Journal: Nature Communications
Article Title: Deficiency of WTAP in hepatocytes induces lipoatrophy and non-alcoholic steatohepatitis (NASH)
doi: 10.1038/s41467-022-32163-w
Figure Lengend Snippet: a Heatmap of top differentiated genes encoding secreted proteins in the livers of Wtap flox/flox and Wtap- HKO mice ( Wtap- HKO fpkm > 500 and FoldChange > 4.5). Data were presented as log 10 fpkm ( n = 3 for each group). b Relative Igfbp1 mRNA levels in the liver and eWAT ( n = 8 for each group; Liver, P = 0.0014; eWAT, P = 0.547). c IGFBP1 protein levels in the liver and eWAT were measured by immunoblotting ( n = 4 for each group). The samples were derived from the same experiment and the blots were processed in parallel. d Serum IGFBP1 protein levels were measured by ELISA ( n = 11 for each group; P = 0.0011). e – p Wtap- HKO mice at 7 weeks old were intravenously injected with a control antibody (IgG) and an anti-IGFBP1 neutralizing antibody per day, respectively, for 5 days. Serum FFA levels were measured ( e ) (IgG, n = 7; Anti-IGFBP1, n = 8; P = 0.0455). p-HSL, HSL, p-PKA substrate, ATGL and GAPDH protein levels in eWAT were measured by immunoblotting and quantified by Image J ( f ) ( n = 3 for each group; p-HSL/HSL, P = 0.0445; ATGL/GAPDH, P = 0.0335; p-PKA substrate/GAPDH, P = 0.0499). cAMP levels were measured by ELISA ( g ) (IgG, n = 7; Anti-IGFBP1, n = 8; P = 0.00998). The ADCY3, ADCY4 and ADCY6 protein levels in the eWAT were measured by immunoblotting and quantified by Image J ( h ) ( n = 3 for each group; ADCY3, P = 0.0009; ADCY4, P = 0.0318; ADCY6, P = 0.00918). serum ALT activity were measured ( i ) ( n = 6 for each group; P = 0.0031). Cleaved caspase 3 levels ( j ) ( n = 3 for each group; P = 0.0012), liver TAG levels ( k ) ( n = 6 for each group; P < 0.0001), hepatic lipid droplets ( l ), TUNEL-positive cells ( m , n ) ( n = 5 for each group; P = 0.0129), and cytochemokine mRNA levels ( o ) ( n = 5 for each group; Tnfα , P = 0.0294; Il1β , P = 0.0133; Il6 , P = 0.0267; iNos , P = 0.3452; Infg , P = 0.6684; Cd14 , P = 0.0111; Csf1 , P = 0.0292; Ccl2 , P = 0.0172; Ccl3 , P = 0.0824; Ccl5 , P = 0.0025; Ccl22 , P = 0.0722; Ccr2 , P = 0.0969; Cxcl2 , P = 0.0693; Cxcl5 , P = 0.092; Cxcl10 , P = 0.0747; Cx3cl1 , P = 0.1067) were measured in the liver. Serum cytokine levels were measured by ELISA ( p ) (IgG, n = 7; Anti-IGFBP1, n = 8; TNFα, P = 0.000178; IL1β, P = 0.0323; CCL2, P = 0.0456). For immunoblotting, the samples were derived from the same experiment and the blots were processed in parallel. n was the number of biologically independent mice. Data represent the mean ± SEM. Significance was determined by unpaired two-tailed Student’s t test analysis. * p < 0.05. ** p < 0.01. Source data are provided as a Source Data file.
Article Snippet: Antibody dilutions were as follows: WTAP (10200-1-AP, Proteintech), 1:2500; METTL3 (96391, Cell Signaling Technology), 1:2500; LaminB1 (12987-1-AP, Proteintech), 1:5000; Tubulin (sc-5286, Santa cruz), 1:5000; β-Actin (60008-1-Ig, Proteintech), 1:5000; GAPDH (60004-1, Proteintech), 1:5000; CDK9 (11705-1-AP, Proteintech), 1:3000; pCDK9 (2549, Cell Signaling Technology), 1:3000; CD36 (18836-1-AP, Proteintech), 1:5000; CCL2 (66272-1-Ig, Proteintech), 1:2500; FLAG (F1804, Sigma), 1:5000; Phosphoserine (AB1603, Sigma), 1:1000; MYC (16286-1-AP, Proteintech), 1:5000; ATGL (55190-1-AP, Proteintech), 1:5000; HDAC1 (5356, Cell Signaling Technology), 1:2500; ADCY3 (bs-20272R, Bioss), 1:3000; ADCY4 (bs-3921R, Bioss), 1:3000; ADCY6 (14616-1-AP, Proteintech); IGFBP1 (A11672, ABclonal), 1:3000; HSL (A15686, ABclonal), 1:5000; phospho-HSL-S563 (AP0851, ABclonal), 1:5000; Phospho-(Ser/Thr) PKA Substrate (9621, Cell Signaling Technology), 1:3000; and Cleaved caspase 3 (9661, Cell Signaling Technology), 1:2500.
Techniques: Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Injection, Activity Assay, TUNEL Assay, Two Tailed Test
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