Fig. 3. CLIC1 silencing increases human GBM CSC survival in the presence of metformin.

A and B) GBM1 CSCs were transiently infected with a plasmid carrying validated shRNA for both Luciferase (shLuc, as a silencing control) and CLIC1 (shCLIC1) and tested for CLIC1 expression. In shCLIC1 cells, protein levels were highly reduced as assessed by immunocytofluorescence (A, shRNA= green, CLIC1= red) and Western blot analysis (B). Reported blot is representative of 2 independent experiments. C) Metformin inhibition of CLIC1 current in GBM1 CSCs. Dose-response of metformin sensitive current experiments expressed as a percentage of maximal inhibition operated by 100 μM IAA94 (n=3 for 0.1-5-20 mM, n=6 for 1 mM, n=4 for 10 mM), an EC₅₀ of 2.3 mM. D) Perforated patch whole-cell experiments using a voltage protocol of 800 ms, 20 mV steps from −60 to +60 mV, starting from −40 mV holding potential. Left panels depict IAA94- and metformin-sensitive currents in shLuc cells. In shCLIC1-transfected cells (right panels), the current that results sensitive to either metformin or IAA94 treatment is null. E) I/V relationships of metformin (left panel) and IAA94 (right panel) sensitive current densities of shLuc (circle; n=7 for metformin; n=4 for IAA94) and shCLIC1 (squares; n=6 for metformin; n=4 for IAA94). Data are expressed as mean ± sem cells. F and G) Down-regulation of CLIC1 expression slightly affects basal cell proliferation (−14% at 48 hours vs. shLuc cells) but significantly reduces the effects of metformin and IAA94 on CSC viability after 48 hours of treatment, evaluated by MTT assay. Results demonstrate that the expression of CLIC1 is required for the antiproliferative activity of metformin in GBM CSCs. Dotted line represents the respective untreated control value for shLuc and shCLIC1 infected cells, taken as 100%. *p<0.05, **p< 0.01 and ***p<0.0001 (t-test).