Fig. 7

FSTL1 increases CCR2 recycling to the membrane by downregulating ATP6V1G2 expression in a CD14/TLR/NF-κB-dependent manner. a THP-1 cells were subjected to cell fractionation analysis at 48 h after FSTL1 treatment (100 ng/ml). Western blot analysis of CCR2 and endosome markers (Rab4, Rab5, Rab11, EEA1, and Lamp1). b–d HEK293 cells were transfected with the BRET donor CCR2-Nanoluc along with the BRET acceptor per well (HaloTag-Rab4, HaloTag-Rab11 or HaloTag-Rab7). The cells were treated with or without FSTL1 (100 ng/ml) for 20 h. CCL2 (100 ng/ml) was added and kinetic measurements of donor emission (460 nm) and acceptor emission (618 nm) were collected every 5 min for 60 min. e, f RNAseq analysis of THP-1 cells treated with or without FSTL1 for 12 h. Gene differential analysis is shown as a volcano plot (e) or heatmap (f). ATP6V1G2 expression was evaluated via qPCR (g, n = 3) or western blotting (h). i FCM analysis of CCR2 expression after 48 h of different treatments. j Cell fractionation analysis of CCR2 via western blotting in THP-1cells. k NanoBRET assays were used to evaluate the distribution of CCR2 in the recycling or degradation pathway in HEK293 cells treated with or without FSTL1 (100 ng/ml). l CCR2 expression was evaluated via western blotting after NF-κB inhibitor (bortezomib) treatment. EE, early endosome; RE, recycling endosome; LE, late endosome. **p < 0.01. Statistical significance was determined by a two-tailed unpaired t-test (g). Data are presented as the mean ± SEM and were pooled from at least three independent experiments