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. 2017 Nov 27;67(2):265–277. doi: 10.2337/db17-0321

Figure 6.

Figure 6

The Clec16a-Nrdp1-USP8 complex is perturbed by glucolipotoxicity. A: Top, representative deconvolution image of Nrdp1:USP8 Proximity Ligation Assay (PLA, red; DAPI, blue) in Min6 β-cells 72 h after transfection with nontargeting (NT) or Clec16a-specific shRNA plasmids after treatment with BSA control or 0.4 mmol/L palmitate for 48 h; bottom, quantification of relative Nrdp1:USP8 PLA events in Min6 β-cells (fold change vs. NT shRNA BSA controls). (n = 4/group and ∼1,200 PLA events quantified/group per experiment.) B: Top, representative deconvolution image of Nrdp1:USP8 PLA (PLA, red; PDX1, green; and DAPI, blue) in β-cells of dissociated human islets after 48 h treatment with BSA control (BSA) or 0.4 mmol/L palmitate and 0.92% BSA + 20 mmol/L glucose (palmitate); bottom, quantification of relative Nrdp1:USP8 PLA events in β-cells of dissociated human islets after control BSA (white bar) or 0.4 mmol/L palmitate and 0.92% BSA + 20 mmol/L glucose (gray bar) treatment for 48 h (n = 3 donors and ∼900 β-cell PLA events quantified/group per donor). C: Top, representative Western blot (WB) of Nrdp1 levels in isolated mouse islets treated for 48 h with or without 0.4 mmol/L palmitate + 20 mmol/L glucose; bottom, relative quantification (by densitometry) of Nrdp1 levels by Western blot in isolated islets from mice treated for 48 h with (gray bars) or without (white bars) 0.4 mmol/L palmitate and 0.92% BSA + 20 mmol/L glucose. n = 3/group. D: Representative Western blot of in vivo ubiquitination assay of HA-Nrdp1 in Min6 cells transfected with HA-Nrdp1, Myc-tagged ubiquitin (Myc-Ub), and Flag–empty vector or Flag-Clec16a plasmids and treated for 48 h with or without 0.4 mmol/L palmitate/BSA coupling. n = 3/group. E: Representative Western blot after Flag-immunoprecipitation (IP) in Min6 cells transfected with HA-Nrdp1, Flag-USP8, and either GFP–empty vector or GFP-Clec16a plasmids and treated for 48 h with or without 0.4 mmol/L palmitate. n = 4/group. F: Top, representative WB of cleaved caspase-3 levels in Min6 cells transfected with or without GFP-Clec16a and treated for 48 h with BSA or palmitate; bottom, relative quantification (by densitometry) of cleaved caspase-3 levels (normalized to actin loading control) by Western blot in Min6 cells transfected with or without GFP-Clec16a and treated for 48 h with BSA (white bars) or palmitate (gray bars) couplings. n = 5/group. *P < 0.05, ***P < 0.001, nonpaired Student t test. ns, not significant.