Fig. 4

PTPN2 inhibits TG-initiated apoptosis through the Ca²⁺/ ROS/ mitochondrial pathway. (A) The representative fluorescence images of Mito Tracker (green)/ Rhod-2 (red) staining demonstrating Ca²⁺ level in the mitochondria shows that ectopic expression of PTPN2 45 kDa or PTPN2 48 kDa decreases Ca²⁺ level in the mitochondria in TG-treated MDA-MB-231 cells. Hoechst 33,342 staining indicates nuclei. Scale bar = 20 um. All experiments are conducted as a single independent experiment (n = 1). (B) The representative fluorescence images of Mito Tracker (green)/ Rhod-2 (red) staining demonstrating Ca²⁺ level in the mitochondria shows that knockdown of PTPN2 increases Ca²⁺ level in the mitochondria in TG-treated MDA-MB-231 cells. Hoechst 33,342 staining indicates nuclei. Scale bar = 20 um. All experiments are conducted as a single independent experiment (n = 1). (C) The representative fluorescence images of DCFH-DA staining demonstrating ROS level shows that ectopic expression of PTPN2 45 kDa or PTPN2 48 kDa decreases ROS production in TG-treated MDA-MB-231 cells. Hoechst 33,342 staining indicates nuclei. Scale bar = 200 um. All experiments were conducted three times independently (n = 3). (D) The bar graph shows that ectopic expression of PTPN2 45 kDa or PTPN2 48 kDa inhibits ROS production in TG-treated MDA-MB-231 cells by measuring mean fluorescence intensity of green fluorescent cells in 3 different fields under the microscope. (E) The representative fluorescence images of DCFH-DA staining demonstrating ROS level shows that knockdown of PTPN2 increases ROS production in TG-treated MDA-MB-231 cells. Hoechst 33,342 staining indicates nuclei. Scale bar = 200 um. All experiments were conducted three times independently (n = 3). (F) The bar graph shows that knockdown of PTPN2 promotes ROS production in TG-treated MDA-MB-231 cells by measuring mean fluorescence intensity of green fluorescent cells in 3 different fields under the microscope. (G) The western blot shows that ectopic expression of PTPN2 45 kDa or PTPN2 48 kDa decreases expression of CHOP, P-AMPK and Bax, and increases expression of Bcl-2 in TG-treated MDA-MB-231 cells. GAPDH serves as a loading control. All experiments are conducted as a single independent experiment (n = 1). (H) The western blot shows that knockdown of PTPN2 increases expression of CHOP, P-AMPK and Bax, and decreases expression of Bcl-2 in TG-treated MDA-MB-231 cells. GAPDH serves as a loading control. All experiments are conducted as a single independent experiment (n = 1). (I) The representative CHOP immunohistochemical staining images of TG-treated 4T1 tissues in ex vivo. Scale bar = 200 um. Each experimental group has three samples (n = 3). (J) The bar graph demonstrates that knockout of PTPN2 significantly promotes CHOP expression of TG-treated 4T1 tissues in ex vivo. (K) The western blot shows that PTPN2 knockout promotes GRP78 and CHOP expression of TG-treated 4T1 tissues in ex vivo. GAPDH serves as a loading control.