Fig 1: Inactivation of SelK inhibits the myogenic differentiation progression of SCs. (A) Immunofluorescence staining of MyoD (green) and Pax7 (red) in TA muscle of Ctrl and SelK KO mice at 3 d postinjury (N = 3). Nuclei were labelled using DAPI staining. Scale bar = 20 μm. (B) Quantification of the percentages of Pax7+MyoD−, Pax7+MyoD+ and Pax7−MyoD+ cell populations in Ctrl and SelK KO TA muscles at 3 d postinjury. (C) Immunofluorescence staining of MyoD (green) and MyoG (red) in TA muscle of Ctrl and SelK KO mice at 5 d postinjury (N = 3). Nuclei were labelled using DAPI staining. Scale bar = 20 μm. (D) Quantification of the percentages of MyoD+MyoG−, MyoD+MyoG+ and MyoD−MyoG+ cell populations in Ctrl and SelK KO TA muscles at 5 d postinjury. (E) qRT-PCR analysis of myogenic transcription factors (MyoD1, MyoG, MyH1, MyH2 and MyH3) mRNA levels in TA muscle of Ctrl and SelK KO mice at 5 d postinjury (N = 3). (F) and (G) Western blotting analysis of MyoD, MyoG and MyHC protein levels in uninjured and 5 d-postinjured TA muscle of Ctrl and SelK KO mice (N = 3). The results are presented as means ± S.D. *p < 0.05, **p < 0.005, ***p < 0.001, values significantly different from the corresponding control by unpaired t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 2: Schematic diagram of the mechanism of imDCs dysfunction mediated by SELENOK. Under ERS conditions, SELENOK, SELENOS and ERS markers were upregulated, and the phenotypic markers were downregulated. The downregulation of RhoA inhibited migration and increased Ca2+ or CD205 enhanced phagocytosis in WT imDCs. In SELENOK KO imDCs, SELENOS upregulation attenuated ERS levels, decreased phenotypic markers, upregulated RhoA due to increased Ca2+, which promoted migration, increased Ca2+ or decreased CD205, which inhibited phagocytosis. imDCs, immature dendritic cells; SELENOK, selenoprotein K; ERS, endoplasmic reticulum stress; SELENOS, selenoprotein S; RhoA, Ras homolog gene family member A; CD205, cluster of differentiation 205; WT, wild-type; KO, knockout.
Fig 3: SelK is upregulated in during SCs differentiation and C2C12 myoblast differentiation. (A) Immunofluorescence staining of Pax7 (red) and SelK (green) in TA muscle of wild-type mice without 1.2% BaCl2 injection. Scale bar = 50 μm. (B) Immunofluorescence staining of Pax7 (red) and SelK (green) in TA muscle of wild-type mice at 5 d postinjury. Scale bar = 50 μm. (C) Immunofluorescence staining of MyoG (red) and SelK (green) in TA muscles of wild-type mice at 5 d postinjury. Scale bar = 50 μm. (D) Western blot analysis of SelK protein levels in uninjured and 5 d-injured TA muscle. N = 3 mice in each group for A-D. (E) Immunofluorescence staining of SelK in C2C12 myoblasts cultured in growth medium for 1 d or in differentiation medium for 2 d. Scale bar = 50 μm. (F) qRT-PCR analysis of SelK mRNA levels in C2C12 myoblasts cultured in growth medium for 1 or 2 d (G1 or G2) or in differentiation medium for 1, 3, 5 or 7 d (D1, D3, D5 or D7). (G) and (H) Western blotting analysis of SelK protein levels in C2C12 myoblasts cultured in growth or differentiation medium for 1 or 2 d (G1 or G2) or in differentiation medium for 1, 3, 5 or 7 d (D1, D3, D5 or D7). N = 3 in each group for E-H. The results are presented as means ± S.D. ***p < 0.001, values significantly different from the corresponding control by unpaired t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 4: SelK accelerates the cargo CD36 integration into COPII vesicles(A and B) HepG2 cells were infected with lentivirus expressing SelK or SelK-knockdown were set up as in Fig. 3. (A) Co-immunoprecipitations of CD36 - Sec24 in HepG2 cells (n = 3). (B) Dual-immunoflorescent staining of CD36 (green) and Sec24 (red) in cells (n = 5). (C–E) HepG2 cells were transfected with wild-type SelK lentivirus (SelK Full-length) or SelK lacking the SH3-binding domain lentivirus (SelK SH3BD TRUNC). (C) Cells were harvested and subjected to an Acyl-RAC assay (n = 3). Input and eluted fractions were separated on SDS gels and subjected to immunoblotting with anti-CD36 antibodies. (D) Cells were harvested and subjected to surface biotinylation. Equal volume of input and eluted fraction for each treatment was loaded on an SDS-PAGE gel and immunoblotted with ß-actin antibody. (E) Deletion of the SH3 binding region in SelK decreased the endogenous CD36 that are enriched in the Golgi apparatus. On Day 0, HepG2 cells transduced with SelK full-length or SelK SH3BD truncated lentivirus were set up as described in (A). On Day 2, cells were incubated with serum-free medium for 12 h followed by 24 h of 160 µM PA treatments. On Day 3, cells were subjected to immunofluorescence assay using the anti-CD36 (green) and anti-GM130 (red, left) or anti-Calnexin (red, right) antibodies. (F) Quantification of CD36 palmitoylation level in cells. (G) Quantification of surface biotinylation results of the CD36 in cells. (H) Pearson's correlation of CD36 and Golgi apparatus (left) or ER (right). All data are shown as means ± s.d., *P < 0.05, ***P < 0.001, ****P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig 5: SelK mediates the subcellular localization of CD36On Day 0, HepG2 cells were set up in 60-mm dishes. On Day 2, cells were transfected with lentivirus expressing SelK (OE SelK) or knockdown SelK (shSelK) (MOI = 10). Lentivirus vectors expressing negative controls (NC or shNC) respectively. (A) Knockdown of SelK reduced the endogenous CD36 that are enriched in the Golgi apparatus. At 36 h after the transfection, the cells were switched to serum-free medium for 12 h, followed by 24 h of PA treatment. Representative dual-immunoflorescent staining image of CD36 (green) and Golgi apparatus (left, red) or ER (right, red) in shSelK HepG2 cells. (B) The histogram of (A), data are shown as means ± s.d., **P < 0.01, ***P < 0.001. (C) Overexpression of SelK enhanced endogenous CD36 in the Golgi apparatus. Analysis of CD36 subcellular localization by fractionation. SelK gene were transfected into HepG2 cells. Cells were switched to serum-free medium for 12 h followed by 24 h of PA treatments, and then subjected to homogenization and cell fractionation by gradient centrifugation. The relative distribution of CD36 protein in different fractions is shown in the following panel after densitometry analysis of the blots (n = 3). (D) Cells were treated with 5 µg/ml BFA or 160 µM PA for 16 h before harvested for surface biotinylation assay as described in Materials and method. Equal volume of input and eluted fraction for each treatment was loaded on an SDS–PAGE gel and immunoblotted with ß-actin antibody (n = 6). Data are shown as means ± s.d., ****P < 0.0001, nsP > 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Supplier Page from Abcam for Anti-SELK antibody