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Figures
- Fig. 1.
Stk40-KO enhances the adipocyte differentiation of MEFs and fetal liver stromal cells. (A) Oil Red O staining of MEFs (passage <4) derived from E14.5 WT or KO embryos after 8 days of differentiation. DMSO was used as a vehicle; MD, IBMX plus dexmathasone; MDI, IBMX , dexmathasone and insulin. The cells of each genotype were pooled from at least ten embryos. WT, Stk40 wild type; KO, Stk40 knockout. Scale bars 100 µm. (B) mRNA levels of adipogenic markers increased Stk40 in KO MEFs at day 8 after MDI induction. Results are mean±s.d. **P<0.01; ***P<0.001 (Student's t-test). (C) Oil Red O staining of fetal liver stromal cells derived from E14.5 WT or KO embryos after 12 days of differentiation. Scale bars: 100 µm. (D) Oil Red O staining of MEFs after 8 days of differentiation. Stk40 overexpression represses adipocyte differentiation in Stk40-KO MEFs. MOI, multiplicity of infection. (E) mRNA levels of adipogenic markers decreased in Stk40-KO MEFs with Stk40 overexpression (KO/Stk40) after 8 days of differentiation. Results are mean±s.d. **P<0.01; ***P<0.001 (Student's t-test).
- Fig. 2.
Stk40 knockdown enhances adipocyte differentiation in mouse BM MSCs and C3H10T1/2 cells. (A) Knockdown of Stk40 in BM MSCs. Ctrli, control short hairpin RNA (shRNA); Stk40i, Stk40 shRNA. α-tubulin was used as a loading control. Positions of protein molecular mass markers are indicated on the right. (B) Oil Red O staining of BM MSCs after 9 days of differentiation. Scale bars: 50 µm. (C) mRNA levels of adipogenic markers were increased in Stk40-KD BM MSCs at the indicated time points. Results are mean±s.d. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). (D) Oil Red O staining of C3H10T1/2 cells after 8 days of differentiation with or without BMP4 pretreatment. (E) The relative mRNA levels of Stk40 in control or Stk40-KD C3H10T1/2 cells treated with or without BMP4 treatment after 8 days of differentiation. Results are mean±s.d. ***P<0.001 (Student's t-test). (F) mRNA levels of adipogenic markers were increased in Stk40-KD C3H10T1/2 cells after 8 days of differentiation. Results are mean±s.d. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). (G) Stk40 KD did not promote adipocyte differentiation of 3T3-L1 cells. Oil Red O staining was performed after 8 days of differentiation. Scale bars: 50 µm. (H) The relative mRNA levels of Stk40 and adipogenic markers in 3T3-L1 cells at indicated time points. Results are mean±s.d.
- Fig. 3.
C/EBPβ is indispensible for Stk40-deficiency-caused enhancement of adipogenesis. (A) Protein levels of adipogenic markers increased in Stk40-KO MEFs at the indicated time points. LAP*, full-length active C/EBPβ (36 kDa); LAP, active C/EBPβ (34 kDa); LIP, truncated C/EBPβ (19 kDa); +/+, Stk40 wild type; −/−, Stk40 knockout; MDI, IBMX+dexmathasone+Insulin. α-tubulin was used as a loading control. Positions of protein molecular mass markers are indicated on the right. (B) Protein levels of C/EBPβ and C/EBPδ decreased in MEFs with Stk40 overexpression. α-tubulin was used as a loading control. (C) Knockdown of C/EBPβ in Stk40-KO MEFs by lentiviral-mediated C/EBPβ short hairpin RNA (C/EBPβi). α-tubulin was used as a loading control. (D) Oil Red O staining of Stk40-KO MEFs after 8 days of differentiation. C/EBPβ knockdown abrogated increased adipocyte differentiation in Stk40-KO MEFs. −/−, Stk40 knockout; Ctrli, control shRNA; C/EBPβi, C/EBPβ shRNA. Scale bars, 50 µm. (E) Protein levels of adipogenic markers increased in Stk40-KD C3H10T1/2 cells at indicated time points. Ctrli, control shRNA; Stk40i, Stk40 shRNA. (F) Knockdown of C/EBPβ in C3H10T1/2 cells rescued Stk40 protein expression. N.S, non-specific band recognized by Stk40 antibody as a sample loading control. Ctrli, Control shRNA; Stk40i, Stk40 shRNA; C/EBPβi, C/EBPβ shRNA. (G) mRNA levels of adipogenic genes decreased in C/EBPβ-KD C3H10T1/2 cells after 8 days of differentiation. C/EBPβ KD abolished increased adipocyte differentiation in Stk40-KD C3H10T1/2 cells. Ctrli, control shRNA; Stk40i, Stk40 shRNA; C/EBPβi, C/EBPβ shRNA. Results are mean±s.d. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). (H) Oil Red O staining of C3H10T1/2 cells treated as in G after 8 days of differentiation. Ctrli, control shRNA; Stk40i, Stk40 shRNA; C/EBPβi, C/EBPβ shRNA.
- Fig. 4.
C/EBPβ and C/EBPδ are post-transcriptionally regulated and their degradation is not impaired in Stk40-null MEFs. (A) mRNA levels of C/EBPβ and C/EBPδ decreased in Stk40-KO MEFs at the indicated time points after MDI induction. Results are mean±s.d. *P<0.05; **P<0.01; ***P<0.001 (Student's t-test). (B) Protein levels of C/EBPβ and C/EBPδ increased in Stk40-KO MEFs at the indicated time points after MDI induction. LAP*, full-length active C/EBPβ (36 kDa); LAP, active C/EBPβ (34 kDa); +/+, Stk40 wild type; −/−, Stk40-KO; MDI, IBMX+dexmathasone+insulin. α-tubulin was used as a loading control. Positions of protein molecular mass markers are indicated on the right. (C) C/EBPβ and C/EBPδ were degraded through 26S proteasome pathway. MEFs were treated with 30 µM MG132 for 4 h before harvest. +/+, Stk40 wild type; −/−, Stk40 knockout; DMSO was used as a vehicle. α-tubulin was used as a loading control. (D) The degradation of C/EBPβ and C/EBPδ was comparable in wild-type and Stk40-KO MEFs. MEFs were treated with 100 µM CHX for indicated time before harvest. CHX, cyclohexamide. (E) The half-life of C/EBPβ and C/EBPδ proteins was comparable in wild type and Stk40-KO MEFs as measured from blots for the experiments shown in D. The gray density of blots was measured by software ImageJ. The levels of C/EBPβ and C/EBPδ at 0.5 h after cyclohexamide treatment were set as 100. LAP*, full length active C/EBPβ (36 kDa); LAP, active C/EBPβ (34 kDa). Results are mean±s.d.
- Fig. 5.
Cap-dependent translation of C/EBPs is enhanced in Stk40-KO and -KD cells. (A) The levels of proteins involved in protein translation in Stk40-KO MEFs. Phosphorylation of 4E-BP1 and S6K1 was increased in Stk40-KO MEFs. α–γ isoforms represent the phosphorylation status of 4E-BP1; α, hypo-phosphorylated isoform; β, intermediate-phosphorylated isoform; γ, hyper-phosphorylated isoform. Thr37/46, Ser65 and Thr70, antibodies against 4E-BP1 phosphorylated at indicated sites. α-tubulin was used as a loading control. (B) The levels of proteins involved in protein translation in Stk40-KD C3H10T1/2 cells. Phosphorylation of 4E-BP1 and S6K1 was increased in Stk40-KD (Stk40i) C3H10T1/2 cells compared with control (Ctrli, control shRNA). α-tubulin was used as a loading control. (C) Stk40 overexpression abolished the increased phosphorylation of 4E-BP1 in Stk40-KO MEFs. GFP or Stk40 was delivered by retroviral vectors. α-tubulin was used as a loading control. (D) Stk40 overexpression abolished the increased phosphorylation of 4E-BP1 in Stk40-KD C3H10T1/2 cells. GFP or Stk40 was transduced by retroviral vectors. α-tubulin was used as a loading control. (E) Cap-dependent translation assays were conducted with a dual Renilla and firefly luciferase system with the human hepatitis C virus IRES driving firefly luciferase expression. Cap-dependent translation was increased in Stk40-KO MEFs (lower left) and Stk40-KD C3H10T1/2 cells (lower right). Cassettes of the dual Renilla and firefly luciferase plasmid were indicated in schema (upper). Results are mean±s.d. *P<0.05; ***P<0.001 (Student's t-test). (F) Protein levels of C/EBPβ and C/EBPδ decreased after eIF4E KD in C3H10T1/2 cells. α-tubulin was used as a loading control. (G) mRNA levels of C/EBPβ and C/EBPδ did not alter after eIF4E KD in C3H10T1/2 cells. Results are mean±s.d.
- Fig. 6.
Fetal organs of Stk40-KO mice display enhanced adipogenic gene expression and 4E-BP1 phosphorylation. (A) Venn diagram of differentially expressed genes (DEGs) in Stk40-KO lungs and in Stk40-KO livers at E18.5. FC, fold change. (B) Gene ontology (GO) analyses of DEGs from a ranking by enrichment scores. Adipogenesis- or lipid-related terms are indicated by asterisks. Enrichment scores were calculated as −log10 (P-value). (C) mRNA levels of adipogenic markers in Stk40-KO livers were increased. +/?, Stk40 wild-type or heterozygous, n=8; −/−, Stk40 knockout, n=8. Results are mean±s.d. ***P<0.001 (Student's t-test). (D) The protein levels of 4E-BP1 and S6K1 in livers at E18.5. Adipocyte marker aP2 as well as phosphorylation of 4E-BP1 (p4E-BP1) and S6K1 (pS6K1) increased in Stk40-KO livers. α-tubulin was used as a loading control. (E) Relative protein levels of p4E-BP1, pS6K1 and aP2 increased in Stk40-KO livers. +/?, Stk40 wild type or heterozygous, n=15; −/−, Stk40 knockout, n=14. Results are mean±s.d. *P<0.05; ***P<0.001 (Student's t-test).
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