Fig 1: Inhibition of autophagy attenuated FGF21‐induced foam cell cholesterol efflux. A, The foam cells were treated with FGF21 (200 ng/mL) for 24 h with a 3MA (5 mmol/L) pre‐treatment. Oil Red O staining was carried out to determine cholesterol accumulation. Scale bar, 20 μm. The stained cells were examined using light microscopy (400×). B, The foam cells were transfected with scrambled (neg) or ATG5 siRNA and then incubated with 200 ng/mL FGF21 for 24 h. Oil Red O staining was carried out to determine cholesterol accumulation. The stained cells were examined using light microscopy. Scale bar, 30 μm. Measurements were taken at least three times with similar results. C, The foam cells were treated with or without 5 mmol/L of 3MA (for 6 h) pre‐treatment or transfected using scrambled (neg) or ATG5 siRNA, followed by incubation with 200 ng/mL of FGF21 for 24 h. Cholesterol efflux assay was then performed. Results are presented as mean (±SD) of 3 experiments (one‐way ANOVA). **P < .01 vs control; &P < .05 vs FGF21 + 3MA; #P < .05 vs FGF21 + ATG5 siRNA
Fig 2: FBXW11 mediated FGF21 regulation of OPA1 degradation.A Representatives immunoblot images and quantification of the OPA1, and FGF21 in siNC or siFGF21 with or without CHX (20 μg/ml, 6 h) treated HL-1 cells. n = 3 per group. B Representatives immunoblot images and quantification of the OPA1, and FGF21 in siNC or siFGF21 with or without MG132 (100 μM, 6 h) treated HL-1 cells. n = 3 per group. C Representatives immunoblot images and quantification of the OPA1, FBXW11, and FGF21 in adCon or adFGF21 (transfected 24 h) treated HL-1 cells with or without CoCl2 (200 nm, 24 h) stimulation. n = 3 per group. D Representatives immunoblot images and quantification of the OPA1, and FBXW11 in pECMV or FBXW11 plasmid (pFBXW11) treated HL-1 cells. n = 3 per group. E Representatives immunoblot images and quantification of the OPA1, and FBXW11 in siNC or siRNA targeting FBXW11 (siFBXW11) treated HL-1 cells. n = 3 per group. F, G IP analysis showed that FBXW11 could interacted with OPA1 and increased OPA1 ubiquitination. Data are expressed as the mean ± SEM, with individual data points. Data were analyzed by ordinary one-way ANOVA with Tukey’s comparisons test (A–C, E), two-tailed unpaired Student’s t test (D).
Fig 3: Fibroblast growth factor 21‐induced autophagy increased foam cell cholesterol efflux via RACK1 expression. A‐C, The foam cells were transfected using scrambled (neg) or RACK1 siRNA for 12 h and then incubated with 200 ng/mL of FGF21 for 24 h. LC3 and p62 were measured using Western blotting. D, Punctuated GFP‐LC3 protein was examined using laser scanning microscopy. Scale bar, 5 μm. E, Autophagosomes were assessed using MDC staining and fluorescence microscopy, with representative cell images at the indicated time‐points. Scale bar, 10 μm; white arrow, autophagosome. F, Autophagosomes were assessed using TEM, with images of representative cells shown from indicated time points. Scale bar, 0.5 μm; white arrow, autophagosome. G, Cholesterol accumulation was determined using Oil Red O staining, followed by light microscopic examination. Scale bar, 15 μm; original magnification, 400×. Measurements were conducted three or more times and gave similar results. H, Effect of RACK1 siRNA on FGF21‐induced cholesterol efflux in foam cells. All results are presented as means (±SD) of 3 experiments (one‐way ANOVA). **P < .01 vs controls; #P < .05 vs FGF21 + RACK1 siRNA
Fig 4: The role of RACK1 in FGF21‐mediated autophagy. ApoE−/− mice were fed a HFD with FGF21 or FGF21 + Ad‐RACK1 shRNA for 12 wk. A‐C, The protein expression levels of LC3, beclin‐1 and p62 protein were detected using Western blotting. Data are presented as mean ± SD of 3 experiments (one‐way ANOVA). **P < .01 vs AS3. D, E, Representative images of H & E and Oil Red O staining of an aortic lesion. Scale bar, 50 μm. Data are expressed as mean ± SD (n = 10 per group, SNK post hoc multiple comparison tests); *P < .05 vs HFD group; #P < .05 vs AS3 + LV‐RACK1 shRNA
Fig 5: Overexpression of OPA1 restore the excess mitochondrial fission caused by FGF21 deficiency or under CoCl2 stimulation.A, B Representative confocal images and quantification of MitoTracker and OPA1 staining of siFGF21 HL-1 cells then transfect OPA1 plasmid (pOPA1) and HL-1 cells transfected pOPA1 then treated with CoCl2 (200 nm 24 h). C, D Quantification of mitochondrial network, branch length, and network branches was analyzed by MiNA in ImageJ. Summary statistics for all cells (30 cells from 5 experiments were analyzed, 10 cells per experiment), box plots show median (horizontal lines), first to third quartile (box), and the most extreme values (vertical lines). E Representative oxygen consumption curves in siFGF21 HL-1 cells then transfect OPA1 plasmid (pOPA1) and HL-1 cells transfected pOPA1 then treated with CoCl2 (200 nm 24 h). Basal respiration rate was measured followed by proton leak after the addition of oligomycin (1.5 μM), maximal respiration was measured after the addition of FCCP (0.5 μM), and non-mitochondrial respiration after the addition of rotenone and antimycin (0.5 μM). F, G Quantification analysis of maximal respiratory and spare capacity. n = 5 per group. H, I Representatives immunoblot images and quantification of the OPA1, PINK, PARKIN, and p-PARKIN in siFGF21 HL-1 cells then transfect OPA1 plasmid (pOPA1) and HL-1 cells transfected pOPA1 then treated with CoCl2 (200 nm 24 h). n = 3 per group. Data are expressed as the mean ± SD (E) and the mean ± SEM, with individual data points (B, F, G, I). Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons test (B–D, F, G, I).
Supplier Page from Abcam for Recombinant Mouse FGF21 protein