7L2 or JMJD6-overexpressing cells were treated with the indicated concentrations of OTX015 for 48 hours. Then, the percentage of annexin V–positive, apoptotic cells from each group was determined by flow cytometry.

Knockout of TCF7L2 and JMJD6 depletes target gene expression, reduces cell viability, and resensitizes OTX-P/R cells to the lethal effects of BETi treatment. (A) SET-2 and SET-2-OTX P/R cells were transfected with negative control sgRNA or sgRNAs against TCF7L2 exon 3 or exon 6 and incubated for 5 days. Then, cells were cytospun onto glass slides and stained with anti-β-catenin, TCF7L2, or TBL1 antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Confocal microscopy analysis was performed (original magnification ×100). Representative images are shown for each condition. (B) Representative immunoblot analysis of SET-2 and SET-2-OTX P/R cells transfected with negative control sgRNA or sgRNAs against TCF7L2 and incubated for 5 days. The expression levels of β-actin in the cell lysates served as the loading control. KO, knockout. (C) SET-2 and SET-2-OTX P/R cells were transfected with negative control sgRNA or sgRNAs against TCF7L2. Following this, the percent cell viability was monitored over 8 days. **P < .01; ***P < .005 for cell viability values significantly less in SET-2-OTX P/R cells transfected with TCF7L2 sgRNA compared with SET-2 parental cells transfected with TCF7L2 sgRNA. (D) SET-2 and SET-2-OTX P/R cells were transfected with negative control sgRNA or sgRNA against JMJD6 and incubated for 5 days. Immunoblot analysis was conducted on the total cell lysates. The expression levels of β-actin in the cell lysates served as the loading control. (E) SET-2 and SET-2-OTX P/R cells were transfected with negative control sgRNA or sgRNAs against JMJD6. Following this, the percent cell viability was monitored over 10 days. Asterisks (***) indicate cell viability values significantly less (P < .005) in SET-2-OTX P/R cells transfected with JMJD6 sgRNA compared with SET-2 parental cells transfected with JMJD6 sgRNA. (F) SET-2-OTX P/R and HEL-OTX P/R cells were transfected with negative control sgRNA or TCF7L2 or JMJD6 sgRNAs and incubated for 3 days. Following this, cells were treated with the indicated concentrations of OTX015 for 48 hours. The percentage of annexin V–positive, apoptotic cells were determined by flow cytometry. (G) SET-2 cells were transfected with a vector for overexpression of TCF7L2 or JMJD6. The overexpression was confirmed by immunoblot analysis. The expression levels of β-actin in the cell lysates served as the loading control. (H) SET-2 cells were transfected with a vector for overexpression of TCF7L2 or JMJD6. Parental and TCF7L2 or JMJD6-overexpressing cells were treated with the indicated concentrations of OTX015 for 48 hours. Then, the percentage of annexin V–positive, apoptotic cells from each group was determined by flow cytometry.

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