Journal: Cell Death & Disease
Article Title: FAM3A drives uncoupling of muscle lipid accumulation and insulin resistance depending on insulin receptor
doi: 10.1038/s41419-025-08298-1
Figure Lengend Snippet: a Western blot images showing the presence of FAM3A in different cellular compartments in soleus muscles and heart tissues. b Levels of the FAM3A protein along with positive controls (TSG101 and CD9) and the negative control (calnexin) in plasma-derived exosomes from patients chosen at random. c Western blot images and quantification of the levels of p-AKT (thr308) and p-insR-β (tyr1150/1151) in cells treated with different concentrations (ng/ml) of rcFAM3A for 8 h (n = 4 biologically independent samples/group; quantitative comparisons between samples were run on the same gel). d Western blot images and quantification of the expression levels of the indicated proteins in soleus muscles from the mice treated with bms536924 and fed an HFD for 10 weeks (n = 6 biologically independent animals/group; quantitative comparisons between samples were run on the same gel). e Western blot images and quantification of the expression levels of the indicated proteins in cells pre-treated with bms536924 or transfected with the insR siRNA and then stimulated with rcFAM3A (200 ng/ml; n = 4 biologically independent samples/group; quantitative comparisons between samples were run on the same gel). The lipid droplets detected by oil red O staining ( f ) and TG content ( g ) were measured in soleus muscles from the mice treated with bms536924 and fed an HFD for five weeks and the results were plotted (n = 6 biologically independent animals/group). Scale bar: 200 μm, insets: 100 μm in ( f ). C2C12 cells were treated with rcFAM3A (200 ng/ml) for 12 hours. Cellular De novo FA synthesis ( h ) and TG content ( i ) were measured and graphed (n = 3 biologically independent samples/group). j , k Body adiposity and body weight gain were measured in mice treated with bms536924 and fed an HFD for 10 weeks and the results were plotted (n = 7 biologically independent animals/group). The data are presented as the means ± SEMs. Statistical significance was determined with a two-tailed independent t test, and P values are indicated ( ns P ≥ 0.05). Source data are provided as a file.
Article Snippet: Anti-FAM3A (Origene, #TA324017; 1:1000), anti-adiponectin (Cell Signaling Technology, #2789; 1:1000), anti-PPARα (Santa Cruz, #sc-398394; 1:500), anti-srebp1 (Santa Cruz, #sc-13551; 1:500), anti-fas (Cell Signaling Technology, #3180; 1:1000), anti-acly (Cell Signaling Technology, #4332; 1:1000), anti-acc (Cell Signaling Technology, #3676; 1:1000), anti-p65 (Santa Cruz, #sc-8008; 1:500), anti-pcna (Santa Cruz, #sc-25280; 1:500), anti-p-AKT (thr308) (Cell Signaling Technology, #9275; 1:1000), anti-AKT (Cell Signaling Technology, #9272; 1:1000), anti-p-insR-β (tyr1150/1151) (Cell Signaling Technology, #3024; 1:1000), anti-insR-β (Cell Signaling Technology, #3025; 1:1000), anti-Calnexin (Abcam, #ab75801; 1:1000), anti-TSG101 (Abcam, #ab125011; 1:1000), anti-CD9 (Abcam, #ab92726; 1:1000), anti-β-actin (LABLEAD, #A0101; 1:4000), anti-GAPDH (Cell Signaling Technology, #5174; 1:4000), and HRP-conjugated secondary antibodies were commercially obtained (Supplementary Table ).
Techniques: Western Blot, Muscles, Negative Control, Clinical Proteomics, Derivative Assay, Expressing, Transfection, Staining, Two Tailed Test
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![Effects of Pas2r12 or Pas2r12–cargo protein complex-mediated stimulation on <t>INSR/IGF1R</t> and ERK1/2. HEK293 cells were pretreated with dimethyl sulfoxide (linsitinib [−]) or with linsitinib (linsitinib [+]) and subsequently stimulated with Pas2r12, Pas2r12–EGFP, Pas2r12–IgG, or insulin for 2 min ( A – C ) or 10 min ( D – F ). Panels A and D show representative Western blot images. Panels B and E display the phosphorylation levels of INSR/IGF1R (pINSR/pIGF1R); panels C and F show the phosphorylation levels of ERK1/2 (pERK1/2); all values were normalized to GAPDH. Phosphorylation levels were analyzed and compared with the solvent control (DMEM only, linsitinib [−]) using Student’s t -test. For each treatment condition, linsitinib (+) was compared with the corresponding linsitinib (−) condition. Statistically significant differences compared with the control are indicated on the bar graphs; significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Statistical comparisons were performed against control cells using Student’s t -test. Error bars indicate the standard error of the mean (SEM). * p < 0.05 and ** p < 0.01. N = 4.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6253/pmc12736253/pmc12736253__pharmaceuticals-18-01885-g001.jpg)
![Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in IGF1R-overexpressing cells. ( A ) HEKI#66 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Level of phosphorylated INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R, normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated HEKI#66 control (linsitinib [−]), and, for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, *** p < 0.001. N = 3.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6253/pmc12736253/pmc12736253__pharmaceuticals-18-01885-g005.jpg)
![Effects of Pas2r12 or Pas2r12–cargo protein complex stimulation on INSR/IGF1R in INSR-overexpressing cells. ( A ) IN#1 cells pretreated with DMSO (linsitinib [−]) or linsitinib (linsitinib [+]) were stimulated with Pas2r12, Pas2r12–EGFP, or insulin for 2 min. Phosphorylation of INSR/IGF1R (pINSR/pIGF1R) was assessed by Western blot. ( B ) Relative levels of pINSR/pIGF1R were normalized to GAPDH, corresponding to the data in panel A. Phosphorylation levels were analyzed using Student’s t -test. Each treatment condition was compared with the solvent-treated IN#1 control (linsitinib [−]), and for each treatment, phosphorylation levels in the linsitinib (−) and linsitinib (+) conditions were also compared. Statistically significant differences compared with the solvent-treated control are indicated on the bar graphs, and significant differences between linsitinib (+) and linsitinib (−) treatments are marked by horizontal lines. Error bars indicate the standard error of the mean (SEM). * p < 0.05, ** p < 0.01, and *** p < 0.001. N = 3.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6253/pmc12736253/pmc12736253__pharmaceuticals-18-01885-g008.jpg)
