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. Author manuscript; available in PMC: 2022 Sep 30.
Published in final edited form as: Nat Metab. 2022 Mar 31;4(4):444–457. doi: 10.1038/s42255-022-00551-7

Extended Data Fig. 1. Additional experiments documenting the efferocytosis-Ptgs2/COX2-TGFβ1 pathway in macrophages. Related to Fig. 1.a–d.

Extended Data Fig. 1

BMDMs were incubated ± ACs, after which noninternalized ACs were removed by rinsing. The cells were assayed for Ptgs2 mRNA after an additional 1 h incubation and COX2 protein after 3 h (a); PGE2 in the media after 3 h (b); and TGFβ1 in the media after 18 h (c). For (d), experiments similar to those in panel a were analyzed for mouse and human Ptgs2/PTGS2 or Tgfb1/TGFβ1 mRNA to prove that mRNA being measured is not residual human mRNA derived from human apoptotic Jurkat cells. e, BMDMs were transfected with scrambled RNA (Scr) or siRbcn and then, after 72 h, assayed for Rbcn mRNA. f, BMDMs were transfected with Scr or siRbcn or treated with vehicle or bafilomycin A1 and then incubated with PKH26-labeled ACs for 45 min, followed by rinsing and quantification of percent PKH26+ macrophages of total macrophages. g, BMDMs were transfected with Scr or siPtgs2 and then, after 72 h, assayed for Ptgs2 mRNA. h, BMDMs were incubated ± ACs for 45 min, after which noninternalized ACs were removed by rinsing. The cells were assayed for Ptges mRNA after an additional 1 h incubation (left). i, BMDMs were transfected with scrambled RNA (Scr) or siPtges and then, after 72 h, assayed for Ptges mRNA. j-k, BMDMs were transfected with Scr, siPtger4, or siPtger2 and then, after 72 h, assayed for Ptger4 or Ptger2 mRNA, respectively. l, BMDMs were transfected with Scr or siTgfb1. After 72 h, the cells were incubated ± ACs for 45 min, after which noninternalized ACs were removed by rinsing. After an additional 1 h of incubation, the cells were assayed for Ptgs2 mRNA. m, BMDMs were transfected with Scr or siTgfb1 and then, after 72 h, assayed for Tgfb1 mRNA. All mRNA data are expressed relative to the first control group. Values are means ± SEM. ns, not significant (P > 0.05); n = 3 biological replicates. Two-sided P values were determined by a Student’s t-test for two groups or one-way ANOVA with Fisher’s LSD posthoc analysis for three or more groups.