(A) Generation and characterization of stable C3aR1 KD cell lines. Shown is qPCR analysis of C3aR1 mRNA in 3 independent shRNA lines in comparison with the scramble shRNA C3aR1 KD cell line expressing normal leptin and PPAR-γ levels and normal differentiation using oil red O staining.
(B) Free glycerol release in C3aR1 KD and control cells. C3aR1 KD prevents TLQP-21 (100 nM) potentiation of isoproterenol (ISO, 50 nM)-induced lipolysis (cell line x treatment F(3,58) = 5.07, p < 0.005, N = 4–14).
(C) Western blot analysis of pERK and pHSL in C3aR1 KD and control adipocytes incubated for 5 min with ISO (50 nM) or TLQP-21 (10 μM). C3aR1 KD prevented TLQP-21-induced phosphorylation of HSL (cell line × treatment F(3,24) = 4.7, p < 0.01, N = 4) and ERK (cell line × treatment F(3,24) = 26.1, p < 0.00001, N = 4).
Tukey’s post hoc tests; NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Data are expressed as average and SEM.
See also Figure S1.