Journal: medRxiv
Article Title: The congenital multiple organ malformation syndrome, Ritscher-Schinzel syndrome is an endosomal recyclinopathy
doi: 10.1101/2024.08.17.24311658
Figure Lengend Snippet: Loss of membrane proteins such as LRP4 and FGFR2 involved in disease pathogenesis in skeletal system in Ritscher-Schinzel syndrome. (A) Representative blots of Co-expression of mCherry-tagged SNX17 with GFP-tagged cytoplasmic tail of Human-LRP4 from three independent experiments. The interaction between mCherry-SNX17 and wild-type GFP-tagged cytoplasmic tails of LRP4 was significantly decreased when NPxY was substituted with NPxA. Bar graphs show relative density of the bands in immunoprecipitated proteins from three independent experiments. (B) Representative blots of LRP4 in cell surface protein fraction obtained from H4 cell line. N-Cadherin was used for loading control. Bar graph shows relative intensity of knock-out cells to their rescue or parental cells among three independent experiments. (C) Volcano plots of transmembrane proteins with decreased (blue circles) or increased (red circles) cell surface abundance in TMT-based proteomics in sh-SNX17 comparing to sh-SCR in MC3T3-E1 mouse osteoblast cells from three independent experiments. Two independent sh-RNAs for SNX17 were used to avoid off-target effects. (D) Representative blots for FLAG-nanotrap of FLAG-SNX17 under co-overexpression of chimeric constructs of FGFR2 cytoplasmic tail from three independent experiments. NxxY motifs were mutated to NxxA in the mutant. (E) Representative blots for FGFR2 in MC3T3-E1. Cell surface protein fraction was obtained, and N-Cadherin were used for loading control. Bar graphs show relative values of sh-SNX17 or sh-VPS35L knock-down cells compared to sh-SCR control cell (n=3). (F) Representative blots for ERK and pERK under stimulation of FGF2 in MC3T3-E1 from three independent experiments. Cells were cultured with 5ng/ml of FGF-2 overnight, then media was replaced with fresh media with 100ng/ml of FGF-2 for 7 min. Cells were lysed and analysed by western blotting. (G) Representative pictures and scatter plots of their body weight in VPS35L Prx1 -cKO mice and their littermate controls. [Control; n=12, Vps35l-cKO Prx1 ; n=9]. (H) Representative images and graph showing length of tibiae in mice at 8 weeks with indicated genotype. [Control; n=12, Vps35l-cKO Prx1 ; n=9]. (I) Gene enrichment analysis of downregulated genes in Vps35l-cKO Prx1 compared to their littermate controls was performed using RNA sequencing analysis data. Total RNA was extracted from E16.5 mice chondrocytes. [Control; n=5, Vps35l-cKO Prx1 ; n=3]. (J) Gene enrichment analysis of gene sets, where genes upregulated by FGF2 stimulation in littermate controls but not in Vps35l-cKO Prx1 were included. Tibiae obtained from E16.5 mouse embryos of either Vps35l-cKO Prx1 or their littermate controls were cultured for four days with or without FGF2, followed by RNA extraction for RNA sequencing analysis. [Control; n=3, Vps35l-cKO Prx1 ; n=4]. (A, B, D, E, F) Error bars represent mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Article Snippet: The following antibodies were used in this study (WB: western blot, IF: immunofluorescence): rabbit anti-SNX17 (Proteintech, 10275-1-AP, WB), mouse anti-GFP (Roche, 11814460001, WB), rabbit anti-GFP (GeneTex, GTX30738, WB), mouse anti-mCherry (antibodies.com, A85305, WB), rabbit anti-mCherry (antibodies.com, A85306, WB), rabbit anti-CCDC22, (Proteintech, 16636-1-AP, WB), mouse anti-CCDC93, (Origene, CF800568, WB), rabbit anti COMMD4 and rabbit anti COMMD9 (kind gift from Prof. Ezra Burstein, WB) rabbit anti-C16orf62 (Abcam, ab97889, WB), rabbit anti-C16orf62 (Pierce, PA5-28553, IF), rabbit anti-DSCR3 (Merck Millipore, ABN87, WB), rabbit anti-integrin-β1 (Abcam, ab52971, WB), goat anti-VPS35 (antibodies.com, A83699, IF), mouse anti-VPS29 (Santa Cruz, sc-398874, WB), rabbit anti-KIAA1033 (Proteintech, 51101-1-AP, WB), mouse anti-Strumpellin (Santa Cruz, sc-377146, WB), mouse anti-β actin (Sigma, A1978, WB), rabbit anti-LRP1 (Abcam, ab92544, WB), rabbit anti-LRP2 (Proteintech, 19700-1-AP, WB), rabbit anti-LRP2 (prepared as described in , IHC), rabbit anti-LRP4 (SIGMA, HPA012300, WB), rabbit anti-LRP8 (Abcam, ab108208, WB), mouse anti-VLDLR (Santa Cruz, sc-18824, WB), rabbit anti-APP (Abcam, ab32136, WB), rabbit anti-APLP2 (Proteintech, 15041-1-AP, WB), mouse anti-AP (Thermo Fisher, MA1-20245, WB), rabbit anti-Dab1 (kind gift from Dr. M Hattori, IP/WB) , mouse anti-Phosphotyrosine (Merck, 05-321, WB), rabbit anti-GLUT1 (Abcam, ab115730, IF/WB), mouse anti-N-cadherin (Cell signalling technology, 14215S, WB), anti-PSD95 (Merck, MAB1596, WB), mouse anti-FLAG (SIGMA, F1804, WB), rabbit anti-FGFR2, (Proteintech, 13042-1-AP, WB), rabbit anti-ERK1/2 (Cell Signaling Technology, 9102, WB), rabbit anti-phospho-ERK1/2 (Cell Signaling Technology, 9101, WB), anti Ctip2 (Abcam, ab18465, IHC), anti Tbr1 (Abcam, ab275960, IHC), anti Neun (Cell signalling technology, 12943, IHC).
Techniques: Membrane, Expressing, Immunoprecipitation, Control, Knock-Out, Over Expression, Construct, Mutagenesis, Knockdown, Cell Culture, Western Blot, RNA Sequencing Assay, RNA Extraction
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![Loss of membrane proteins such as <t>LRP4</t> and FGFR2 involved in disease pathogenesis in skeletal system in Ritscher-Schinzel syndrome. (A) Representative blots of Co-expression of mCherry-tagged SNX17 with GFP-tagged cytoplasmic tail of Human-LRP4 from three independent experiments. The interaction between mCherry-SNX17 and wild-type GFP-tagged cytoplasmic tails of LRP4 was significantly decreased when NPxY was substituted with NPxA. Bar graphs show relative density of the bands in immunoprecipitated proteins from three independent experiments. (B) Representative blots of LRP4 in cell surface protein fraction obtained from H4 cell line. N-Cadherin was used for loading control. Bar graph shows relative intensity of knock-out cells to their rescue or parental cells among three independent experiments. (C) Volcano plots of transmembrane proteins with decreased (blue circles) or increased (red circles) cell surface abundance in TMT-based proteomics in sh-SNX17 comparing to sh-SCR in MC3T3-E1 mouse osteoblast cells from three independent experiments. Two independent sh-RNAs for SNX17 were used to avoid off-target effects. (D) Representative blots for FLAG-nanotrap of FLAG-SNX17 under co-overexpression of chimeric constructs of FGFR2 cytoplasmic tail from three independent experiments. NxxY motifs were mutated to NxxA in the mutant. (E) Representative blots for FGFR2 in MC3T3-E1. Cell surface protein fraction was obtained, and N-Cadherin were used for loading control. Bar graphs show relative values of sh-SNX17 or sh-VPS35L knock-down cells compared to sh-SCR control cell (n=3). (F) Representative blots for ERK and pERK under stimulation of FGF2 in MC3T3-E1 from three independent experiments. Cells were cultured with 5ng/ml of FGF-2 overnight, then media was replaced with fresh media with 100ng/ml of FGF-2 for 7 min. Cells were lysed and analysed by western blotting. (G) Representative pictures and scatter plots of their body weight in VPS35L Prx1 -cKO mice and their littermate controls. [Control; n=12, Vps35l-cKO Prx1 ; n=9]. (H) Representative images and graph showing length of tibiae in mice at 8 weeks with indicated genotype. [Control; n=12, Vps35l-cKO Prx1 ; n=9]. (I) Gene enrichment analysis of downregulated genes in Vps35l-cKO Prx1 compared to their littermate controls was performed using RNA sequencing analysis data. Total RNA was extracted from E16.5 mice chondrocytes. [Control; n=5, Vps35l-cKO Prx1 ; n=3]. (J) Gene enrichment analysis of gene sets, where genes upregulated by FGF2 stimulation in littermate controls but not in Vps35l-cKO Prx1 were included. Tibiae obtained from E16.5 mouse embryos of either Vps35l-cKO Prx1 or their littermate controls were cultured for four days with or without FGF2, followed by RNA extraction for RNA sequencing analysis. [Control; n=3, Vps35l-cKO Prx1 ; n=4]. (A, B, D, E, F) Error bars represent mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.](https://med-rxiv-images-cdn.bioz.com/dois_ending_with_58/10__1101_slash_2024__08__17__24311658/10__1101_slash_2024__08__17__24311658___F4.large.jpg)