Fig. 6

miR-135 knockdown leads to increased glutamine dependence. a Schematic representation of ¹³C- glucose metabolism. b Heatmap of significantly changed labeled metabolites in glycolysis, amino acids and tricarboxylic acid cycle in control and miR-135 knockdown MIA PaCa-2 cells analyzed by LC-MS. c Labeled pyruvate, lactate, serine and glycine were analyzed in control (anti-NC), miR-135 knockdown (anti-miR-135) and miR-135/PFK1 double knockdown MIA PaCa-2. Fold change is calculated by the labeled metabolites normalized to control MIA PaCa-2 cells. Each value represents mean ± SD in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. d Labeled and unlabeled α-ketoglutarate, succinate, fumarate, and glutamate were analyzed in control (anti-NC) and miR-135 knockdown (anti-miR-135) MIA PaCa-2 cells by using LC-MS. Fold change is calculated by the labeled or unlabeled metabolites divided by pool and normalized to control MIA PaCa-2 cells. Metabolite data are shown as mean ± SD in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-tailed Student’s t test. e Control (anti-NC), miR-135 knockdown (anti-miR-135) and miR-135/PFK1 double knockdown MIA PaCa-2 cells were cultured in complete medium for 24 h. Glutamine uptake was measured using the Nova Bioprofile 100 analyzer. f Control (anti-NC), miR-135 knockdown (anti-miR-135), shPFK1 and miR-135/PFK1 double knockdown MIA PaCa-2 cells were cultured in glutamine-free medium for indicated time points. Cell viability was assessed by Trypan blue exclusion. Each value represents mean ± SD in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. Cells were lysed and immunoblotting was performed with indicated antibodies