Menin-regulated Pbk controls High fat diet-induced compensatory beta cell proliferation (2021)
Figure 1

Figure 1. Pbk kinase expression is upregulated in pancreatic beta cells of HFD-induced diabetic mice with beta cell compensatory proliferation

A.Volcano plot showing the fold change (y-axis) versus adjusted (adj.) p-value (x-axis) of the beta cell transcriptomes between chow and HFD-fed mice (16 weeks). Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj. P value < 0.01 (two-tailed paired student's t-test). Genes with >10 fold upregulated expression are labeled with their name. Pbk gene expression is indicated by the arrow. RNAseq data was available from https://doi.org/10.1371/journal.pone.0213299.t004.

B. qPCR for detecting Pbk mRNA level in different metabolically active organs from DIO and control mice (n=3 per group). **P = 0.0029 (islets), ***P = 0.0007 (Muscle) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

C. PBK mRNA level comparisom in human islets from obese (BMI>30) or lean donors (BMI<25) (n=3 per group). *P = 0.0261 (two-tailed unpaired student's t-test).

D. Representative images of insulin (green) and Pbk (red, indicated by white arrow) double immuno-staining in pancreas of DIO mice with 12 weeks HFD feeding (60% fat diet, n=4) or age-matched control mice (10% fat diet, n=4). Nuclei were labeled by DAPI (blue). Islet area was circled with white dashed line. Scale bar: 50 μm.

E. Quantification of the percentage of Pbk positive beta cells. Four mice for each group, and 10 islet images per mouse were analyzed. *P = 0.0150 (two-tailed unpaired student's t-test).

F. WB data showed an upregulation of Pbk expression level in islets of mice on HFD compared with that of control lean mice.

G. Beta cell mass comparison between DIO and control mice (n=4 per group). *P = 0.0419 (two-tailed unpaired student's t-test).

H. Representative images of insulin (green) and BrdU (red, indicated by white arrow) double immuno-staining in pancreas of DIO mice with 12 weeks HFD feeding (60% fat diet, n=4) or age-matched control mice (10% fat diet, n=4). Nuclei were labeled by DAPI (blue). Islet area was circled with white dashed line. Scale bar: 50 μm.

I. Quantification of the percentage of BrdU positive β-cells. Four mice for each group, and 5-10 islet images per mouse were analyzed. *P = 0.0230 (two-tailed unpaired student's t-test).

J. The representative image of co-staining of Pbk and BrdU in pancreatic sections. Islet area was circled with white dashed line. Pbk and BrdU co-staining are denoted by white arrows. Scale bar: 50 μm.

K. Quantification of the percentage with pbk and BrdU co-staining cells among total Pbk positive cells in HFD-fed mouse islets. The mean value of four mice from 5 sections for each mouse were presented.

L. Pbk KD in INS-1 cells suppresses cell growth. Western blot data showed the decreased Pbk expression level in INS-1 cells with Pbk-targeted shRNA transduction. Cell growth curve was from three independent experimets (n=3). ***P < 0.0001 (Vector:shRNA-#1), *P = 0.0181 (Vector:shRNA-#5) , (Two way ANOVA).

M. The role of ectopic expression of Pbk-WT and Pbk-Mut on INS-1 cell growth. Western blot data showed the overexpression of V5-flaged wild type and mutant Pbk in INS-1 cells. Cell growth curve was from three independent experimets (n=3). **P = 0.0069 (Two way ANOVA). ns, not statistically significant difference.

N. Cell cycle analysis via PI staining followed by flow cytometry for the cells with Pbk overexpression. PIME cells streadily transduced with vector, Pbk-WT, and Pbk-Mut expression plasmids.

O. Purified recombinant His-Erk2 protein incubated with the purified PBK in the presence or absence of ATP (100 μM ) or menin inhibitor (1 μM or 0.1 μM), as indicated. After SDS-PAGE, the phosphorylation of Erk2 was detected with the pERK1/2 antibody. Anti-ERK1/2 antibody showed that equal amount of Erk2 was used for the kinase assay.

P. Purified recombinant His-Erk2 proteins were incubated with IP-ed Pbk from PIME cells (WT or menin-/-) for the kinase activity assay. The phosphorylation of Erk2 was detected with the anti- pERK1/2 antibody. The Western blot incubated with the anti-ERK1/2 antibody showed that an equal amount of Erk2 was loaded for the kinase assay.

Data information: Data are represented as mean ± SEM.