Journal: bioRxiv

Article Title: RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

doi: 10.1101/2025.02.10.637381

Figure Lengend Snippet: (A-B) Fibroblasts isolated from skin biopsy from a CS patient bearing the RAB23 biallelic p.(L145*) nonsense mutation was reprogrammed into iPSC clones. (A) Top : A representative phase contrast image showing successfully reprogrammed-iPSC clone displaying a normal karyotype (bottom), and pluripotency characterized by positive (B) co-immunostainings of human stem cell markers SSEA4, NANOG, OCT3/4 and TRA-1-60 respectively. (C) Representative co-immunocytochemistry images depicting positive expression of NESTIN (red) and SOX2 (green) on neural progenitor stem cells induced from iPSCs. (D) Representative co-immunocytochemistry images depicting healthy adult and CS patient iPSC-derived human neural progenitor cells (hNPCs) co-immunostained for SOX2 (magenta), NESTIN (red) and primary cilia marker ARL13B (green). (E) Graph depicts the quantification of the percentage of ciliation in hNPCs. Box plot represents data from four independent experiments. n.s.= not significant. Unpaired Student’s t-test. (F-G) Box plots depict the measurements of (F) cilia length and (G) cilia volume on hNPCs. Data represents the measurements of ∼65–80 cilia in each genotype obtained from three independent experiments. **** P value ≤ 0.0001, * P value ≤ 0.05 Unpaired Student’s t-test. (H-I) Representative co-immunocytochemistry images depict MAP2+ (red) neurons differentiated from iPSCs of healthy adults (control) and CS patients (p.(L145*)). Primary cilia were labelled by AC3 (green). (I) Graph depicts the percentage of ciliation, i.e. quantification of the proportion of ciliated neurons (AC3+ MAP2+) in the MAP2 positive neuronal population. A significant reduction in ciliation was observed in neurons bearing p.(L145*) mutation. Box plot represents data from five independent experiments. ** P value ≤ 0.01 Unpaired Student’s t-test. (J) Graph depicts the quantification of the percentage of ciliation in primary fibroblast cells. The primary cilia in human fibroblast were visualised by immunostaining of acetylated-alpha-tubulin and ARL13B. Primary fibroblasts cultured from a healthy donor and a CS patient with p.(L145*) mutation show comparable percentages of ciliation. Box plot represents data from four independent experiments. n.s.= not significant. Unpaired Student’s t-test.

Article Snippet: The primary antibodies and dilution factors used were: anti-rabbit Arl13b (Proteintech, 17711-1-AP 1:1500), anti-mouse Arl13b (DSHB/NeuroMAB clone N295B/66 1:800), anti-rabbit AC3 (Santa Cruz discontinued 1:1500), anti-mouse Nestin (Sigma/Millipore MAB5326 1:1000), anti-mouse Map2 (Sigma, M9942 1:1000), anti-rabbit Pax6 (Biolegend/Covance, PRV-278P 1:1000), anti-mouse NeuN (Sigma/Millipore, MAB377 1:800), anti-rabbit Tbr1 (Abcam, AB31940 1:500), anti-mouse acetylated alpha tubulin (Sigma Aldrich, T6793 1:2000).

Techniques: Isolation, Mutagenesis, Clone Assay, Immunocytochemistry, Expressing, Derivative Assay, Marker, Control, Immunostaining, Cell Culture

Journal: bioRxiv

Article Title: RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

doi: 10.1101/2025.02.10.637381

Figure Lengend Snippet: (A) Representative immunocytochemistry images depict control and Nes-CKO cortical NPCs immunostained for Nestin (red) and primary cilia marker Arl13b (green). (B) Graphs depict the quantification of the percentage of ciliation, ciliary volume and length in mouse cortical neural progenitor cells culture. Each dot represents the average percentage value of each independent experiment. * P- value ≤ 0.05, ** P- value ≤ 0.01, **** P -value ≤ 0.0001, Unpaired Student’s t-test. For cilia volume and length measurement; Control n = 230, Nes-CKO n = 211, measured from three independent experiments. (C) Graphs depict the (left) 7Gli:GFP reporter signals and the (right) relative fold-change in control and Nes-CKO cortical NPCs treated with 200 nM SAG and DMSO respectively for 24 hours. Data points represent triplicate readings from three independent experiments. Left: **** P -value ≤ 0.0001, ** P -value ≤0.01, n.s.= not significant, two-way ANOVA. Right: **** P -value ≤ 0.0001, Unpaired Student’s t-test. (D) Graph depicts the relative gene expression level of control and Nes-CKO cortical NPCs quantified by real-time qPCR. **** P -value≤0.0001, n.s.= not significant, two-way ANOVA. (E) Graphs depict the relative gene expression levels of Gli1 and Rab23 in wild-type cortical NPCs overexpressing wild-type and mutant forms of Rab23 at day 6 post-transduction. NPCs were transduced with lentiviruses carrying different overexpression construct i.e. Rab23WT, Rab23Q68L, Rab23SN respectively. The gene expression levels of Gli1 and Rab23 at day 6 post-transduction were quantified by real-time qPCR. ** P- value ≤ 0.01, n.s.= not significant, one-way ANOVA.

Article Snippet: The primary antibodies and dilution factors used were: anti-rabbit Arl13b (Proteintech, 17711-1-AP 1:1500), anti-mouse Arl13b (DSHB/NeuroMAB clone N295B/66 1:800), anti-rabbit AC3 (Santa Cruz discontinued 1:1500), anti-mouse Nestin (Sigma/Millipore MAB5326 1:1000), anti-mouse Map2 (Sigma, M9942 1:1000), anti-rabbit Pax6 (Biolegend/Covance, PRV-278P 1:1000), anti-mouse NeuN (Sigma/Millipore, MAB377 1:800), anti-rabbit Tbr1 (Abcam, AB31940 1:500), anti-mouse acetylated alpha tubulin (Sigma Aldrich, T6793 1:2000).

Techniques: Immunocytochemistry, Control, Marker, Expressing, Mutagenesis, Transduction, Over Expression, Construct