Fig 2: Cav1 improves cellular viability of Atorvastatin (ATV)-treated TKI-resistant NSCLC cells and is associated with GLUT3-mediated glucose uptake. A. Cell viability assay (n=3) demonstrates the effects of HALO-tagged Cav1 expression in ATV-treated cells. Cells were seeded in 96-well format prior adding 1 µM of ATV for 12 h. This was followed by transfection of Cav1 constructs ranging from 0.1 to 0.5 µg in increments of 0.1 µg for additional 60 h (total 72 h). Significance in differences in viability indexes, in which vehicle acted as control, was determined by t test. B. Glucose uptake measurement (n=3) in tumor cells treated with vehicle, ATV (at 1 µM) or a combination of ATV and Cav1-HALO (2 µg) vector for 72 h using a 6-well plate format. Glucose uptake is presented as percentages and normalized to vehicle. Significance in differences in glucose uptake, in which vehicle or ATV acted as controls, was determined by t test. C. Western blotting on Cav1, GLUT1, and GLUT3 protein expression from cells exposed to vehicle, ATV (at 1 µM), ATV and EV (6 ug) combination, or ATV and Cav1-HALO (6 µg) vector transfection combination for 72 h. Black arrow indicates Cav1-HALO, and white arrow indicates endogenous Cav1 proteins. EV - empty vector. Protein size is indicated in kDa. D. Cav1 was immunoprecipitated from tumor cell extracts and levels of co-precipitated GLUT1 and GLUT3 was determined with respective antibodies. Protein size is indicated in kDa. E. Representative fluorescence images of Duolink Proximity Ligation assays to detect protein interaction between Cav1 and GLUT3 in NSCLC cells. Fluorescence intensities were quantified in 10 cells using Image J software and significance in differences in fluorescence intensities, in which PC-9P acted as control, was determined by t test.

Fig 3: The effect of high glucose and halofuginone or tryptophanol on glucose transporters. Mesothelial cells were cultured under normal or high-glucose conditions in the presence or not of halofuginone or tryptophanol. GLUT-1, GLUT-3, and SGLT-1 levels were assessed with Western blotting. Nine experiments were performed for each substance; three of them are depicted in panels (A) and (E). High glucose increased the expression of all glucose transporters (A–H). Halofuginone reduced the expression of GLUT-1, GLUT-3, and SGLT-1 (B–D). Tryptophanol decreased the level of GLUT-1 (F), and, to a lesser extent, SGLT-1 (H), while it left GLUT-3 unaffected (G). *, #, +, and ^ indicate p < 0.05 compared to first, second, third, or fourth depicted conditions. Error bars correspond to standard error of means. The number inside each bar corresponds to the mean fold-change compared to the control.

Fig 4: Schematic diagram of the proposed model depicting how Atorvastatin, an anti-cholesterol drug, suppresses growth of TKI-resistant NSCLC tumors. Atorvastatin inhibits Cav1, which regulates GLUT3 signalling, supports tumor growth by modulating cellular energy homeostasis through glucose uptake.

Fig 5: TAT-Cx43266–283 maintains its antitumour effect under several nutrient-deprived conditions. (a) G166 GSCs were recorded by time-lapse microscopy in medium containing only glucose or only amino acids (Videos 1 and 2, respectively). After 4 days, these media were changed to complete medium and cells were recorded for a further 24 h (nutrient replenishment). Note that cells that appeared dead (bright, round shape) could survive and proliferate after nutrient replenishment, which was not true for cells maintained in medium without any nutrients (Supplementary Fig. 3 and Video 3). Scale bar: 100 µm. (b–d) GSCs were treated with 50 µM TAT or TAT-Cx43266–283 in complete medium for 24 h. Then, the medium was replaced with medium containing glucose (glucose-only medium), amino acids (amino acid-only medium), or both glucose and amino acids, as well as 50 µM of each peptide, for another 24 h. For comparison, cells were treated in parallel with complete medium and 50 µM of peptides. Then, the indicated assays were performed. (b) MTT oxidation of GSCs under the indicated conditions. (c) GSCs were recorded by time-lapse microscopy in complete or glucose-only medium (Videos 4–9; the images are the last photomicrograph from each video) or photographed under the indicated conditions. Scale bar: 100 µm. (d) Representative western blots of the indicated proteins under the indicated conditions. GSCs treated with TAT-Cx43266–283 in glucose-only medium exhibited decreased levels of HIF-1?, GLUT-3, HK- 2, G6PD, and Sox-2, but not HK-1 (left panel). However, GSCs treated in complete medium showed only slight decreases (right panel). GAPDH and a-tubulin blots are shown as loading controls. All data are mean ± s.e.m. and were obtained from at least three independent experiments with at least two technical replicates (???P < 0.001 vs control; ###P < 0.001 vs TAT; +++P < 0.001 TAT-Cx43266–283 in glucose-only medium vs in complete medium). Numbers under the bars indicate the number of biological replicates in (b).