Fig 1: Dyrk1b is upregulated in the liver of mice with diet-induced fatty liver disease and in human NASH.(A) Dyrk1b transcripts normalized to Hprt in the indicated conditions. Henceforth, each dot in the dot plots represents biological replicates; n > 8 mice each, unpaired t test, 2-tailed. (B and C) Western blot (WB) analysis and quantification of specified liver proteins in mice fed CD or HCD. Henceforth, each lane of WBs represents biological replicates; n = 4 mice each, unpaired t test, 2-tailed. (D) Correlation analysis between NASH score and Dyrk1b expression in the liver of HCD-fed mice. (E) WB and quantification of Dyrk1b levels in the nuclear (top) and cytoplasmic extracts (bottom) in the liver of mice fed with CD or HCD; n > 6 each, unpaired t test, 2-tailed. (F and G) Dyrk1b protein expression visualized by immunofluorescence in the liver of CD- or HCD-fed mice. IgG was used as control (top row); n = 5 mice each group. Arrows indicate cytoplasmic expression. Unpaired t test, 2-tailed. (H and I) Representative images and quantification of DYRK1B expression in the liver biopsies of patients with NASH versus controls; n = 20 controls, n = 27 NASH samples, unpaired t test, 2-tailed, Welch-corrected. (J) Correlation analysis between NASH score and percentage Dyrk1b expression in the human NASH samples (n = 22) from I. Scale bars: 20 µm. *P = 0.05, **P = 0.01, ***P = 0.001.
Fig 2: Dyrk1b increases membrane DAG and impairs insulin receptor activation.(A and B) Glucose (A) and insulin (B) during intraperitoneal glucose tolerance test (IPGTT) in Dyrk1bAAV-WT mice after 6-hour fast; n > 8 mice per group, 1-way ANOVA, Holm-Šidák post hoc test, P (area under the curve) = 0.0025. (C and D) Insulin tolerance test (ITT) on Dyrk1bAAV-WT (C) and Dyrk1bAAV-shRNA mice (D). After 6 hours of fasting, fast-acting Humulin (0.75 U/kg) was injected i.p., and glucose was measured at indicated time points. n > 8 mice per group, 1-way ANOVA, Holm-Šidák post hoc test. (E and F) Liver glycogen quantification in the indicated mice after fast/refeed for 6 hours; n > 13 mice each group for E, n > 9 for F, unpaired t test, 2-sided. (G) Plasma membrane sn-1,2-, sn-1,3-, and sn-2,3-DAG levels in Dyrk1bAAV-WT mice; n > 6 mice each group, unpaired t test, 2-sided. (H) Membrane PKCe levels after fractionation into membrane and cytosolic fractions; n > 6 mice each group, unpaired t test, 2-sided. (I) p-IRKT1150 enrichment by immunoprecipitation in liver lysates of the indicated mice; n > 4 mice each group, unpaired t test, 2-sided. (J and K) p-IRKY1162 in liver samples from Dyrk1bAAV-WT mice only fasted (J) or fasted and stimulated with insulin for 15 minutes (K); n > 3 mice each group, unpaired t test, 2-sided. (L) Plasma membrane sn-1,2-, sn-1,3-, and sn-2,3-DAG levels in Dyrk1bAAV-shRNA mice; n > 6 mice each, unpaired t test, 2-sided. (M) p-IRKT1150 enrichment by immunoprecipitation in the indicated liver lysates; n > 4 mice each group, unpaired t test, 2-sided. (N and O) p-IRKY1162 in liver samples from fasted (N, n = 3 mice each group) and insulin-stimulated (O, n > 5 mice each group) Dyrk1bAAV-shRNA mice; unpaired t test, 2-sided. *P = 0.05, **P = 0.01.
Fig 3: Analysis of globally altered proteomes in the liver upon changes in Dyrk1b levels.(A and B) Representative heatmaps displaying hierarchical clustering of significantly altered proteome in the liver of designated mice; n = 2 are displayed for each condition; n = 3 biological replicates were analyzed for each genotype. The fold changes relative to respective controls were calculated, and the significantly altered proteins were determined using the following thresholds: P < 0.05, unpaired t test, FDR < 0.05, Benjamini-Hochberg corrected. The heatmaps were generated by Qlucore Omics software. The tissues were processed by liquid chromatography–tandem mass spectrometry. (C and D) The altered functions predicted by IPA for Dyrk1bAAV-WT (C) and Dyrk1b–/– (D). (E and F) Top selected upstream regulators of proteins that are altered in Dyrk1bAAV-WT (E) and Dyrk1b–/– (F) liver predicted by IPA.
Fig 4: Knockdown of Dyrk1b protects against hepatic steatosis and hyperlipidemia.(A) Schematic representation of knockdown of Dyrk1b using shRNAs targeted to the liver by AAV8. (B–D) Dyrk1b expression in Dyrk1bAAV-shRNA versus scrambledAAV visualized by HRP-mediated IHC in the liver (B and C) and WB (D); n > 5 mice each for B and C. The mice were fed HCD for 3 months. Unpaired t test, 2-tailed. (E–G) ORO staining, counterstained with hematoxylin, and total hepatic TAG (G) in the liver of indicated mice, fed HCD for 3 months and fasted for 6 hours; n > 10 each, unpaired t test, 2-tailed. (H) Plasma TAG after 6-hour fast in Dyrk1bAAV-shRNA versus scrambledAAV mice; n > 7 each, unpaired t test, 2-tailed. (I) Primary hepatocytes transduced by AAV8 containing either scrambled or Dyrk1b shRNA at an MOI of 60; n = 11 per group, unpaired t test, 2-tailed. Scale bars: 150 µm. *P = 0.05.
Fig 5: Dyrk1b increases FA uptake in the hepatocytes.(A–H) Representative confocal images from littermate controls and Dyrk1b–/– hepatocytes at indicated time points after addition of BODIPY-FA; n = 5 mice each genotype, n = 4 technical replicates. (I) Quantification of intracellular fluorescence in controls and Dyrk1b–/– hepatocytes measured by the microplate reader after cell lysis at the indicated time points. The background fluorescence (prior to addition of BODIPY-FA) was subtracted and normalized to the total protein content. Unpaired t test, 2-sided, n = 5 mice each genotype, n = 4 technical replicates. (J–L) Representative confocal images showing BODIPY-FA uptake 1 minute after addition in Dyrk1b–/– hepatocytes transduced with AAV8 containing empty vector (AAVcontrol) or Dyrk1bWT or Dyrk1bkin.def virus, at an MOI of 60. The FA uptake experiment was performed 72 hours after virus transduction, to allow for sufficient transcription. n = 2 mice each condition, n = 4 technical replicates. (M) Quantification of intracellular fluorescence, in the indicated genotypes; 1-way ANOVA, Tukey’s post hoc test. White arrows indicate the intracellular fluorescence detected by BODIPY-FA. Scale bars: 150 µm. **P = 0.01, ***P = 0.001.
Supplier Page from Abcam for Anti-Dyrk1B/MIRK antibody [EPR5617] Blocking peptide