Extended Data Fig. 4: Lipid peroxidation-ALDH2 pathway is responsible for acetate production in TAMs.
a, b, The protein levels of ALDH2 and ALDH1B1 in TAMs generated from THP-1 cell-derived MΦs or RAW264.7 cells. c, d, Confirmation of ALDH2 or ALDH1B1 knockdown in THP-1 cells stably expressing with shRNA targeting ALDH2 (shALDH2) or ALDH1B1 (shALDH1B1). e, Immunoblots of ALDH2 protein in BMDM or liver isolated from Aldh2-flox or Aldh2-cKO mice. f, g, Confirmation of Aldh2 or Aldh1b1 knockdown in RAW264.7 cells stably expressing shRNA targeting Aldh2 (shAldh2) or Aldh1b1 (shAldh1b1). h, i, Secreted acetate levels in TAMs with silenced Aldh2 or Aldh1b1 (h) or in TAMs treated with ALDH2 inhibitor (i). j, k, Effects of silencing of ALDH2 on the mRNA levels of IL10 and ARG1 in TAMs and corresponding MΦs. l–q, MDA levels (l, m), Cox1 and Cox2 mRNA levels (n, o) and the lipid peroxidation levels (p, q) in TAMs and corresponding MΦs. Quantitation of the lipid peroxidation levels are shown in q. r, s, Effects of Fer-1 (2 μM) or NAC (5 mM) on the lipid peroxidation levels in TAMs (r) and primary TAMs (s). t, Effects of Aspirin (10 mM) on the MDA generation in TAMs. u–z, As depicted in u and v, TAMs generated from RAW264.7 cells were incubated with exogenous [U-13C]-LA for 36 h with or without etomoxir or NAC, and were subjected to determine labeled carbons in LA (w), AA (x) and secreted acetate (y). The proportion of labelled secreted acetate to labelled AA is shown in z. Scale bar, 20 μm (p, r and s). Data are shown as three biologically independent cell cultures (h, i, l, m, t, w and x), four biologically independent cell cultures (y, z), mean ± s.d. of triplicate experiments (j, k, n and o), or mean ± s.d. of 5 representative pictures (q), images are representative of three independent experiments (a–g, q–s). P values were determined by one-way ANOVA with Tukey’s multiple comparisons test (h, i and t) or unpaired two-tailed Student’s t-tests (j–o and q).
