Journal: Investigative Ophthalmology & Visual Science

Article Title: Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish

doi: 10.1167/iovs.16-19898

Figure Lengend Snippet: arl13b is not required for zebrafish retinal development. ( A , B ) Lateral view of larvae at 5 dpf. ( C – F ) Methylene blue-stained plastic sections of 5 dpf wild-type siblings ( arl13b +/? ) and arl13b −/− mutant retinas. The outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GC) were present in wild-type and mutant retinas. ( G , H ) Transmission electron micrographs of 5 dpf retinas do not show any evidence of photoreceptor outer segment disorganization or ciliary defects. Scale bars : 200 μm ( A , B ), 20 μm ( C , D ), 10 μm ( E , F ), and 2 μm ( G , H ).

Article Snippet: Human ARL13b cDNA was obtained from OriGene (Rockville, MD, USA) in the pCMV6-AC-GFP shuttle vector, which expresses a turbo green fluorescent protein (tGFP).

Techniques: Staining, Mutagenesis, Transmission Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish

doi: 10.1167/iovs.16-19898

Figure Lengend Snippet: Ultrastructural analysis of photoreceptor connecting cilia by transmission electron microscopy. ( A , B ). Horizontal sections through photoreceptors of wild-type and arl13b −/− larvae at 5 dpf show the connecting cilia in the vicinity of the mitochondria (M). Calyceal processes surround the cilium ( arrows ). ( C , D ) Higher magnification images revealed no obvious defects in microtubule organization in arl13b −/− mutants. Scale bars : 0.5 μm ( A , B ) and 0.2 μm ( C , D ).

Article Snippet: Human ARL13b cDNA was obtained from OriGene (Rockville, MD, USA) in the pCMV6-AC-GFP shuttle vector, which expresses a turbo green fluorescent protein (tGFP).

Techniques: Transmission Assay, Electron Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish

doi: 10.1167/iovs.16-19898

Figure Lengend Snippet: arl13b −/− mutants have shorter photoreceptor outer segments and cilia at 4 dpf. ( A , B ) Immunohistochemistry on transverse cryosections from 4 dpf wild-type and arl13b −/− mutant retinas stained for rhodopsin ( red ). ( C ) Quantification of the length of rhodopsin staining along the long axis of the photoreceptor. ( D , E ) Immunohistochemistry for acetylated α-tubulin ( red ) and Ift88 ( green ). ( F ) Quantification of average cilia length. All sections were counterstained with DAPI to label nuclei. Scale bars : 10 μm. Error bars : ± SEM. * P < 0.0001 using Student's t -test with Welch's correction.

Article Snippet: Human ARL13b cDNA was obtained from OriGene (Rockville, MD, USA) in the pCMV6-AC-GFP shuttle vector, which expresses a turbo green fluorescent protein (tGFP).

Techniques: Immunohistochemistry, Mutagenesis, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish

doi: 10.1167/iovs.16-19898

Figure Lengend Snippet: Progressive degeneration of arl13b −/− photoreceptors. ( A ) Overview of the blastula transplant strategy. Heterozygous arl13 +/− fish were crossed to the TαC:GFP ucd1 transgenic line to produce arl13b +/− ; TαC:GFP ucd1 animals. Progeny from heterozygous crosses were injected with rhodamine-dextran and donor cells transplanted into the animal pole of unlabeled wild-type hosts. At 4 dpf, arl13b −/− ;TαC:GFP ucd1 mutants were identified phenotypically ( top ). Wild-type hosts containing photoreceptors from mutant donors were identified by mosaic GFP fluorescence within the eye ( bottom ). ( B , C ) Cryosections of 14 dpf larvae showing GFP+ clones populating retinas bilaterally and unilaterally. ( D , E ) Immunofluorescence image of 16 dpf larval retinas immunolabeled for rhodopsin ( red ). Cone photoreceptors from donor embryos express GFP ( green ). Transplanted cone photoreceptors from an arl13b −/− ; TαC:GFP ucd1 mutant donors exhibited partial rhodopsin mislocalization within the donor clone ([ E ], arrows ). Sections were counterstained with DAPI to show nuclei. ( E ) Graph showing the percentage of hosts ( n > 11 retinas per genotype) still containing GFP-positive cells from wild-type or arl13b −/− mutant donors at specified time points. Scale bar : 50 μm.

Article Snippet: Human ARL13b cDNA was obtained from OriGene (Rockville, MD, USA) in the pCMV6-AC-GFP shuttle vector, which expresses a turbo green fluorescent protein (tGFP).

Techniques: Transgenic Assay, Injection, Mutagenesis, Fluorescence, Clone Assay, Immunofluorescence, Immunolabeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish

doi: 10.1167/iovs.16-19898

Figure Lengend Snippet: Suppression of arl13b and PCP components result in shortened photoreceptor cilia and outer segments. ( A – D ) Immunohistochemistry using antibodies against acetylated α-tubulin ( red ) to label cilia in retinal cryosections of 5 dpf zebrafish larvae. ( E – H ) Immunohistochemistry using rhodopsin antibodies to label photoreceptor outer segments in retinal cryosections of 5 dpf larvae. ( I ) Quantification of cilia length. ( J ) Quantification of outer segment area. All sections were counterstained with DAPI. Results are significant (**** P < 0.0001) using a 1-way ANOVA with Tukey's multiple comparisons test correction. Error bars : ± SEM. Scale bar : 10 μm.

Article Snippet: Human ARL13b cDNA was obtained from OriGene (Rockville, MD, USA) in the pCMV6-AC-GFP shuttle vector, which expresses a turbo green fluorescent protein (tGFP).

Techniques: Immunohistochemistry

Journal: Investigative Ophthalmology & Visual Science

Article Title: Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish

doi: 10.1167/iovs.16-19898

Figure Lengend Snippet: Arl13b biochemically interacts with Vangl2. ( A ) Protein extracts from HEK293-T cells transiently transfected with Vangl2 and/or Arl13b and immunoprecipitated (IP) with antibodies against Vangl2 (lanes 1–3) or Arl13b (4–5) and subsequently immunoblotted (IB) with antibodies against Arl13b. Immunoblots of input samples show the presence of Arl13b from different extracts used for IP (lanes 6–8). Black arrows indicate bands corresponding to Arl13b, while open arrows indicate nonspecific bands. ( B ) Protein extracts from HEK293-T cells transiently transfected with Vangl2 and/or Arl13b and immunoprecipitated with antibodies against Arl13b (lanes 1–3) or Vangl2 (lanes 4–5) and subsequently subjected to IB with antibodies against Vangl2. Immunoblot of input samples to show the presence of Vangl2 in different protein extracts (lanes 6–8, black arrow ). The high molecular weight band ( white arrow ) likely represents Vangl2 homodimers. Molecular mass of protein markers is denoted in kDa.

Article Snippet: Human ARL13b cDNA was obtained from OriGene (Rockville, MD, USA) in the pCMV6-AC-GFP shuttle vector, which expresses a turbo green fluorescent protein (tGFP).

Techniques: Transfection, Immunoprecipitation, Western Blot, High Molecular Weight

Journal: Molecular Medicine

Article Title: Glucagon secretion by pancreatic alpha-cells requires an intact tubulin-cytoskeleton-primary cilium axis

doi: 10.1186/s10020-026-01428-1

Figure Lengend Snippet: Impaired ciliogenesis in ARL13B-knock-down and IFT88-knock-down alphaTC1.9 cells. A Representative western-blot analysis of ARL13B after 48 h of siRNA-ARL13B (ARL13B-KD) and siRNA-scramble (control) transfection. B Quantification of ARL13B/GAPDH in ARL13B-KD and control cells, N = 9. C Quantification of Arl13b gene expression by RT-PCR, N = 6. D Representative immunostaining of acetylated tubulin staining in ARL13B-KD and control cells. E Quantification of cilia number and, F cilia size, N = 9. G Representative western-blot analysis of IFT88 after 48 h of siRNA-IFT88 (IFT88-KD) and siRNA-scramble (control) transfection. H Quantification of IFT88/GAPDH in IFT88-KD and control cells, N = 15. I Quantification of Ift88 gene expression by RT-PCR, N = 6. J Representative immunostaining of acetylated tubulin staining in IFT88-KD and control cells. K Quantification of cilia number and, L cilia size, N = 8–10. ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM. Graph bar= 10 μm

Article Snippet: RNA was extracted using Trizol (Invitrogen, USA), reverse transcription was performed with iScript cDNA Synthesis Kit (Biorad, USA) and qPCR was carried on with the following Taqman (Applied Biosystems, USA): Dync1h1 (Mm00466548_m1), Ide (Mm00473077_m1), Arl13b (Mm01349328_m1), Ift88 (Mm01313467_m1), Insr (Mm01211875_m1).

Techniques: Knockdown, Western Blot, Control, Transfection, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Staining

Journal: Molecular Medicine

Article Title: Glucagon secretion by pancreatic alpha-cells requires an intact tubulin-cytoskeleton-primary cilium axis

doi: 10.1186/s10020-026-01428-1

Figure Lengend Snippet: Impaired glucagon secretion in ARL13B-KD and IFT88-KD cells. A Low glucose (1mM) stimulates glucagon secretion in alphaTC1.9 cells meanwhile high glucose (16mM) inhibits it in control cells, meanwhile, ARL13B-KD cells have lost glucagon secretion capacity at low glucose, N = 9. B IFT88-KD cells have lost glucagon secretion capacity at low glucose, N = 6. * p < 0.05. Data are represented as mean ± SEM

Article Snippet: RNA was extracted using Trizol (Invitrogen, USA), reverse transcription was performed with iScript cDNA Synthesis Kit (Biorad, USA) and qPCR was carried on with the following Taqman (Applied Biosystems, USA): Dync1h1 (Mm00466548_m1), Ide (Mm00473077_m1), Arl13b (Mm01349328_m1), Ift88 (Mm01313467_m1), Insr (Mm01211875_m1).

Techniques: Control

Journal: Molecular Medicine

Article Title: Glucagon secretion by pancreatic alpha-cells requires an intact tubulin-cytoskeleton-primary cilium axis

doi: 10.1186/s10020-026-01428-1

Figure Lengend Snippet: Impaired insulin action and insulin receptor levels in ARL13B-KD and IFT88-KD cells. A Insulin inhibits glucagon secretion at 1mM glucose in alphaTC1.9 cells, meanwhile insulin inhibition is lost in ARL13B-KD cells, N = 9. B Insulin inhibits glucagon secretion at 1mM glucose in alphaTC1.9 cells, meanwhile insulin inhibition is lost in IFT88-KD cells, N = 5. C Insulin receptor levels are decreased in ARL13B -KD cells in respect to control. D Representative western-blot of IR in ARL13B -KD and control cells. E Quantification of Insr gene expression by RT-PCR, N = 6. F Insulin receptor levels are decreased in IFT88-KD cells in respect to control, N = 15. G Representative western-blot of IR in IFT88-KD and control cells. H Quantification of Insr gene expression by RT-PCR, N = 6. ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM

Article Snippet: RNA was extracted using Trizol (Invitrogen, USA), reverse transcription was performed with iScript cDNA Synthesis Kit (Biorad, USA) and qPCR was carried on with the following Taqman (Applied Biosystems, USA): Dync1h1 (Mm00466548_m1), Ide (Mm00473077_m1), Arl13b (Mm01349328_m1), Ift88 (Mm01313467_m1), Insr (Mm01211875_m1).

Techniques: Inhibition, Control, Western Blot, Gene Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular Medicine

Article Title: Glucagon secretion by pancreatic alpha-cells requires an intact tubulin-cytoskeleton-primary cilium axis

doi: 10.1186/s10020-026-01428-1

Figure Lengend Snippet: IDE and primary cilium dynamics under glucose-regulated glucagon secretion and inhibition. A Glucagon secretion in wild type cells (alphaTC1.9) under conditions of low (1mM) and high (16mM) glucose. B Representative western-blots for IDE, acetylated tubulin (AcTub), tubulin, ARL13B, insulin receptor (IR) and the nuclear protein p84. C Quantification of IDE protein levels, tubulin acetylation, alpha-tubulin, ARL13B, IR and p84 (housekeeping), N = 9. * p < 0.05 ** p < 0.01, *** p < 0.001. Data are represented as mean ± SEM

Article Snippet: RNA was extracted using Trizol (Invitrogen, USA), reverse transcription was performed with iScript cDNA Synthesis Kit (Biorad, USA) and qPCR was carried on with the following Taqman (Applied Biosystems, USA): Dync1h1 (Mm00466548_m1), Ide (Mm00473077_m1), Arl13b (Mm01349328_m1), Ift88 (Mm01313467_m1), Insr (Mm01211875_m1).

Techniques: Inhibition, Western Blot

Journal: Molecular Medicine

Article Title: Glucagon secretion by pancreatic alpha-cells requires an intact tubulin-cytoskeleton-primary cilium axis

doi: 10.1186/s10020-026-01428-1

Figure Lengend Snippet: Impaired ciliogenesis in pancreatic alpha-cells of A-IDE-KO mice. A Representative pictures of insulin (green), glucagon (red), acetylated tubulin (white) and DAPI (blue) in A-IDE-WT ( N = 4) and A-IDE-KO ( N = 4) mouse pancreata. B Quantification of cilia number per alpha-cell number. C Quantification of cilia number per beta-cell number. D Representative western-blot analysis of ARL13B, acetylated tubulin (AcTUB), alpha-tubulin and actin in A-IDE-KO isolated islets and controls. E Quantification of ARL13B, F AcTUB, and, G alpha-tubulin western-blots (normalized to actin), A-IDE-WT ( N = 6) and A-IDE-KO ( N = 8). * p < 0.05. Data are represented as mean ± SEM. Graph bar= 10 μm

Article Snippet: RNA was extracted using Trizol (Invitrogen, USA), reverse transcription was performed with iScript cDNA Synthesis Kit (Biorad, USA) and qPCR was carried on with the following Taqman (Applied Biosystems, USA): Dync1h1 (Mm00466548_m1), Ide (Mm00473077_m1), Arl13b (Mm01349328_m1), Ift88 (Mm01313467_m1), Insr (Mm01211875_m1).

Techniques: Western Blot, Isolation