Figure 5

Effect of GABRP knock-down in vitro and in vivo. (A) Proliferation of 50 nM scrambled control siRNA (siControl) or GABRP siRNA (siGABRP) treated cells was assessed by CellTiter Glo luminescent viability assay after 72 hours and indicated as percentage of respective control for each cell line. Each vertical bar represents the percent cell growth measured as mean luminescence ± SEM in each treatment group. *P < 0.05 as compared to respective siRNA control group. (B) GABRP was assessed in vector control and GABRP CRISPR KO MDA-MB-468 (KO-1) cells using FLAG tagged ECD-binding monoclonal Fab and anti-FLAG-PE antibody by flow-cytometry. GABRP protein expression was assessed by immunoblotting and densitometric analysis was performed in ImageJ software. Empty control (no guide sequence), Vector control (scrambled sgRNA sequence), and GABRP KO-1 MDA-MB-468 cells were harvested for genomic DNA isolation and analyzed by T7 endonuclease assay. Pound sign (#) indicated undigested DNA and stars (*) indicate digested products. (C) Proliferation of 50 nM scrambled control siRNA (siControl) or GABRP siRNA (siGABRP) treated vector control and CRISPR KO-1 cells was assessed by CellTiter Glo luminescent viability assay after 72 hours and indicated as percentage of siRNA control-treated vector control MDA-MB-468 cells. Vertical bar represents the percent cell growth measured as mean luminescence ± SEM in each treatment group. *P < 0.05 as compared to siRNA control-treated vector control MDA-MB-468 cells. D. Proliferation of control siRNA (siControl) or GABRP siRNA (siGABRP) treated or untreated MDA-MB-468 (parent) cells was assessed by counting cells after trypan blue exclusion assay over 96 hours; data points represent mean viable count per well ± SD in each group. Cells from these treatment groups were collected 96 h after transfection and probed for GABRP protein levels by Western blotting (below). (E) The stable GABRP knockdown cell line (shGABRP), shRNA control (shControl) and parent MDA-MB-468 cell lines were allowed to form colonies in soft agar. Vertical bars indicate average number of colonies in three replicates compared to parent cell line. P-values were calculated by Student t test. *P < 0.05. (F) Tumor volume of stable GABRP knockdown (shGABRP), stable control (shControl) and untreated MDA-MB-468 (parent) xenograft in female athymic Nude-Foxn1 nude mice (Nu/Nu) in (N = 5 in each group). P-values were calculated by Student t test. *P < 0.05. Tumors shown below were isolated at the end 10-weeks inoculation period.