Fig. 4: IP3R-mediated elevation in Ca²⁺ levels is required for TBC1D9 recruitment to bacteria and TBK1 activation.

a Representative trace of Fura 2-AM ratio during GAS WT or Δslo infection. b–d HeLa cells expressing EmGFP-LC3 and mRuby2-TBC1D9 and treated with or without BAPTA-AM (10 μM) were infected with GAS for 4 h. c Confocal images of TBC1D9 recruitment during infection and d quantification of cells harboring TBC1D9-positive bacteria, and d quantification of colocalization between LC3 and TBC1D9. e Effects of BAPTA-AM treatment on TBK1 phosphorylation during GAS infection. f Effects of IP3Rs depletion on TBK1 phosphorylation during GAS infection. g, h Recruitment of ULK1 to intracellular GAS in IP3Rs-KD cells. HeLa cells transfected with mClover-ULK1 and the indicated siRNAs were infected with GAS, fixed at 4 h, and stained for endogenous ubiquitin. g Representative confocal images and h the frequency of ULK1-positive bacteria. i, j HeLa cells transfected with EmGFP-LC3 and the indicated siRNAs were infected with GAS for 4 h. i Representative confocal images and j the frequency of cells with LC3-positive bacteria. k HeLa cells transfected with the indicated siRNAs were infected with GAS and intracellular bacterial CFU was determined at 6 h p.i. Data in (c) (n > 200 cells per condition), (d) (n > 5 images per condition), (h) (n > 50 bacteria per condition), (j) (n > 200 cells per condition), and (k) represent the mean ± SEM of three independent experiments. P values calculated by two-tailed Student’s t test. **P value < 0.01, ***P value < 0.001.