Fig. 6: TBK1 is required for mTORC1 activation and glycolysis in physiological conditions.

a Immunoblots of 786-O or A498 cells expressing doxycycline-inducible control shRNA (Ctrl), or TBK1 shRNAs (82, 85) grown in DMEM or HPLM medium. b Immunoblots of 786-O or UMRC2 cells treated with TBK1 PROTAC grown in DMEM or HPLM medium. c Immunoblots of A498 cells expressing empty vector (EV), wild type TBK1, or S172D mutant grown in DMEM or HPLM medium. d Immunoblots of 786-O cells treated with TBK1 CMPD1 grown in DMEM or HPLM medium. e, f Measurement of extracellular acidification rate (ECAR) in A498 cells expressing doxycycline-inducible control (Ctrl) (n = 6 independent cell cultures) or TBK1 shRNAs (n = 7 independent cell cultures) (e) or expressing EV, S172D mutant (n = 7 independent cell cultures for each group) (f) grown in HPLM. The median value (center line), lower quartile and upper quartile (box edges) and maximum and minimum value whiskers are indicated in the boxplot. Each experiment has been repeated two times with similar results. g Representative soft agar images (left) and colony quantification (right) of A498 cells expressing Ctrl or TBK1 shRNA 85 grown in DMEM or HPLM medium. n = 4 biological cell cultures for each group. This experiment has been repeated two times with similar results. Scale bar, 1 mm. Subcutaneous xenograft tumor growth (n = 6 mice for each group) (h), immunoblot with corresponding gel quantifications (n = 6 paired tumors, each mouse was inoculated with Ctrl and sh85 cells at both flanks) (i) of A498 cells with TBK1 depletion by shRNA 85. Doxycycline (Dox) chow was treated at the indicated time point. qRT-PCR of glycolytic genes in A498 xenograft tumors (n = 4 biological tumor samples per group) (j) or in vitro cells grown in DMEM (n = 3 biological cell samples per group) (k) expressing Ctrl, or TBK1 shRNA 85. Each experiment has been repeated two times with similar results. Statistical analysis was conducted by two-way ANOVA followed by Tukey’s multiple comparison test (h) or two-tailed Student’s t test (e, f, g, j, and k, unpaired; i, paired). Error bars represent SEM, NS denotes no significance. Representative immunoblots and gels shown in figures were repeated at least two times independently with similar results. Source data are provided as a Source Data file.