Fig 1: Effect of WLS deficiency on BMDCs. (A) Colony formation of BMDCs with wild-type, heterozygous, or wls-null (wlsfx/fx) genotypes (n = 8). (B and C) Percentage of apoptotic and autophagic BMDCs with wild-type, heterozygous, or wls-null genotypes (n = 8). (D) Western blots of autophagic markers (LC3B, PIK3C3, ATG5, ATG12 and ATG16L1) in lysates of wild-type and wls-null BMDCs (n = 3; p< 0.001). (E) Percentage of apoptotic BMDCs with wild-type and wls-null genotypes by WNT1 and WNT3A treatment. (F) Percentage of autophagic BMDCs with wild-type and wls-null genotypes after WNT1 and WNT3A treatment. (G) Level of cytokine-expressing cells in wild-type or wls-null (wlsfx/fx) BMDCs. (H) Levels of IL12A, IL6, and IL10 secreted by wild-type and wls-null BMDCs, or (I) IFNG and TNF secreted by cocultured CD4+ T cells in the presence or absence of LPS. (J) Spleen size in wild-type and DC-specific wls-null mice with or without LPS treatment. Flow cytometric analysis of the total number of (K) splenocytes and (L) DCs in wild-type and DC-specific wls-null mice after LPS treatment (n = 8; p< 0.001)
Fig 2: WLS-mediated molecular chaperone supercomplex modulates protein glycosylation, cytokine secretion, and cell fate in BMDCS. (A) Total glycosylation levels as measured by ELISA in BMDCs with wild-type and wls-null genotypes after TM, BFA, or DTT treatment. (B) Glycosylation levels of different kinds of glycan modification of total proteins determined by phenol-sulfuric acid method. (C) Heatmap of 507 biomarkers in wild-type and wls-null (wlsfx/fx) BMDCs; (D) Comparison of the number of hypoglycosylated proteins between wild-type and wls-null cells. #, hypo-glycosylation, no significance (*, p< 0.05); (E) GO biological process over-representation analysis with 15 signal pathways between wild-type and wls-null cells; (F) Glycosylation levels of CDH5/VE-Cadherin, BMPR1B, TGFBR1, LRP6, FZD1/Frizzled-1, FZD3, FZD4, FZD5, FZD6, and CTNNB1/ß-catenin in wild-type and wls-null cells. (G) Glycosylation levels as measured by ELISA in anti-WNT1, WNT3A, and WNT5A immunoprecipitates of wild-type and wls-null BMDC. (H) Levels of WNT1, WNT3A, and WNT5A as measured by ELISA in wild-type and wls-null BMDCs. (I) Glycosylation levels of INS (insulin), IGF1, GRB2, IGF2R, INSR, GCG (glucagon), SLC2A1/GLUT1, SLC2A2, SLC2A3, and SLC2A5 in wild-type and wls-null cells. (J-K) Levels of GCG, glucose, and ATP as measured by ELISA in wild-type and wls-null BMDCs. (L) Glycosylation levels of CD14, TLR1, TLR2, TLR3, TLR4, IFNB, IL6, and TNF in wild-type and wls-null cells. (M) Glycosylation levels of IFNA, IFNG, IFNAR1, IFNGR2, IL10, IL12A, IL23A and TGFA in wild-type and wls-null cells
Fig 3: WLS deficiency results in ER stress and the loss of ER quality control. (A) Western blot analysis of UPR sensors, EIF2AK3-ElF2A signals, and calcium regulators in wild-type (+/+), heterozygous (fx/+), and wls-null (fx/fx) Itgax-Cre BMDCs. (B) TEM analysis of the ultrastructure and translational ribosome complex in wild-type and wls-null BMDCs. Red arrow, translational ribosomal complex. (C) intracellular calcium levels in wild-type and wls-null BMDCs following treatment with CCL2 (0.7 nM). (D-F) Western blotting analysis of WLS, ERN1, EIF2AK3, ATF6, HSPA5 CANX, and CALR in anti-WLS, anti-ERN1, and anti-EIF2AK3 immunoprecipitates probed with the respective antibodies in wild-type or wls-deficient BMDCs. Data were generated from three independent experiments. (G) Confocal immunofluorescent imaging of WLS, ERN1, EIF2AK3, and ATF6 in wild-type or wls-null BMDCs. (H-I), Western blot analysis of WLS, ERN1, EIF2AK3, ATF6, HSPA5, CANX, and CALR in anti-WLS immunoprecipitates of BMDCs treated with WNT1 and WNT3A, respectively
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