Journal: The FEBS journal

Article Title: Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

doi: 10.1111/febs.17024

Figure Lengend Snippet: Fig. 2. Effects of serum and cell density on ciliogenesis in mouse embryonic fibroblasts (MEFs). (A) MEFs (MEF-WT and MEF-STIM1/) were seeded at low or high density, followed by incubation with or without serum for 24 h. Immunofluorescence images of the cilia marker ARL13B (green) and nuclei (blue). (B) Percentages of ciliated cells are presented as means SEM of the n = 3 independent experiments. **P < 0.01; ***P < 0.001 by one-way ANOVA. (C, D) MEF-STIM1/ cells were incubated in culture medium without serum with (C) 1 lM ionomycin or (D) 0.1 lM BAPTA-AM at low density for 1 h. Images show immunofluorescence staining for ARL13B (green) and nuclei (blue). Percentages of ciliated cells are presented as means SEM of the (C) n = 3 and (D) n = 4 independent experiments. *P < 0.05; **P < 0.01 by one-way ANOVA. Yellow stars indicate ciliated cells. Scale bar: 20 lm.

Article Snippet: After CAS blocking, the cells were incubated with the primary antibody against ARL13B (#17711-1-AP; Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Incubation, Marker, Staining

Journal: The FEBS journal

Article Title: Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

doi: 10.1111/febs.17024

Figure Lengend Snippet: Fig. 4. Effects of serum and cell density on ciliogenesis in Hs 578T cells. (A) Hs 578T cells were seeded at a low or high density, followed by incubation with or without serum for 24 h. Immunofluorescence images of the cilia marker ARL13B (green) and nuclei (blue). (B) Percentages of ciliated cells are presented as means SEM of the n = 3 independent experiments. *P < 0.05; ***P < 0.001 by one-way ANOVA. (C, D) Hs 578T cells were incubated in culture medium without serum with (C) 1 lM ionomycin or (D) 0.1 lM BAPTA-AM at low density for 1 h. Images show immu- nofluorescence staining for ARL13B (green) and nuclei (blue). Percentages of ciliated cells are presented as means SEM of the (C) n = 5 and (D) n = 6 independent experiments. *P < 0.05; ***P < 0.001 by one-way ANOVA. Yellow stars indicate ciliated cells. Scale bar: 20 lm.

Article Snippet: After CAS blocking, the cells were incubated with the primary antibody against ARL13B (#17711-1-AP; Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Incubation, Marker, Staining

Journal: The FEBS journal

Article Title: Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

doi: 10.1111/febs.17024

Figure Lengend Snippet: Fig. 5. STIM1 negatively regulates ciliogenesis in Hs 578T cells. (A) Hs 578T cells were transfected with mOrange-STIM1 to overexpress STIM1 proteins, followed by incubation without serum at low density. Immunofluorescence images of STIM1 (red), ARL13B (green), and nuclei (blue). (B) Hs 578T cells were transfected with shSTIM1-GFP to knock down STIM1 proteins, followed by incubation with 10% FBS at low density. Immunofluorescence images of shSTIM1 (green), ARL13B (red), and nuclei (blue). (C) Western blots show STIM1 expression after siRNA targeting STIM1. (D) Hs 578T cells were transfected with siSTIM1 for 72 h. Immunofluorescence images of ARL13B (green) and nuclei (blue). Percentages of ciliated cells are presented as means SEM of the (A) n = 6, (B) n = 4, (C) = 3 and (D) n = 3 independent experiments. **P < 0.01 by one-way ANOVA. Yellow stars indicate ciliated cells. Scale bar: 20 lm.

Article Snippet: After CAS blocking, the cells were incubated with the primary antibody against ARL13B (#17711-1-AP; Proteintech, Rosemont, IL, USA) overnight at 4 °C.

Techniques: Transfection, Incubation, Knockdown, Western Blot, Expressing

Journal: bioRxiv

Article Title: Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

doi: 10.1101/2021.03.02.433576

Figure Lengend Snippet: A . Confocal images of sections of the dorsal striatum from 13-month G2019S LRRK2 KI mice; astrocyte marker, Glial fibrillary acidic protein (GFAP) (magenta, white dotted outline), cilia marker, ADP-ribosylation factor-like protein 13B (Arl13B) (green, white dashed box), and DAPI (blue). B ., C ., Quantitation of the percentage of astrocytes containing a cilium and astrocyte ciliary length from sections described in A. D . Confocal images of sections of the dorsal striatum from 10-month G2019S LRRK2 BAC Tg mice, antibody labeled for Glial fibrillary acidic protein (GFAP, magenta, white dashed box), Arl13B (green, white box), and stained with DAPI (blue). E, F . Quantitation of the percentage of astrocytes containing a cilium and astrocyte ciliary length from sections described in D. Scale bars, 10 µm. Values represent 4-5 brains per group, 2-3 sections per mouse, and >30 astrocytes per mouse. Significance was determined by t-test; B, **, P = 0.0011; E, ** P = 0.0054; F, **** P < 0.0001.

Article Snippet: Afterwards, the cells were incubated in blocking solution containing primary antibodies for 1 hr at RT (EnCOR Chicken-anti-GFAP at 1:2000, Abcam Rabbit-anti-pRab10 at 1:1000, Neuromab Mouse-anti-Arl13B at 1:2000).

Techniques: Marker, Quantitation Assay, Labeling, Staining

Journal: bioRxiv

Article Title: Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

doi: 10.1101/2021.03.02.433576

Figure Lengend Snippet: Mice (8-weeks old) were fed MLi-2 LRRK2 inhibitor-containing chow or control chow for two consecutive weeks prior to perfusion and staining. A . Confocal images of sections of the dorsal striatum from 8 week R1441C LRRK2 KI mice; ChAT (green, white dotted outline); Adenylate cyclase 3 (AC3) (magenta), and DAPI (blue). B . Quantitation of the percentage of ChAT + and ChAT - neurons containing a cilium. C . Percentage of ChAT + neurons containing a cilium ± MLI-2. D . Quantitation of ChAT + neuron ciliary length. E . Confocal images of Astrocytes identified by antibodies to GFAP (magenta, white dotted outline), Arl13B (green), and DAPI (blue), ± MLi-2. F ., G . Quantitation of the percentage of total astrocytes (F) or GFAP/S100B + astrocytes (G) containing a cilium. Scale bars, 10 µm. Significance was determined by t-test. B, *, P =0.0417.

Article Snippet: Afterwards, the cells were incubated in blocking solution containing primary antibodies for 1 hr at RT (EnCOR Chicken-anti-GFAP at 1:2000, Abcam Rabbit-anti-pRab10 at 1:1000, Neuromab Mouse-anti-Arl13B at 1:2000).

Techniques: Control, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

doi: 10.1101/2021.03.02.433576

Figure Lengend Snippet: A . Confocal images of sections from 6-month PPM1H -/+ or age-matched WT mice; ChAT (green, white dotted outline); AC3 (magenta, yellow arrowhead), and DAPI (blue). B . Percentage of ChAT + interneurons containing a cilium. Wild type, light gray; PPM1H -/+ , dark gray as indicated. C . Confocal images of sections of the dorsal striatum from 6-month PPM1H -/+ or age-matched WT mice; GFAP (magenta, white dotted outline), Arl13B (green, yellow arrowhead), and DAPI (blue). D . Percentage of GFAP + astrocytes containing a cilium. Wild type, light gray; PPM1H -/+ , dark gray as indicated. Scale bars, 10 µm. Significance was determined by t-test; (B) **, P = 0.0058; (D) ***, P = 0.0002. Values represent the data from individual brains, analyzing 4 brains per group, 2-3 sections per mouse, and >30 cells per mouse. E . Wildtype and PPM1H -/+ knock-out mice were treated with vehicle (40% (w/v) (2-hydroxypropyl)-β-cyclodextrin) or 30 mg/kg MLi-2 dissolved in vehicle by subcutaneous injection 2 hr prior to tissue collection. 40 µg brain tissue extract was analyzed by quantitative immunoblotting analysis with the indicated antibodies. Each lane represents extract from a different mouse.

Article Snippet: Afterwards, the cells were incubated in blocking solution containing primary antibodies for 1 hr at RT (EnCOR Chicken-anti-GFAP at 1:2000, Abcam Rabbit-anti-pRab10 at 1:1000, Neuromab Mouse-anti-Arl13B at 1:2000).

Techniques: Knock-Out, Injection, Western Blot

Journal: bioRxiv

Article Title: Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

doi: 10.1101/2021.03.02.433576

Figure Lengend Snippet: A , B . 10-month R1441C LRRK2 KI mouse dorsal striatum was subjected to in situ hybridization using a Gli1 probe (gray dots, highlighted with yellow arrowheads). Astrocytes were detected with (A) anti-GFAP or (B) anti-S100B (green, white dashed outline); primary cilia were detected with anti-Arl13B (magenta, white arrows). C , D . Average numbers of Gli1 dots from (C) GFAP and (D) S100B + astrocytes with (+) or without (-) primary cilia. Values represent the mean ± SEM from (C) 4 WT and 3 R1441C brains each containing >35 cells (D) 3 WT and 3 R1441C brains each containing >21 cells. (C) WT cilia (-) vs WT cilia (+); *,P =0.020, R1441C cilia (-) vs R1441C cilia (+); **, P =0.0032. (D) WT cilia(-) vs WT cilia(+); P =0.12, R1441C cilia(-) vs R1441C cilia(+); *,P = 0.017. Significance was determined by unpaired t-test. Arrows indicate primary cilia from GFAP or S100B + astrocytes. Scale bars, 10µm.

Article Snippet: Afterwards, the cells were incubated in blocking solution containing primary antibodies for 1 hr at RT (EnCOR Chicken-anti-GFAP at 1:2000, Abcam Rabbit-anti-pRab10 at 1:1000, Neuromab Mouse-anti-Arl13B at 1:2000).

Techniques: In Situ Hybridization

Journal: bioRxiv

Article Title: Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

doi: 10.1101/2021.03.02.433576

Figure Lengend Snippet: BAC Transgenic G2019S +/- LRRK2 rat astrocytes were dissected from P5 pups and cultured for 1 week ± 200 nM MLi-2. A . Astrocytes were labeled with anti-GFAP and DAPI (blue); rabbit-anti-phospho-Rab10 (white), and anti-Arl13B (green). The dashed line-boxed, numbered regions were enlarged and are shown at the lower right as numbered. Quantitation of the percent of cells displaying primary cilia in cultures of G2019S +/- or WT astrocytes after 24 hrs ± HbEGF ± 200 nM MLi-2 as indicated. C . Quantitation of pRab10 intensity in ciliated or non-ciliated G2019S +/- astrocytes as in B. (B) HbEGF starved G2019S +/- vs. HbEGF starved G2019S +/- plus MLi-2; **, P=0.0017, HbEGF starved G2019S +/- vs. HbEGF fed G2019S +/- ; **, P=0.0041, HbEGF starved G2019S +/- vs. HbEGF fed G2019S +/- plus MLi-2; ***, P=0.0002, HbEGF starved G2019S +/- vs. HbEGF starved WT; ***, P=0.0003, HbEGF starved G2019S +/- vs. HbEGF fed WT, *, P=0.0112. (C) Non-ciliated G2019S +/- vs Ciliated G2019S +/- ; ***, P=0.0009, Non-ciliated G2019S +/- vs MLi-2 treated Non-ciliated G2019S +/- ; ****, P<0.0001; Non-ciliated G2019S +/- vs MLi-2 treated ciliated; ****, P<0.0001; ns = not statistically significant. Significance was determined either by student’s t-test or by Ordinary one-way ANOVA using Dunnett’s multiple comparisons test. Scale bars, 10µm.

Article Snippet: Afterwards, the cells were incubated in blocking solution containing primary antibodies for 1 hr at RT (EnCOR Chicken-anti-GFAP at 1:2000, Abcam Rabbit-anti-pRab10 at 1:1000, Neuromab Mouse-anti-Arl13B at 1:2000).

Techniques: Transgenic Assay, Cell Culture, Labeling, Quantitation Assay

Journal: Cilia

Article Title: A novel ICK mutation causes ciliary disruption and lethal endocrine-cerebro-osteodysplasia syndrome

doi: 10.1186/s13630-016-0029-1

Figure Lengend Snippet: Mutations found in ICK affect ciliogenesis and ciliary localization. a , b The cellular localization of ICK was analyzed in HEK293T cells overexpressing wild-type or two mutated forms of mRFP-ICK: p.R272Q (positive control, mutation previously studied in ECO infants) and p.G120C (new mutation detected in the current study). Nuclei were stained with DAPI (blue) and mRFP-ICK proteins are shown in red. mRFP-ICK with the p.G120C mutation showed the same nuclear localization as wild-type mRFP-ICK transfected cells, while nuclear import was completely lost when mRFP-ICK with the p.R272Q mutation was expressed (Fisher’s exact test two-tailed p < 0.0001, >35 cells counted per condition). c , d The ciliary localization of ICK was studied in mIMCD3 cells transiently transfected with wild-type or two mutant forms of mRFP-ICK: p.R272Q or p.G120C. Wild-type mRFP-ICK mostly localized to the ciliary axoneme and was often enriched at the ciliary base, while both mRFP-ICK mutants enriched at ciliary tips. Ciliary axonemes were visualized with anti-ARL13B ( green ), ciliary transition zones that are present at the ciliary base were marked with anti-RPGRIP1L ( pink ), mRFP-ICK constructs were shown in red , and nuclei were stained with DAPI ( blue ). Per construct >50 transfected, ciliated cells were counted. e , f Cilium presence was studied in serum-starved fibroblasts derived from an ECO patient with a homozygous missense mutation in ICK (c.815G > A; p.R272Q) and compared to fibroblasts derived from two healthy unrelated controls. Control I represents a non-Amish individual, while control II is from the Amish community. Ciliogenesis was significantly reduced in fibroblasts from the ECO patient compared to the controls (Fisher’s exact test two-tailed p < 0.0001 for both). At least 80 cells were counted per condition. Ciliary axonemes were visualized with anti-ARL13B ( green ), ciliary transition zones were marked with anti-RPGRIP1L ( red ), and nuclei were stained with DAPI ( blue )

Article Snippet: The following primary antibodies were used: ARL13B (rabbit polyclonal, Proteintech Group, Manchester, United Kingdom, 1:500) and RPGRIP1L (custom-made guinea pig polyclonal; SNC039, 1:500).

Techniques: Positive Control, Mutagenesis, Staining, Transfection, Two Tailed Test, Construct, Derivative Assay, Control

Journal: Molecular cancer research : MCR

Article Title: Inhibition of Ciliogenesis Promotes Hedgehog Signaling, Tumorigenesis, and Metastasis in Breast Cancer

doi: 10.1158/1541-7786.MCR-17-0034

Figure Lengend Snippet: A, ICC staining of Ift88fl/fl/PyMT+ mammary tissues sections for ARL13b (cilia marker; red), γ-tubulin (centrosome marker; green), CK5 (myoepithelial cell marker; white) and Hoechst (nuclei). The lumen (lum) and stromal (str) regions of the mammary gland are separated by a dashed line. Arrowheads point to primary cilia in Ad-GFP-treated glands, and to centrosomes without cilia in Ad-Cre-GFP-treated glands. B, The bar graph represents quantification of the percent ciliated epithelial cells (basal (bas), luminal (lum), total epithelial cells (all)) from Ift88fl/fl/PyMT+ (Ad-GFP (blue), n=3; Ad-Cre-GFP (red), n=3) and Ift20fl/fl/PyMT+ (Ad-GFP, n=4; Ad-Cre-GFP, n=4) mammary glands. Error bars indicate SE; P values were determined by the Student t-test (paired, two-tailed); *P<0.05. C, Kaplan-Meier curves for tumor-free survival and D, average tumor volume versus time graphed for transplants of Ift88fl/fl/PyMT+ (n=9) and Ift20fl/fl/PyMT+ (n=6) mammary epithelial cells treated with Ad-Cre-GFP (red) and Ad-GFP (blue). Error bars indicate SE; log-rank test and mixed model P-value displayed. Representative images of E, whole mammary tumors, and F, H&E staining of mammary tumor sections from end stage mammary tumors. H&E staining depicts a Grade 1 tumor from an Ad-GFP treated transplant and a Grade 3 tumor from an Ad-Cre-GFP treated transplant. G, The bar graph represents the percent of tumors that were graded as in situ, grade 1, 2 or 3. Ift88fl/fl/PyMT+ (Ad-GFP, n=9; Ad-Cre-GFP, n=9) and Ift20fl/fl/PyMT+ (Ad-GFP, n=6; Ad-Cre-GFP, n=6).

Article Snippet: For all immunofluorescent staining, the primary antibodies used included: ARL13b (1:400, mouse monoclonal IgG 2a , UC, Davis/NIH NeuroMab Facility, clone N295B/66), acetylated tubulin (1:1000, mouse monoclonal IgG 2B , Sigma, Cat. # T7451, clone 6–11B-1), γ-tubulin (mouse monoclonal IgG1, Sigma, Cat. # T5326, clone GTU-88), CK5 (1:400, rabbit polyclonal, Abcam, Cat. # ab53121).

Techniques: Staining, Marker, Two Tailed Test, In Situ

Journal: Cell reports

Article Title: Primary cilia control oligodendrocyte precursor cell proliferation in white matter injury via Hedgehog-independent CREB signaling

doi: 10.1016/j.celrep.2023.113272

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-Arl13b (Proteintech, 17711–1-AP, 1:1000), rat monoclonal anti-Pdgfra (BD Biosciences, 558774, 1:200), rabbit anti-Pdgfra (gift from W. Stallcup), rat monoclonal anti-MBP (Bio-Rad, MCA409S, 1:1000), and chicken polyclonal anti-GFP (Aves Labs, GFP-1020, 1:1000).

Techniques: Recombinant, cDNA Synthesis, Control, Gene Expression, Sequencing, Software, Imaging, Light Microscopy