Fig. 4

Cacna2d3 regulates survival of Mtb via Ca²⁺-mediated autophagy. a, b Bacterial survival of Mtb in (a) primary macrophages treated with Cacna2d3 siRNA; (b) macrophages from WT and Cacna2d3 mutant (Cac3^PB/PB) mice, after infection with Mtb (MOI = 5) for 24 h, compared to 2 h. c, d WT or Cac3^PB/PB macrophages were infected with Mtb, and then subjected to (c) TEM analysis; red arrow indicated the bacteria. (d) IB of lysates. e–h Confocal detection of mRFP-GFP-LC3B spots in Cacna2d3-siRNA-treated macrophages (e, f) or Cac3^PB/PB macrophages, after infection with Mtb (MOI = 5) for 6 h (g, h); Bar, 5 μM. i Calcium assay in WT or Cac3^PB/PB macrophages infected with Mtb (MOI = 5) for the duration of 1 min. j, k CFU assay (j) and intracellular survival of Mtb (k) in WT or Cac3^PB/PB macrophages pretreated with Bepridil (BPD) (10 μM), and then infected with Mtb (MOI = 5) for 2 h or 24 h. l, m Confocal detection of mRFP-GFP-LC3B spots in WT or Cac3^PB/PB macrophages pretreated with DMSO or BPD infected with Mtb at MOI 5 for 6 h. Bar, 5 μM. n, o IB of lysates for phosphorylated-CaMKKβ (n) and phosphorylated-ULK1 (o) from WT or Cac3^PB/PB macrophages infected with Mtb for indicated times. NS, not significant (p > 0.05), * p < 0.05 and ** p < 0.01 by the unpaired t-test (f, h) or Mann-Whitney U test (a, b, j). Data are from three independent experiments with biological duplicates in each (a, b, f, h, j, k, m; mean ± s.e.m. of n = 3 duplicates) or representative of three independent experiments (c–e, g, i, l, n, o)