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. Author manuscript; available in PMC: 2021 Sep 17.
Published in final edited form as: Mol Cell. 2020 Aug 20;79(6):934–949.e14. doi: 10.1016/j.molcel.2020.08.002

Figure 6. Interplay between H2B Glu35 PARylation and Ser36 phosphorylation controls adipogenesis.

Figure 6.

(A) Expression of the adipocyte marker genes Pparg and Adipoq in 3T3-L1 cells expressing Dox-inducible Wt, S36A mutant, or E35A mutant H2B during a time course of differentiation with MDI, as determined by RT-qPCR. Ectopic H2B expression was induced at differentiation day 1. Each point represents the mean + SEM (n = 7). Asterisks indicate significant differences from the corresponding Wt (two-way ANOVA followed by Dunnetts’s multiple comparisons test; ** p < 0.01, and **** p < 0.0001).

(B) Oil red O staining of lipid droplets in 3T3-L1 cells expressing Dox-inducible Wt, S36A mutant, or E35A mutant H2B after 6 days of differentiation with MDI. Ectopic H2B expression was induced at differentiation day 1.

(C) Western blots showing the levels of H2B-Ser36p, PAR, and PARP-1 in visceral white adipose tissue (WAT) from C57BL/6 wild type (Wt) or Parp1 null (Parp1−/−) mice. β-actin was used as a loading control.

(D) Quantification of the H2B-Ser36p levels shown in (C). Each bar represents the mean + SEM (n = 6). Asterisks indicate significant differences from Wt (Student’s t-test; ** p < 0.01).

(E) Imaging of fresh visceral WAT from Dox-inducible Mural Chaser (PdgfrbrtTA; TRE-Cre; Rosa26RmT/mG) Parp1+/+ or Mural Chaser Parp1loxp/loxp mice treated with Dox for 9 days, followed by 4 weeks of high-fat/high sucrose diet. (Top panels) Representative confocal fluorescence images collected at 10x magnification. Membrane-anchored Tomato fluorescent protein is shown as red. Membrane-anchored GFP fluorescent protein is shown as green. Scale bars represent 50 μm. (Bottom panels) Representative images collected at 63x magnification of immunohistochemical staining of H2B-Ser36p in visceral WAT. Scale bars represent 50 μm.

(F) Quantification of the percent of newly formed adipocytes from the Pdgfrβ+ preadipocytes shown in top panels of (E). Each bar represents the mean + SEM (n = 9 in Parp1+/+ group, and n = 6 in Parp1loxp/loxp group). Asterisks indicate significant differences from Parp1+/+ (Student’s t-test; ** p < 0.01).

[See also Figure S7]