Fig. 6. PKM2 siRNA-mediated knockdown reduces cell growth/viability, protects against shikonin-induced PDAC cell death and inhibits PMCA activity.

a Treatment of Mia PaCa-2 cells with PKM2 siRNA reduces the expression of PKM2 as assessed using western blotting with PKM2-specific antibody (ai, aii) and qPCR (aiii). As additional controls, the same samples were western blotted with a PKM1-specific antibody and a pan-PKM1/2 antibody and compared with lysates from non-cancerous human pancreatic ductal epithelial cells (HPDE) and human pancreatic stellate cells (HPSC). Mouse skeletal muscle (SkM) lysates were used as a positive control for PKM1 expression (ai). GAPDH was used as a loading control. aii PKM2 protein expression from PKM2 siRNA-treated cells was quantified by densitometry by normalising PKM2 band intensity to the GAPDH loading control before normalising to the band intensity of the scrambled siRNA-treated cells (siRNA) in each gel (n = 3 separate experiments). b Effect of PKM2 siRNA vs scrambled siRNA with and without shikonin on cell growth/viability using CCK-8 assay (b and inset figure) over 96 h. Maximum rate of growth (48–72 h) was compared for each treatment (inset figure). c Western blot showing shikonin-induced PARP1 cleavage following 3–24-h treatment with 5 µM shikonin. β-actin was used as a loading control. d Representative in situ Ca²⁺ clearance assays to assess PMCA activity in fura-2-loaded MIA PaCa-2 cells treated with scrambled siRNA (siSCR) (di) or PKM2 siRNA (siPKM2) (dii) for 72 h. Cells were first perfused with HEPES-PSS containing 0 Ca²⁺/1 mM EGTA and 30 μM CPA (arrow) before adding 20 mM Ca²⁺ to induce Ca²⁺ entry. Subsequent removal of external Ca²⁺ (0 Ca²⁺/1 mM EGTA) led to rapid Ca²⁺ clearance and the falling phase of this initial Ca²⁺ clearance phase was fitted to a single exponential to yield a time constant (τ) as a measure of clearance rate. diii Expanded time course superimposed for siSCR (black) and siPKM2 (grey). div Mean time constant for siSCR (black box) and siPKM2 (grey box). An unpaired t-test was used to determine statistical significance. n = 7–9 (8–18 cells analysed per experiment) *p ≤ 0.01. Using a paired experimental design, the effect of shikonin during the second Ca²⁺ clearance phase (R₂) was compared with the initial Ca²⁺ clearance phase (R₁) for both scrambled siRNA (siRNA) and PKM2 siRNA-treated cells (siPKM2). div Mean normalised linear Ca²⁺ clearance (% R₂/R₁) measured from the same starting value (as indicated by the dashed lines). *p < 0.05, statistical significance was determined using a Kruskal –Wallis test with Tukey’s multiple comparisons. e ATP was measured using a 96-well plate based on firefly luciferase luminescence assay in scrambled siRNA vs PKM2 siRNA-treated Mia PaCa-2 cells treated with or without 5 μM shikonin for 6 h. Raw luminescence counts were normalised to protein assessed using a BCA assay prior to normalising to vehicle controls and expressed as a percentage. *p < 0.05, statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test prior to normalisation.