Fig. 3

miR-135 promotes PDAC survival upon glutamine deprivation. a–c MIA PaCa-2 cells (a), PANC-1 (b), and BxPc-3 (c) were transiently transfected with inhibitor control or hsa-miR-135 inhibitor. 48 h posttransfection, cells were cultured in glutamine-free medium. Relative miR-135b expression was assessed by qPCR. Cell viability was assessed by DAPI exclusion flow cytometry at the indicated time points. d MIA PaCa-2 cells were transfected with microRNA inhibitor control clone (anti-NC) or miR-135 inhibitor clone (anti-miR-135). miR-135b expression was assessed by qPCR. e MIA PaCa-2 cells expressing anti-NC or anti-miR-135 were cultured in glutamine-free medium. Cell viability was assessed by DAPI exclusion flow cytometry at the indicated time points. Cells were also lysed and immunoblotting was performed with indicated antibodies (f). Each value represents the mean ± SD in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. g MIA PaCa-2 cells were transfected with microRNA scramble control clone or miR-135a precursor clone. miR-135a relative expression was assessed by qPCR. Each value represents the mean ± SD in four independent experiments. ***p < 0.001, Student’s t test. h MIA PaCa-2 cells expressing scramble or miR-135a were cultured in glutamine-free medium. Cell viability was assessed by DAPI exclusion flow cytometry at the indicated time points. Each value represents the mean ± SD in three independent experiments. ***p < 0.001, Student’s t test. i Cells were also lysed and immunoblotting was performed with indicated antibodies (CM: complete medium; No Gln: glutamine-free medium). PDAC: pancreatic ductal adenocarcinoma