Fig 2: MEK1 keeps SIRT1 protein stability through proteasomal degradation inhibitory(A) We co-cultured proteasome inhibition, MEK1 inhibition or knockdown liver CSCs with CHX (10 ng/ml) for indicated times. Western blots analyzed expression of SIRT1. Grey level was measured triplicated independently. (B) Analysis of SIRT1 expression in liver CSCs by western blots. MEK1 deletion or inhibition (U0126, 5 μM) CSCs was cultured for 48 hours, then combined with or without Proteasome inhibitor (MG132, 10 μM) for 8 hours, before harvested. Those proteins were compared with CSCs of DMSO treatment with or without MG132. Grey level was measured and marked. (C) MEK1 inhibition or knockdown CSCs (Huh7- and PLC/PRF/5-NanogPos cells) were treated with 10 μM MG132 for 8 hours before harvest. Total protein extracts from an equivalent number of seedlings were prepared for Co-IP in same conditions and analyzed using immunoblot with the Poly-Ub antibody.

Fig 3: MEK1/2 deletion in mature osteoblasts promotes bone formation in mice.(A) Map2k1 (MEK1) and Map2k2 (MEK2) mRNA levels in the tibial bones of Map2k1fl/fl;Map2k2+/+ (WT) and Map2k1fl/fl;Dmp1Cre;Map2k2−/− (dKODmp1) mice were measured by RT-PCR analysis. (B, C) MicroCT analysis showing femoral bone mass in 8-week-old Map2k1fl/fl;Map2k2+/+ (WT), Map2k1fl/fl;Dmp1Cre;Map2k2+/+ (Map2k1fl/fl;Dmp1Cre), Map2k1fl/fl;Map2k2−/− (Map2k2−/−), and Map2k1fl/fl;Dmp1Cre;Map2k2−/− (dKODmp1) mice. 3D reconstruction (B) and the relative quantification (C) are displayed. Trabecular bone volume/total volume (Tb. BV/TV), trabecular thickness (Tb. Th), trabecular number Tb. (N), and cortical thickness (Ct.Th). Scale bar, 1 mm (B). MicroCT analysis of 8-week-old WT and dKODmp1 skull (D) and vertebrae (E). Quantification of skull thickness Th (D), and trabecular bone mass (E, bottom) and 3D reconstruction of lumbar 4 (L4) vertebrae (E, top) are shown. Scale bar, 200 µm (E). (F) Biomechanical properties of 8-week-old WT and dKODmp1 femurs, including maximum moment, load at failure, and bending stiffness were quantified using three-point bending test. (G) Dynamic histomorphometry analysis of 8-week-old WT and dKODmp1 femurs. Quantification (top) and images of calcein/alizarin labeling (bottom) are displayed. BFR/BS, bone formation rate per bone surface; MAR, mineral apposition rate. Scale bar, 50 µm (bottom). (H, I) MicroCT analysis of femoral bone mass in 17-week-old Map2k1fl/fl;Map2k2−/− and Map2k1fl/fl;BglapCreErt;Map2k2−/− mice treated with vehicle or tamoxifen; vehicle-treated Map2k1fl/fl;Map2k2−/− (Ctrl) and Map2k1fl/fl;BglapCreErt;Map2k2−/− (Bglap-Ctrl) mice, tamoxifen-treated Map2k1fl/fl;Map2k2−/− (Ctrl) and Map2k1fl/fl;BglapCreErt;Map2k2−/− (dKOBglap) mice. 3D reconstruction (H) and the relative quantification (I) are displayed. Scale bar, 1 mm (H). (J) TUNEL (top) and hematoxylin and eosin (H&E) (bottom)-stained longitudinal sections of 8-week-old WT and dKODmp1 femurs. Scale bars, 50 µm (top) and 20 µm (bottom). (K) Numbers of osteocytes/bone area (N.Ot/B.Ar) and empty lacunae in the cortical bones of 8-week-old WT and dKODmp1 femurs. (L) Transmission electron microscopy (TEM) images of osteocytes in the cortical bones of 8-week-old WT and dKODmp1 femurs. 1, Ot in osteoids; 2, Ot in mineralized bone matrix; 3, apoptotic Ot in bone matrix. Ot, osteocyte; Oc, osteoclast; Ob, osteoblast. Scale bar, 5 µm. Data are representative of three independent experiments (A, B, E) [top], (G) [bottom], (H, J, L) or pooled from two experiments C, D, E [bottom], F, G [top], (I, K). A two-tailed unpaired Student’s t-test for comparing two groups (A, D–G, I, K) or ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (C) (A, C–G, I, K; error bars represent the standard deviation [SD of biological replicates]). Figure 2—source data 1.Source data for Figure 2A, C–G1, K.

Fig 4: mTORC2/SGK1 activation is important for MEK1/2 deficiency-induced osteoblast differentiation.(A) Immunoblot analysis showing phosphorylation levels of mechanistic target of rapamycin (mTOR) signaling components in Ctrl and dKO Obs. GAPDH was used as a loading control. (B–D) Ctrl and dKO Obs expressing sh-Scr or sh-Sgk1 shRNAs were cultured under osteogenic conditions. Alkaline phosphatase (ALP) activity (B), osteogenic markers expression (C), and mineralization (D) were assessed after 6 or 18 days of culture, respectively. (E) Conditioned medium (CM) collected from Ctrl and dKO Obs expressing sh-Scr or sh-Sgk1 shRNAs 6 days after osteogenic culture was added to transwell migration of mouse endothelial cells (EPOCs) and migrated cells were assessed 12 hr after incubation. (F) Mouse bone marrow-derived mesenchymal stromal cells (BMSCs) were cultured under osteogenic condition in the presence of CM, and ALP activity and Cola1 mRNA level were analyzed at day 6 of the culture. (G) Vehicle- or trametinib-treated Obs expressing sh-Scr or sh-Sgk1 shRNAs were treated with Mitotracker or TMRE 4 days after osteogenic culture and mitochondrial mass and membrane potential were assessed using flow cytometry, respectively. (H) Oxygen consumption rate (OCR) in Ctrl and dKO Obs expressing sh-Scr or sh-Sgk1 shRNAs after 6 days of culture. (I) Diagram showing the molecular actions of extracellular signal-regulated kinase (ERK) on bone formation. Data are representative of two or three independent experiments (A–H). A two-tailed unpaired Student’s t-test for comparing two groups (B–D) or ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test (E, F, H; B–F, H; error bars, standard deviation [SD] of biological replicates). Figure 6—source data 1.Full immunoblots for Figure 6A. Figure 6—source data 2.Source data for Figure 6B–F, H.

Fig 5: MEK1 signaling activity is required for the maintenance of liver CSC self-renewal(A) Huh7-NanogPos cells were co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) in sphere-forming conditions for 7 days, counted at the same magnification. (B) Huh7-NanogPos cells were treated with different concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) and grown for 14 days. Cells were stained with crystal violet and counted. (C) Western blot analysis of stemness protein expression in Huh7- and PLC/PRF/5-NanogPos cells, which co-cultured with various concentrations of U0126 (0 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) for 48 hours. (D) Huh7-NanogPos cells were treated with 5 μM U0126 for 14 days, while the negative control treated with DMSO for 14 days. Then we subcutaneous injected 1 × 102, 1 × 103, 1 × 104 cells into NOD-SCID mice. After 30 days, we harvested and counted the tumors. Extreme Limiting Dilution Analysis was acquired from http://bioinf.wehi.edu.au/software/elda/.