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. 2020 Feb 6;3(3):e201900576. doi: 10.26508/lsa.201900576

Figure S6. Role of PERK in thermogenesis.

Figure S6.

(A, B) Gross appearance (A) and haematoxylin and eosin staining (B) of interscapular BAT (iBAT) derived from PERK+/+ to PERK−/− newborn mice within 12 h after birth. (A) Freshly harvested iBAT were rinsed with PBS and macroscopic images were taken (A). (B) Isolated iBATs were fixed with 4% paraformaldehyde and embedded in paraffin, and the tissue sections were stained with haematoxylin and eosin (B). Representative images were shown in this Figure. (A, B) Scale bar, 5 mm (A) and 40 μm (B). White squares denote magnified regions. (C) Mitochondrial stress-induced phosphorylation of PERK at Ser715, Ser717, and/or Ser719 in differentiating cells. Brown preadipocytes were stimulated with 10 μM CCCP for the indicated time points on day 2. The cell lysates were analysed by immunoblotting (IB) with the indicated antibodies. An arrowhead indicates the phosphorylated PERK at Ser715, Ser717, and/or Ser719. The asterisk indicate a nonspecific band. (D) Requirement of PERK for CCCP stimulation-induced intracellular thermogenesis. siRNA-transfected brown adipocytes were injected with a nanogel thermometer, stimulated with 10 μM CCCP and observed by inverted microscopy. The intracellular temperature was analysed using ImageJ software. Data are shown as the fold change relative to the value at 0 min. P = 0.0019 (repeated measures ANOVA, sictrl, five individual cells; siPERK, five individual cells). (E) Involvement of PGC-1α in PERK-mediated mitochondrial inner membrane and cristae protein biogenesis in brown adipocytes. siRNA-transfected cells were infected with the indicated retroviruses, lysed on day 6, and analysed by IB or immunoprecipitation-IB with the indicated antibodies. The expression of Cyt C and PGC-1α were calculated and is shown as the ratio relative to the expression of actin (average band intensity, n = 3).