Fig. 4.

SINGD contributes to β cell dysfunction in diabetes. a Immunoblot of indicated proteins using lysates of isolated human islets treated with DMSO or PKD inhibitor CID755673 (30 μM) for 24 h. GAPDH was used as a loading control. Quantification of Proinsulin/GAPDH and Insulin/GAPDH ratios, *P < 0.05, N = 3, one-tailed, paired t-test. b Quantitative RT-PCR using mRNA isolated from pancreatic islets of 8-week-old BTBR +/+ and BTBR ob/ob mice using primer pairs amplifying cDNA of PKD1, PKD2 and PKD3. The expression values were normalized to housekeeping genes HPRT and GAPDH. N_+/+ = 8; N_ob/ob = 5; *P < 0.05, two-tailed t-test. c Immunoblot of PKD using lysates of islets isolated from 8-week-old BTBR +/+ and BTBR ob/ob mice. Tubulin was used as a loading control. Quantification of PKD/Tubulin ratio, *P < 0.05, N = 3 per group, two-tailed t-test. d Compound A activates PKD through inhibition of p38δ. e Imaging of INS1^PGCD cells endogenously expressing Phogrin-GFP(Phogrin-GFP^endo) and CD63-DsRed (CD63-DsRed^endo) treated with Glc/Pal for 24 h in the presence of DMSO or Compound A, 100 nM. Arrowheads point to Phogrin-GFP^endo/CD63-DsRed^endo co-localization. Scale bar, 10 μm. Quantification of co-localization between CD63-DsRed^endo and Phogrin-GFP^endo using Manders coefficients. N_Glc/Pal,DMSO = 58; N_{Glc/Pal,Compound A} = 59 from three independent experiments. *P < 0.01, two-tailed t-test. f, g Immunoblots of indicated proteins using lysates of isolated human islets treated with DMSO or Compound A for 48 h. GAPDH was used as a loading control. h Immunoblot of indicated proteins using lysates of human islets isolated from a T2D donor. Islets were treated with DMSO or Compound A for 48 h. GAPDH was used as a loading control. i Time-course of fasting blood glucose levels of BTBR ob/ob mice of indicated ages implanted with osmotic pumps containing control solvent (DMSO) or Compound A. N_DMSO = 5; N_{Compound A} = 7; *P < 0.05, two-tailed t-test. j Time-course of fasting blood glucose levels of BTBR ob/ob mice of indicated ages implanted with osmotic pumps containing control solvent (50% DMSO) or CID755673. N_{50% DMSO} = 5; N_CID755673 = 5; **P < 0.01, two-tailed t-test. In a, b, c, e, i, and j data are shown as mean ± SEM, source data are provided as a Source Data file