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. 2018 May 25;9(40):26072–26085. doi: 10.18632/oncotarget.25437

Figure 3. Expression of Nur77 derived peptide in H460 cells induces apoptosis preferentially in paclitaxel resistant H460 lung cancer cells.

Figure 3

(A) Annexin V staining of H460 parental and paclitaxel resistant cells after expression of control GFP or GFP-Nur77 peptide for 72 hours. Histogram gate indicates difference in annexin V positive cells between H460 parental (P) (blue) and paclitaxel resistant (R) (orange) cells. Data is representative of 3 independent experiments and quantification of apoptosis is shown in the right panel. (B) Annexin V staining of H460 parental (P) (blue) and resistant (R) (orange) cells after expression of control GFP or GFP-Nur77 peptide for after 96 hours. Histogram gate indicates difference in annexin V positive cells between H460 parental and paclitaxel resistant cells. Data is representative of 3 independent experiments and quantification of apoptosis is shown in the right panel. (C) Annexin V staining of H460 parental and resistant cells after expression of GFP-Nur77 peptide with or without co-treatment with paclitaxel (10 nM). Histogram gates indicate difference in annexin V positive cells between vehicle and paclitaxel 10 nM (Upper panels), Nur77 peptide expression alone and Nur77 expression combined with paclitaxel 10 nM (Lower panels). Data is representative of 3 independent experiments and quantification of apoptosis is shown in the right panel. (D) Annexin V staining of H69AR multidrug resistant lung cancer cells after expression of control GFP (black) or GFP-Nur77 peptide (blue), histogram gate indicates difference in annexin V positive cells between GFP and GFP-Nur77 peptide expression. The right panel is the quantification of percentage of apoptotic cells from the histograms. (E) Expression of Nur77 peptide induces cell death in a Bcl-2-dependent manner in multidrug resistant H69AR lung cancer cells. Flow cytometry-based analysis of cell death in Bcl-2 knockout H69AR CRISPR lines after expression of GFP-Nur77 peptide for 36 hours relative to control GFP expression. Suppression of Bcl-2 expression is confirmed by Western blot analysis of H69AR CRISPR lines expressing control CRISPR plasmid (Bcl-2 WT) and Bcl-2 targeted gRNA CRISPR plasmid (Bcl-2 KO). Results are the of mean±s.d. unpaired two-tailed t-test ****=P<0.0001.