Journal: Human molecular genetics

Article Title: Essential role of nephrocystin in photoreceptor intraflagellar transport in mouse.

doi: 10.1093/hmg/ddp068

Figure Lengend Snippet: Figure 5. Localization of KIF3A and IFT88 in the photoreceptors of Nphp1-targeted mutants and control littermates at postnatal day 14. Indirect anti-KIF3A (red) and anti-IFT88 (red) immunofluorescence in retinal cryosection of the targeted mutants (del20/del20) and control littermates (þ/þ) shown in middle panel. The position of the connecting cilium in the cryosection was revealed by double-immunolabeling with anti-acetylated a-tubulin (green) antibodies (left panel). The DAPI was used to stain the nuclei (blue). The red/green merged images are shown in right panel. OS, outer segment; IS, inner segment. Scale bars: 5 mm.

Article Snippet: A purified goat antibody to IFT88 was purchased from Imgenex (San Diego, CA, USA).

Techniques: Control, Immunolabeling, Staining

Journal: Human molecular genetics

Article Title: Essential role of nephrocystin in photoreceptor intraflagellar transport in mouse.

doi: 10.1093/hmg/ddp068

Figure Lengend Snippet: Figure 8. Colocalization of EmGFP-tagged nephrocystin and acetylated a-tubulin to the photoreceptor connecting cilia of the rescued mutants at p60. Local- ization of EmGFP-tagged nephrocystin in the photoreceptor cryosection by direct EmGFP fluorescence imaging (green) is shown in (A, D, G and J). Cryosec- tions were double-immunolabeled with anti-Bbs2 (red in B), anti-g-tubulin (red in E), anti-IFT88 (red in H) or anti-acetylated a-tubulin (red in K) to reveal the position of basal body, microtubule organization center, distal end of connecting cilium or the connecting cilium, respectively. The red/green merged images are shown in the right panel (C, F, I and L). The colocalization of these immunolabeled markers (red) with EmGFP-tagged nephrocystin (Green) indicates yellow. OS, outer segment; IS, inner segment. Scale bars: 5 mm.

Article Snippet: A purified goat antibody to IFT88 was purchased from Imgenex (San Diego, CA, USA).

Techniques: Imaging, Immunolabeling

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A) Effect of ATP in cell migration comparing normal ciliated cholangiocytes (NHC SCR), normal deciliated cholangiocytes (NHC IFT88) and the CCA cell line HUCCT1. Representative images obtained from the wound healing assay and bar graphs showing the distance migrated by the cells relative to control (vehicle) in 24 h are depicted (**p<0.01, n=3). (B) Cell migration analysis by wound healing assay showing the effects of ATP, ADP, Apyrase and their combinations. Bar graph shows the distance migrated by the cells relative to control (vehicle) in 24 h (*p<0.05, **p<0.01, n=3). (C) Invasion assay, representative pictures and bar graph showing the percentage of invasion in 24 h (**p<0.01, n=3). (D) Proliferation rates were assessed in real time using IncuCyte or (E) by MTS assay. Results are expressed in % proliferation relative to control (vehicle).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Migration, Wound Healing Assay, Invasion Assay, MTS Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A) Expression of LKB1 was evaluated in normal cholangiocytes (NHC, H69), experimentally deciliated cholangiocytes (NHC IFT88, H69 IFT88) and CCA cell lines (KMCH, HUCCT1, OZ, EGI-1) by western blotting. (B) Presence of LKB1 in primary cilia using acetylated α-tubulin or ARL13b as ciliary markers. The expression was assessed by confocal immunofluorescence on scramble normal cholangiocytes (NHC SCR). (C) Western blot comparing the effect of ATP on LKB1 phosphorylation in normal cholangiocyte cell line (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88) and iCCA cell line (HUCCT1) (**p<0.01, n=3). (D) Western blots for p-LKB1 and total LKB1 showing the effect of pre-treatment (30 min) with suramin 100 μM or H89 20 μM on the treatment with ATP for 30 min. (E) NHC cells transfected with shRNA-LKB1 (NHC LKB1) or shRNA-scramble (NHC SCR). Expression levels of LKB1 protein were evaluated by western blot (**p<0.01, n=3) and the effect of ATP on migration was evaluated by wound healing assay (**p<0.01, n=3).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Expressing, Western Blot, Immunofluorescence, Transfection, shRNA, Migration, Wound Healing Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A,B) Western blots showing the effect of ATP for 30 minutes on AKT and PTEN phosphorylation in normal ciliated cholangiocytes (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88), and the iCCA cell line HUCTT1. Bar graph shows densitometry expressed as % of phosphorylated/total ratios in control conditions (*p<0.05, n=3) (**p<0.01, n=3).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A) F-actin and filopodia were evaluated by Phalloidin staining (red) in normal ciliated cholangiocytes (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88), the iCCA cell line (HUCCT1), and normal ciliated cholangiocytes with LKB1 knockdown (NHC LKB1), in the presence or absence of ATP. Nuclei were stained in blue with DAPI (Magnification X600). (B) Quantification of filopodia after treatment with ATP for 30–60 min is shown in bars representing the average number of filopodia per cell (**p<0.01 n=67). (C) The effect of ATP for 60 min on FAK expression was evaluated by western blot (**p<0.01 n=3).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Staining, Expressing, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth

doi: 10.1002/hep.30308

Figure Lengend Snippet: (A) Western blot analysis and graph bar showing the effect of HMC 1 mM for 30 min on LKB1 phosphorylation in normal cholangiocytes (NHC SCR), experimentally deciliated cholangiocytes (NHC IFT88 (shRNA-IFT88)) and iCCA cell lines (HUCCT1) (**p<0.01 n=3). (B) Effect of HMC 1 mM for 24–48 h on proliferation. Rates were assessed by MTS assay (**p<0.01, n=24). (C) Effect of HMC 1mM on migration. Bar graph shows the distance migrated by the cells relative to control (vehicle) in 48 h evaluated in NHC SCR, NHC IFT88 (shRNA-IFT88), and HUCCT1 cells assessed by wound healing assay (**p<0.01 n=3). (D) Effect of HMC 1mM on NHC LKB1 (shRNA-LKB1) migration, bar graph shows the distance migrated by the cells relative to control (vehicle) in 24 h (**p<0.01, n=3). (E) Effect of HMC 1 mM on apoptosis evaluated in 24 h by flow cytometry using (FITC)-annexin V/propidium iodide (PI) staining (**p<0.01 n=3). (F) The anti-tumoral effect of HMC was assessed in vivo using a rat orthotopic syngeneic CCA model. Animals were treated for 8 days with 100mg/kg HMC or vehicle after 6 days of tumor initiation. Bar graph shows tumor/liver rate (%) comparing tumors treated with saline solution (control) and HMC (*p<0.05, n=4). (G) Apoptosis in tumor tissues were assessed by DNA fragmentation detection where green dots are cells in apoptosis. Bar graph shows % nuclei/field in control and treated tumors (*p<0.05, n=4).

Article Snippet: After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight.

Techniques: Western Blot, shRNA, MTS Assay, Migration, Wound Healing Assay, Flow Cytometry, Staining, In Vivo

Journal: Nature communications

Article Title: Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents.

doi: 10.1038/ncomms6308

Figure Lengend Snippet: Figure 6 | Ciliary defects in Goto-Kakizaki rats. (a–d) Examples of image analysis on Wistar (a,b) and GK rats (c,d). b-Cells were identified based on insulin staining (red). We used anti-acetylated a-tubulin to visualize cilia. Insulin-negative tissue was masked (shown in grey) and the analysis of the green channel was limited to the b-cell area. Examples of cilia are emphasized in the enlarged insets corresponding to the areas highlighted by the white boxes. Scale bars, 20 mm. (e,f) The number of ciliated b-cells is reduced in GK compared with age-matched Wistar rats. (e) Average ratio of cilia to b-cell volume in three individual animals per strain (n ¼ 20 per group, P ¼ 0.000 (one-way ANOVA) error bars: s.e.m.). (f) Ratio of cilia number to b-cell volume in pooled islets of each strain (n ¼ 60, P ¼ 0.000, error bars: s.e.m.). (g) Ciliary length is similar in GK and Wistar rats (20 islets per animal were analysed, (one-way ANOVA) error bars: s.e.m.). (h) Basal body and ciliary genes are misregulated in GK compared with Wistar controls (n ¼ 6, *P ¼ 0.021, **Po0.01 (one-way ANOVA), error bars: st. dev.). Pcm1, Bbs4 and Ift88 message were normalized to the 18S ribosomal subunit. (i) Pcm1 and Kif3a protein levels are increased, whereas Stx1a levels are decreased in GK islets compared with Wistar controls (n ¼ 3, loading control: g-tubulin).

Article Snippet: Ciliary and basal body gene expression levels were monitored using TaqMan assays for Ift88 (Rn01489251_m1), Bbs4 (Rn01766633_m1) and Pcm1 (Rn01488002_m1) and normalized to 18S (100 fold dilution of the template) with TaqMan Fast Advanced Master Mix (all BD Biosciences/Applied Biosystems) on an ABI Prism 7900HT system.

Techniques: Staining, Control