Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Physical interaction between CEP290 and the BBSome. (A) Co-immunoprecipitation of CEP290 in HEK293T cells stably expressing FLAG-BBS4 and FLAG-BBS5. Lysates from stable cell lines and control (parental) cells were subjected to immunoprecipitation (IP) with the anti-FLAG antibody and precipitated proteins were analyzed by immunoblotting with indicated antibodies. Normal mouse IgG pull-down was used as a negative control. (B and C) Interaction of endogenous CEP290 and the BBSome in HEK293T cells (B) and mouse retina (C). Lysates from HEK293T cells and mouse retina were subjected to IP using antibodies against CEP290, and precipitated proteins were analyzed by immunoblotting with indicated antibodies. (D) Schematic representation of the CEP290 deletion mutants. Numbers indicate expressed portions of CEP290 in amino acid positions. Known IQCB1-, CC2D2A- and RAB8A-binding domains and the BBSome-interacting region are also summarized. SMC, structural maintenance of chromosomes; MYO-Tail, myosin-tail homology domain. (E) The BBSome binds to the N-terminal part of CEP290. CEP290 deletion mutants (FLAG-tagged) were transfected into HEK293T cells and lysates were analyzed by IP using anti-FLAG antibodies. Untransfected cells were used as a negative control. (F) BBS4 interacts with CEP290. HA-tagged, individual BBSome components were transiently transfected with FLAG-Cep_1 constructs. Lysates were subjected to IP with anti-HA antibodies. (G) PCM1-independent interaction between the BBSome and CEP290. HEK293T cells were co-transfected with Cep_1 fragment and siRNA against PCM1.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Immunoprecipitation, Stable Transfection, Expressing, Control, Western Blot, Negative Control, Binding Assay, Transfection, Construct

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Co-localization of CEP290 and the BBSome. (A) Co-localization of GFP-tagged BBS4 and CEP290 in RPE1 cells (yellow arrowheads). Localization of CEP290 (red) was probed with the anti-CEP290 antibody in hTERT-RPE1 after 24 h of serum withdrawal, whereas BBS4-GFP was probed with the anti-GFP antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Note the variable localization of BBS4, although these cells were cultured in the same condition (from a single well). Scale bar, 5 μm. (B) Co-localization of Bbs4 and Cep290 in the mouse retina. Antibodies against Cep290 (red) and rhodopsin (green; a marker of the OS) were used in WT photoreceptors in the left panel, whereas in the middle and right panels Bbs4 (red) and rhodopsin (green) localizations were probed in WT and Bbs4−/− (4KO) photoreceptors. Both Cep290 and Bbs4 localize to the connecting cilium. CC, connecting cilium; ONL, outer nuclear layer. Scale bar, 10 μm. (C) Co-fractionation of the BBSome and Cep290 in the photoreceptor OS fraction. The photoreceptor OS was isolated from the mouse retina and probed with antibodies against multiple subcellular marker proteins, Bbs4, Bbs7 and Cep290.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Staining, Cell Culture, Marker, Fractionation, Isolation

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: The CEP290 localization in the centriolar satellite and the connecting cilium is BBSome-dependent. (A) The BBSome is required for centriolar satellite localization of CEP290 (red). RPE1 cells were transfected with siRNAs against CEP290, BBS1, BBS4, BBS9 and PCM1. Antibodies against γ-tubulin and acetylated tubulin (green) were used to mark the basal body and cilia, respectively. (B) Quantification of CEP290 mis-localization in BBSome-depleted RPE1 cells. Cells with concentrated CEP290 staining around the centrosome (within 2 μm from the centrosome) were counted as positive, whereas cells with CEP290 only in the TZ and centrosome were considered negative. At least 120 cells per experiment were counted and graphs represent averages of three independent experiments. Error bars represent SEM. One-way ANOVA followed by Tukey's post-test was used for statistical analysis. **P < 0.01 compared with Ctrl KD cells. (C) Localization of Cep290 (green; left) in WT (top), Bbs1M390R/M390R (middle) and Bbs4−/− (bottom) mouse retinas. OS localization of Prph2 (red) with respect to acetylated tubulin (green) is not affected in BBS mutant retinas (right panels). Scale bars, 10 μm.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Transfection, Staining, Mutagenesis

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Increased body weight and higher leptin levels in Bbs4+/−; Cep290+/rd16 mice. (A) Weight gains in male animals versus age (minimum of six animals per group). Values are expressed as mean + SEM. By month 3, double-heterozygous mice are significantly heavier than single-heterozygous littermates. *P < 0.05 versus single-heterozygous animals; **P < 0.01 versus single-heterozygous animals. (B) Serum leptin levels of single- and double-heterozygous mice. One-way ANOVA and t-test were used for statistical analysis. *P < 0.05 versus single-heterozygous animals. (C) STAT3 phosphorylation upon leptin administration was reduced in double-heterozygous mice. Hypothalamic protein extracts were analyzed by western blotting. (D) Quantification of STAT3 phosphorylation. Band intensities of phospho-STAT3 (P-STAT3) were normalized with those of total STAT3 and induction ratios were calculated by comparison with vehicle-injected samples. Data represent mean + SEM. n = 5 for vehicle and n = 10 for leptin. *P < 0.05.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Phospho-proteomics, Western Blot, Comparison, Injection

Journal: Human Molecular Genetics

Article Title: BBS mutations modify phenotypic expression of CEP290 -related ciliopathies

doi: 10.1093/hmg/ddt394

Figure Lengend Snippet: Impaired rhodopsin trafficking and diminished ERG responses in mice with combined loss of Cep290 and Bbs4 alleles. (A) Immunohistochemical analysis of WT, Bbs4+/+;Cep290rd/rd, Bbs4+/−;Cep290rd/rd and Bbs4−/−; Cep290rd/rd retinas at P21 with antibodies against rhodopsin. Scale bar, 5 μm. (B) DA-SCR ERG b-wave amplitudes in the indicated mouse genotypes (at the age of 1 month). Removing one or two Bbs4 alleles on a Cep290rd/rd background results in a lower ERG response. **P < 0.01 versus Bbs4+/+;Cep290rd/rd mice. Error bars are SD.

Article Snippet: Other antibodies used were purchased from the following sources: mouse monoclonal antibodies against ABCA4 (3F4; Abcam), acetylated tubulin (6–11B-1; Sigma), γ-tubulin (GTU-88; Sigma), FLAG (M2; Sigma), HA (F-7; Santa Cruz Biotechnology), GFP (3E6; Invitrogen), beta-actin (AC-15; Sigma), rhodopsin (RET-P1; Santa Cruz Biotechnology), and synaptophysin (Santa Cruz Biotechnology; sc-55507); mouse polyclonal antibodies against IQCB1 (NPHP5; Abcam; ab69927); rabbit polyclonal antibodies against BBS8 (Sigma; HPA003310), BBS9 (Sigma; HPA021289), CEP290 (for immunoprecipitation, immunoblotting and RPE1 cell immunofluorescence microscopy; Bethyl Lab; IHC-00365), HSPA5/GRP78/BiP (Cell Signaling; 3177), PCM1 (Sigma; HPA023374), PDI (Sigma; P7372), PRPH2 (Proteintech Group; 18109), STAT3 (Santa Cruz Biotechnology; SC-483), phospho-STAT3 (Y705; Cell Signaling; 9131) and Tgoln2/Tgn46 (Abcam; ab16059).

Techniques: Immunohistochemical staining

Journal: Experimental & Molecular Medicine

Article Title: Reduced expression of TAZ inhibits primary cilium formation in renal glomeruli

doi: 10.1038/s12276-022-00730-2

Figure Lengend Snippet: a Western blots showing IFT88 and IFT140 expression in cells with YAP or TAZ silencing and subjected to 24 h of serum starvation. b Immunocytochemical analysis of IFT140 (red) and the ciliary basal body (green) in SV40MES13 cells with YAP or TAZ knockdown and subjected to 24 h of serum starvation. c The localization of IFT140 in primary cilia was classified into 4 categories: localized only at the ciliary base, localized at both the base and tip, evenly distributed throughout cilia, and not localized in cilia. d NPHP6 and NPHP9 mRNA expression changed in SV40MES13 cells with YAP/TAZ knockdown and subjected to 24 h of serum starvation. e , f Accumulation of NPHP6 and NPHP9 (red) in primary cilia (green) under identical conditions in SV40MES13 cells. g , h The graph shows the fluorescence intensities of NPHP 6 and 9 localized around primary cilia; fluorescence intensities were measured by ZEN blue edition imaging software (Zeiss). i – l Primary cilia recovery in cells with TAZ silencing and knockdown of either NPHP 6 or 9 after 24 h of serum starvation. i Fluorescence images showing primary cilia in cells with knockdown of TAZ or YAP or double knockdown of TAZ and NPHP 6 or 9 after 24 h of serum starvation. j The graphs show the ratio of the number of ciliated cells to the number of DAPI-stained nuclei per image. k Primary cilia lengths were measured by the ZEN black edition program. l The graphs show the proportions of cilia lengths in three ranges: <2.5 μm, 2.5 μm to 5 μm, and >5 μm. a.u. means arbitrary unit. Mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were transfected with siRNAs targeting Nphp6 (sc-72866, Santa Cruz Biotechnology) at a concentration of 30 nM and Nphp9 (50 nM; sc-61177, Santa Cruz Biotechnology) at a concentration of 50 nM; the rest of the transfection process was the same as described above.

Techniques: Western Blot, Expressing, Knockdown, Fluorescence, Imaging, Software, Staining

Journal: Molecular Biology of the Cell

Article Title: The novel centriolar satellite protein SSX2IP targets Cep290 to the ciliary transition zone

doi: 10.1091/mbc.E13-09-0526

Figure Lengend Snippet: Cep290 recruitment to centrosomal satellites and basal bodies depends on SSX2IP in RPE-1 cells. (a) Cells were transfected with control or SSX2IP siRNA and stained for Cep290 and γ-tubulin using indirect immunofluorescence. (b) Average projection of 50 cells immunostained as shown in a. Centering of single images before projection was based on the γ-tubulin signal. (c, d) Quantification of signal intensities of Cep290 at the basal body (c) or in satellites (d) of cells transfected with control or SSX2IP siRNA. (c, d) Left (bars), mean values of averages ± SEM from three independent experiments ( n = 150) normalized to controls. Right (box-and-whiskers plots), quantification of a single representative experiment. * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student's t test).

Article Snippet: Of importance, mutations in Cep290, also known as BBS14, were previously shown to correlate with several ciliopathies, including Bardet–Biedl syndrome, Meckel–Gruber syndrome, and Joubert syndrome ( Staples et al. , 2012 ).

Techniques: Transfection, Control, Staining, Immunofluorescence, Two Tailed Test