Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured in low or high density. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without serum. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without aphidicolin. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. cGAS in nuclear/cytosolic fractions of indicated cell types. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Western Blot, Cell Culture

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Fluorescence images of GFP‐hcGAS, GFP‐hcGASΔcGAMP, GFP‐hcGASΔDNA, and GFP‐hcGASΔOligo in HEK293 cells cultured with or without aphidicolin. Scale bar: 20 μm. B Corresponding quantification of (A). The nuclear cGAS/total cGAS was calculated from 6 different fields with n > 50 cells. C, D A nuclear export signaling (NES) is not sufficient to dislodge chromatin‐bound cGAS from the nucleus. (C) Fluorescence images of GFP‐hcGAS, GFP‐hcGAS‐NLS, and GFP‐hcGAS‐NES in HEK293 cells. Scale bar: 10 μm. (D) Immunoblots of subcellular fractions of GFP‐hCGAS‐, GFP‐hCGAS‐NLS‐, and GFP‐hCGAS‐NES‐expressing HEK293 cells. Data information: Data are presented as means ± SEM. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS: P > 0.05 and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Fluorescence, Cell Culture, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A, B Micronuclei (indicated by arrowhead) in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells before (0 h) or 24 h after γ‐irradiation (IR; 10 Gy). Scale bar: 10 μm (A). (B) The average MNs/cell. Graphs show mean ± SEM ( n = 3 independent experiments) representing six different microscopic fields with over 200 cells. C IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n = 3 independent experiments. D Experimental outline for micronucleus generation and cell death after γ‐irradiation. E Micronucleus (indicated by arrowhead) and cGAS staining in WT and cGAS −/− BMDMos exposed to γ‐irradiation (10 Gy). Scale bar: 10 μm. F Average MNs/cell in BMDMos. MN graphs show mean ± SEM ( n = 3 independent experiments) representing eight different microscopic fields with over 200 cells. G Cell death in WT and cGAS −/− BMDMos that were first synchronized at G2/M, then γ‐irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ± SD, x biological triplicates ( n = 3) per treatment group are shown. Data information: Statistical significance in (B), (C), and (F) was assessed using unpaired two‐tailed Student's t ‐test. *** P ≤ 0.001 and **** P ≤ 0.0001. Statistical significance in (G) was assessed using two‐way ANOVA test, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Expressing, Irradiation, Transfection, Plasmid Preparation, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Pulsed‐field gel electrophoresis analysis of γ‐irradiated (10 Gy) WT and cGAS −/− BMDMos. B, C Comet assay in GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells γ‐irradiated (IR: 10 Gy) for 15 min (B). RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293T cells stimulated with transfected DNA for 6 h (C). D, E Comet assay of HEK293 cells stimulated with 10 μg/ml cGAMP for indicate periods, then γ‐irradiated and incubated at 37°C for indicated duration (D). (E) Immunoblots of IRF3 phosphorylation in HEK293 cells treated as in (D). F–H Images (F) and quantifications (G) of comet tails 15 min after irradiation of GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells. RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells stimulated with transfected 23 DNA for 6 h (H). I, J Images (I) and quantifications (J) of micronuclei in GFP‐NLS‐ and GFP‐hcGASΔcGAMP‐expressing HEK293 cells 24 h after γ‐irradiation (IR; 10 Gy). DAPI (DNA). Scale bar: 10 μm. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. K Quantifications of comet tails 15 min after irradiation (10 Gy) of GFP‐NLS‐, GFP‐hcGAS‐, or GFP‐mcGAS‐expressing HEK293 cells. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. Data information: Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS P > 0.05, *** P ≤ 0.001, and **** P ≤ 0.0001. Mean ± SEM of n = 3 independent experiments. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Pulsed-Field Gel, Electrophoresis, Irradiation, Single Cell Gel Electrophoresis, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Incubation, Western Blot, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Reporter assays showing the effect of NLS and NES on cGAS‐mediated inhibition of DNA repair. B Both full‐length hcGAS and hcGAS cat (161–522aa) inhibit HR repair. C–E cGAS does not impede ATM activation. ATM phosphorylation in γ‐irradiated (10 Gy) GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells (C), GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells (D), or γ‐irradiated (2.5 Gy) WT, cGAS −/− , and Sting −/− BMDMos (E). Data information: Data are means ± SD, n = 3. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. *** P < 0.001 **** P < 0.0001, NS: P > 0.05. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Irradiation, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A cGAS is not recruited to DSB sites: Confocal microscopic images of GFP‐NLS‐ or GFP‐hcGAS‐expressing U2OS‐DSB reporter cells incubated (or not) with Shield‐1 and 4‐OHT to induce the expression and translocation of mCherry‐LacI‐FokI (red) to specific DSB sites. Scale bar: 10 μm. The arrowheads indicate DSB sites. B cGAS does not co‐localize with γ‐H2AX at DSB sites: GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells exposed (or not) to γ‐irradiation (IR: 10 Gy), then stained for γ‐H2AX. Scale bar: 10 μm. C, D Nuclear cGAS is mainly chromatin‐bound and remains unaltered upon γ‐irradiation. (C) Cytosolic (cyto) and nuclear fractions of γ‐irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules. (D) Cytosolic, soluble nuclear, and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins. E–G cGAS co‐isolates with DNA repair proteins because of bound chromatin bridges. (E) Nuclease digestion abrogates the co‐isolation of cGAS and DNA repair proteins: Lysates of control (−IR) and γ‐irradiated (+IR, 10 Gy, 30 min) GFP‐hcGAS‐expressing HEK293 cells were treated (or not) with benzonase before cGAS immunoprecipitation and analysis for indicated proteins. (F) Agarose gel analysis of DNA in corresponding cell lysates in (E). (G) Co‐isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ‐irradiated HEK293 cells expressing GFP‐hcGAS or GFP‐hcGASΔDNA probed for indicated proteins. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Expressing, Incubation, Translocation Assay, Irradiation, Staining, Isolation, Control, Immunoprecipitation, Agarose Gel Electrophoresis, Binding Assay

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Confocal images of γ‐irradiated GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293 cells stained for RAD51 (red) with or without γ‐irradiation. Scale bar: 10 μm. Schematics of the D‐loop formation assay, including pre‐incubation of template dsDNA with cGAS cat (i) or with cGAS cat being added after RAD51 was bound to dsDNA (ii). Pre‐incubation of dsDNA with mcGAS cat prevents D‐loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D‐loop formed in each reaction (left) was graphed as the average of triplicates ± SD. Schematics of the D‐loop assay. Pre‐incubation of template dsDNA with hcGAS cat blocks subsequent D‐loop formation. The percentage of D‐loop formation (below) was graphed as the average of triplicates ± SD. Data information: Unpaired two‐tailed Student's t ‐test was used for statistical analyses. NS P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Irradiation, Expressing, Staining, Tube Formation Assay, Incubation, Activity Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Negative‐stain electron micrographs of cGAS‐dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm. Effect of indicated hcGAS variants on D‐loop formation when pre‐incubated with dsDNA. Percentage of D‐loop formed in each reaction (left) graphed as the average of triplicates ± SD. Overview of a single 1:1 hcGAS‐DNA complex depicting the location of the Y215 within the cGAS‐dsDNA interface. DR‐GFP assay showing that hcGASΔDNA‐Y215E is impaired in HR inhibition. hcGASΔDNA‐Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24. hcGASΔDNA‐Y215E and hcGASΔcGAMP are defective in synthase activity. Negative‐stain electron micrographs showing that hcGAScat‐ΔDNA‐Y215E is defective in inducing cGAS‐dsDNA complexes. Scale bar: 100 nm. Effect of indicated hcGAS variants on D‐loop formation. Data information: Data are means ± SD, n = 3. Unpaired Student's t ‐test was used for statistical analyses: NS P > 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Staining, Incubation, Inhibition, Activity Assay

Journal: Scientific Reports

Article Title: A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples

doi: 10.1038/s41598-021-95799-6

Figure Lengend Snippet: Limit of detection of the two different RT-LAMP formats and of RT-PCR. ( A ) A known number of copies of in vitro transcribed (IVT) viral RNA (N-gene) were amplified and detected by colorimetric RT-LAMP using the (i) WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs) or (ii) the separate components (enzymes purchased individually and an in-house-made colorimetric buffer). The reactions were incubated at 65 °C for 30 min. ( B ) 10 μL of the RT-LAMP reaction were resolved in an agarose gel (2%) electrophoresis. The ladder pattern corresponds to the expected LAMP amplification pattern. ( C ) Limit of detection of ten replicates of the two test formats. ( D ) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig. ).

Article Snippet: In one format, we used the WarmStart Colorimetric LAMP 2 × Master Mix (New England Biolabs), which includes all the reagent components with the exception of the primers.

Techniques: Reverse Transcription Polymerase Chain Reaction, In Vitro, Amplification, Incubation, Agarose Gel Electrophoresis, Electrophoresis, Generated

Journal: Scientific Reports

Article Title: Metformin inhibits lithocholic acid-induced interleukin 8 upregulation in colorectal cancer cells by suppressing ROS production and NF-kB activity

doi: 10.1038/s41598-019-38778-2

Figure Lengend Snippet: Metformin inhibits LCA-induced IL-8 expression in HCT116 CRC cells. ( A ) Three CRC cell lines, SW480, HT29, HCT116, were treated with 10 mM metformin for 1 h. The cells were then incubated with 30 µM LCA for 4 h. Then, mRNA was extracted, and IL-8 expression was evaluated by RT-PCR. ( B ) HCT116 cells pretreated with 0–20 mM metformin for 1 h were exposed to 30 µM LCA for 4 h. The cells were then extracted for mRNA, and IL-8 expression was evaluated by RT-PCR. ( C ) HCT116 cells transfected with the pGL2-IL-8 plasmid were pretreated with 0–20 mM metformin for 1 h. The cells were then incubated with 30 µM LCA for 12 h, lysed with passive lysis buffer, and submitted for the luciferase assay. ( D ) HCT116 cells were pretreated with 0–20 mM metformin for 1 h and incubated with 30 µM LCA for 24 h. The culture media were harvested and utilized for ELISA to check the levels of secreted IL-8 cytokine. Agarose gel images were cropped for clarity of the presentation. # P < 0.05 versus control; *P < 0.05 versus LCA. The above data represent the means ± SD from triplicate measurements.

Article Snippet: HCT116 human colon carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Lysis, Luciferase, Enzyme-linked Immunosorbent Assay, Agarose Gel Electrophoresis, Control

Journal: Scientific Reports

Article Title: Metformin inhibits lithocholic acid-induced interleukin 8 upregulation in colorectal cancer cells by suppressing ROS production and NF-kB activity

doi: 10.1038/s41598-019-38778-2

Figure Lengend Snippet: Metformin inhibition of LCA-induced IL-8 expression in HCT116 CRC cells is not mediated through MAPK signaling. ( A ) HCT116 cells pretreated with 10 mM metformin for 1 h were exposed to 30 µM LCA for 30 min, 60 min, or 120 min. The cells were then extracted for protein and tested for the levels of phosphorylated Erk1/2, JNK, and P-38. ( B ) HCT116 cells transfected with the pGL3-AP1 promoter and pRLTK plasmids were pretreated with 30 µM of PD, JNKi, or SB for 1 h. The cells were then incubated with 30 µM LCA for 12 h and lysed with passive lysis buffer to check AP-1 activity by dual luciferase assay. ( C ) HCT116 cells transfected with the pGL3-AP1 promoter and pRLTK plasmids were pretreated with 0–20 mM metformin for 1 h. The cells were then incubated with 30 µM LCA for 12 h and checked for AP-1 activity by dual luciferase assay. ( D ) HCT116 cells were pretreated with 10 mM metformin for 1 h and incubated with 30 µM LCA for 0–240 min. The cells were then extracted for protein and checked for total and phosphorylated STAT3 protein. Blot images were cropped for clarity of the presentation. # P < 0.05 versus control; *P < 0.05 versus LCA. The above data represent the means ± SD from triplicate measurements.

Article Snippet: HCT116 human colon carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Inhibition, Expressing, Transfection, Incubation, Lysis, Activity Assay, Luciferase, Control

Journal: Scientific Reports

Article Title: Metformin inhibits lithocholic acid-induced interleukin 8 upregulation in colorectal cancer cells by suppressing ROS production and NF-kB activity

doi: 10.1038/s41598-019-38778-2

Figure Lengend Snippet: Metformin inhibits LCA-induced IL-8 expression in HCT116 CRC cells via NF-κB signaling. ( A ) HCT116 cells were treated with 0–20 µM Bay and incubated with 30 µM LCA for 4 h. The cells were then extracted for mRNA, and IL-8 expression was checked by RT-PCR. ( B ) HCT116 cells were treated with 0–20 µM Bay for 1 h and then incubated with 30 µM LCA. After 24 h, cultured media were harvested and evaluated specifically for human IL-8 by ELISA. ( C ) Dominant mutants of IκBα or IκBβ were cotransfected with the pGL2-IL-8 plasmid and pRLTK plasmid into HCT116 cells. The cells were then incubated with LCA for 12 h, and the IL-8 promoter activity was evaluated by dual-luciferase assay. ( D ) HCT116 cells were pretreated with metformin for 1 h, and then 30 µM LCA was added for 30, 60, or 120 min. The cells were then harvested, and nuclear and cytoplasmic proteins were fractionated to check for P65 and Ikβα by Western blot analysis. ( E ) A 200 bp fragment of the IL-8 promoter immunoprecipitated by an NF-κB P65 specific antibody and the DNA input of CHIP assay were quantitated via PCR. ( E ) HCT116 cells transfected with the pGL3-NF-κB and pRLTK plasmids were treated with 0–20 mM metformin for 1 h. Subsequently, 30 µM LCA was added to the cells for 12 h, and NF-κB promoter activity was tested by dual luciferase assay. Agarose gel and blot images were cropped for clarity of the presentation. # P < 0.05 versus control; *P < 0.05 versus LCA. The above data represent the means ± SD from triplicate measurements.

Article Snippet: HCT116 human colon carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Activity Assay, Luciferase, Western Blot, Immunoprecipitation, Transfection, Agarose Gel Electrophoresis, Control

Journal: Scientific Reports

Article Title: Metformin inhibits lithocholic acid-induced interleukin 8 upregulation in colorectal cancer cells by suppressing ROS production and NF-kB activity

doi: 10.1038/s41598-019-38778-2

Figure Lengend Snippet: Metformin blocks ROS production induced by LCA, in turn inhibiting NF-κB activity to diminish LCA-induced IL-8 expression in HCT116 CRC cells. ( A , B ) Representative images (80x) and statistically quantitative values of ROS production by confocal microscope. Scale bar, 10 µM. ( C ) pGL3-NF-κB plasmid-transfected HCT116 cells were treated with 0–2.5 mM NAC for 1 h. Subsequently, 30 µM LCA was added for 12 h, and the cells were harvested to evaluate the NF-κB promoter activity by dual luciferase assay. # P < 0.05 versus control; *P < 0.05 versus LCA. The above data represent the means ± SD from triplicate measurements.

Article Snippet: HCT116 human colon carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Activity Assay, Expressing, Microscopy, Plasmid Preparation, Transfection, Luciferase, Control

Journal: Scientific Reports

Article Title: Metformin inhibits lithocholic acid-induced interleukin 8 upregulation in colorectal cancer cells by suppressing ROS production and NF-kB activity

doi: 10.1038/s41598-019-38778-2

Figure Lengend Snippet: Metformin suppresses NADPH oxidase, in turn blocking LCA-stimulated ROS production. ( A ) HCT116 cells were treated with 0–30 µM LCA for 4 h and then lysed with NADPH buffer to extract protein for the NADPH oxidase assay. ( B ) HCT116 cells were pretreated with 0–20 mM metformin for 1 h, and then 30 µM LCA was added for 4 h. The cells were then lysed with NADPH buffer, and NADPH oxidase activity was checked. ( C ) HCT116 cells were treated with specific NADPH oxidase inhibitors, DPI and apocynin, for 1 h. The cells were then treated with 30 µM LCA for 4 h and checked for IL-8 expression by RT-PCR. ( D ) HCT116 cells pretreated with 15–30 µM DPI and 2.5–10 µM apocynin for 1 h were incubated with 30 µM LCA for 24 h. The cultured media were then harvested and used for secreted IL-8 quantification by ELISA. Agarose gel images were cropped for clarity of the presentation. # P < 0.05 versus control; *P < 0.05 versus LCA. The above data represent the means ± SD from triplicate measurements.

Article Snippet: HCT116 human colon carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Blocking Assay, Activity Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Agarose Gel Electrophoresis, Control

Journal: Scientific Reports

Article Title: Metformin inhibits lithocholic acid-induced interleukin 8 upregulation in colorectal cancer cells by suppressing ROS production and NF-kB activity

doi: 10.1038/s41598-019-38778-2

Figure Lengend Snippet: Metformin inhibits proliferation and tubelike formation of ECV304 endothelial cells stimulated by CM derived from LCA-treated HCT116 CRC cells. ( A ) ECV304 cells were incubated in DMEM media supplemented with 10% FBS for 24 h. Then, CM derived from LCA-treated HCT116 cells with and without 5–20 mM metformin (Met) pretreatment was added for another 24 h. Cell proliferation was checked by cell viability, proliferation & cytotoxicity assays. ( B , C ) Quantitative data and representative images (10x) of the tube formation assay. ECV304 cells were seeded on Matrigel-coated wells of a 96-well plate and then incubated with CM derived from LCA-treated HCT116 cells with and without 5–20 mM metformin pretreatment. After 12 h, the cells were observed with a Nokia microscope to check the formation of tubelike structures. Scale bar, 500 µM. # P < 0.05 versus control; *P < 0.05 versus LCA. The above data represent the means ± SD from triplicate measurements.

Article Snippet: HCT116 human colon carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD, USA).

Techniques: Derivative Assay, Incubation, Tube Formation Assay, Microscopy, Control

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. Silver staining of the eluate following tandem affinity purification of N-terminally LAP (EGFP-TEV cleavage site-S tag-PreScission cleavage site)-tagged CEP89 expressed in confluent RPE cells. The cell lysates were purified with GFP antibodies and S-protein beads, resolved by Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) and visualized by silver staining. The bands corresponding to S tagged CEP89 (S-CEP89), NCS1, and C3ORF14 are indicated. Molecular weights (kDa) estimated from a protein marker are indicated. Asterisk denotes a band corresponding to TEV protease used for tandem affinity purification. B. Tabulation of peptide-spectrum matches (PSMs), unique peptide counts, coverage, and the length of the amino acids from the mass spectrometry analysis of the eluate shown in (A). C. A cartoon depicting the region of CEP89 important for centrosomal localization or binding to NCS1. Localization data can be found in . D. Immunoblot (IB) analysis of the eluates from a co-immunoprecipitation assay of the full length or the indicated fragments of N-terminally LAP-tagged CEP89 expressed in confluent RPE cells. The cell lysates were purified with GFP antibodies, resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Molecular weights (kDa) estimated from a protein marker are indicated. E-F. Immunoblot (IB) analysis of the eluates from in vitro binding assay of the in vitro translated (IVT) N-terminally HA-tagged CEP89 (E) or C3ORF14 (F) and the indicated N-terminally MYC tagged proteins. The in vitro translated proteins were mixed and captured by HA-agarose beads, resolved by SDS-PAGE and immunoblotted with the indicated antibodies. The CEP350 fragment (2470-2836 a.a.), which binds to FOP efficiently serves as a negative control. Red asterisks indicate non-specific bands, which overlap with the MYC-tagged C3ORF14. Molecular weights (kDa) estimated from a protein marker are indicated. G. The order of binding for CEP89-NCS1-C3ORF14 interaction.

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Silver Staining, Affinity Purification, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Marker, Mass Spectrometry, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Negative Control

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. Immunoblot (IB) analysis of the eluates from co-immunoprecipitation assay of endogenous NCS1 in the control (sgSafe) or NCS1 knockout cells. The eluates were immunoblotted with antibodies against NCS1 or CEP89. Molecular weights (kDa) estimated from a protein marker are indicated. B. Immunofluorescence images taken via wide-field microscopy. RPE cells expressing the indicated N-terminally LAP (EGFP and S) tagged fragments of CEP89 or LAPN (control) were serum starved for 24 hours, fixed, and stained with antibodies against GFP and CEP170. Scale bar: 10 μm.

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Control, Knock-Out, Marker, Immunofluorescence, Microscopy, Expressing, Staining

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. Immunofluorescence images taken via wide-field microscopy. Control (sgGFP) or NCS1 knockout RPE cells were serum starved for 24 hours, fixed, and stained with indicated antibodies. Insets at the right panels are the enlarged images of the mother centriole. Ac-Tub indicates acetylated α-tubulin. Scale bar: 10 μm. B. Immunofluorescence images taken via wide-field microscopy. RPE cells expressing C-terminally LAP (LAPC) tagged C3ORF14 were serum starved for 24 hours, fixed, and stained with indicated antibodies. Scale bar: 10 μm. C. Box plots showing centrosomal signal intensity of NCS1. RPE cells were grown in fetal bovine serum (FBS)-containing media for 24 hours, and then grown in either fetal bovine serum (FBS)-containing media (+ FBS) or serum free media (-FBS) for an additional 24 hours. Cells were fixed and stained with NCS1 antibody. Centrosomal signal intensity of NCS1 was measured from fluorescence images using the method described in Materials and Methods. A.U., arbitrary units. Data are combined from three replicates. Statistical significance was calculated from a nested T-test. The raw data, experimental conditions, and detailed statistics are available in Figure 2C-Source data D-I. Immunofluorescence images taken via 3D-structured illumination microscopy. Side view (D, F, G, and H) or Top view (E and I) is shown. RPE cells were either grown to confluent (H) or serum starved for 24 hours (D-G and I), fixed and stained with indicated antibodies. Each individual image is from a representative z-slice. Scale bar: 1 μm. CEP170: a marker of subdistal appendage and proximal end of the mother centriole. A cartoon at the right of each figure shows estimated positions of each protein at the mother centriole. J. A cartoon depicting the localization of NCS1 relative to RAB34 and CEP89. NCS1 is sandwiched between RAB34 and CEP89. K-M. Immunofluorescence images taken via wide-filed microscopy. Control (sgGFP) or indicated knockout RPE cells were serum starved for 24 hours, fixed, and stained with indicated antibodies. Scale bar: 10 μm. Insets at the right panels are the enlarged images of the mother centriole. Quantification data are available in . N. A cartoon depicting the order of recruitment of the CEP89-NCS1-C3ORF14 complex.

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Immunofluorescence, Microscopy, Control, Knock-Out, Staining, Expressing, Fluorescence, Marker

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A-D. Immunofluorescence images taken via wide-field microscopy. RPE cells expressing N-terminally LAP tagged wilde-type NCS1 (A), N-terminally LAP tagged myristoylation defective mutant (G2A) of NCS1 (B), C-terminally LAP tagged wild-type NCS1 (C), or C-terminally LAP tagged myristoylation defective mutant (G2A) of NCS1 (D) were serum starved for 24 hours, fixed, and stained with indicated antibodies. Scale bar: 10 μm. Insets at the right panels are the enlarged images of the mother centriole. The experimental conditions are available in Figure 2-Figure Supplement 1-Source Data.

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Immunofluorescence, Microscopy, Expressing, Mutagenesis, Staining

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A-C. Box plots showing centrosomal signal intensity of CEP89 (A), NCS1 (B), or GFP-C3ORF14 (C) from the immunofluorescence experiments shown in Fig.2K-M. Data from a representative experiment are shown. A.U., arbitrary units. The raw data and experimental conditions are available in Figure 2-Figure Supplement 2A-C-Source Data. D-E. Immunoblot (IB) analysis of expression of CEP89, NCS1, and LAPC-C3ORF14 in indicated RPE cells. The cells were serum starved for 24 hours, lysed and analyzed by immunoblot. Molecular weights (kDa) estimated from a protein marker are indicated. A.U., arbitrary units; n.s., not significant

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Immunofluorescence, Western Blot, Expressing, Marker

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A-B. Box plots showing centrosomal signal intensity of TTBK2 (A) and KIZ (B) in control (sgGFP), CEP89, NCS1, or C3ORF14 knockout RPE cells. The data combined from three independent experiments. Statistical significance was calculated from nested T test. The raw data, experimental conditions, detailed statistics are available in Figure 2-figure supplement 3A-B-Source data. C-D. Quantification of centrosomal signal intensity of ANKRD26 (C) and NCS1 (D) in indicated RPE cells. The data from the representative experiment are shown. The raw data and experimental condition are available in the source data of Figure 2-figure supplement 3C-D-Source data.

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Control, Knock-Out

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. Time course of cilium formation assay in control (sgGFP) and indicated knockout RPE cells. The indicated cells were serum starved for 12, 24,48,72,96 hours, fixed, stained with α -ARL13B (to mark cilium) and α -CEP170 (to mark centriole), and imaged via wide-field microscopy. Data averaged from four independent experiments. Error bars represent ± SEM. Statistics obtained through comparing between each knockout and control by Welch’s t-test. The raw data, experimental conditions, detailed statistics are available in Figure 3A-Source data. B. Cilium formation assay in control (sgGFP) and indicated knockout RPE cells serum starved for 24 hours. Data averaged from three independent experiments, and each black dot indicates the value from the individual experiment. Error bars represent ± SEM. Statistics obtained by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 3B-Source data. C. Ciliary vesicle recruitment assay in control (sgGFP) or indicated knockout RPE cells grown to confluence (without serum starvation). The data are averaged from 4 ndependent experiments, and each black dot indicates the value from each individual experiment. Error bars represent ± SEM. Statistics obtained through comparing between each knockout and control by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 3C-Source data. D. Transmission electron microscopy analysis of the mother centriole in control (sgGFP) or NCS1 knockout RPE cells serum starved for 3 hours. The representative mages of the mother centrioles without (left) or with (right) ciliary vesicle at the distal appendage are shown. Scale: 200 nm. E. Quantification of the data from the experiments shown in . The raw data and detailed statistics are available in Figure 3E-Source data. This experiment was synchronized with the experiment shown in the of (Tomoharu ), hence the values for sgGFP are exactly the same as the ones shown in Tomoharu ). F. CP110 removal assay in control (sgGFP) and indicated knockout RPE cells serum starved for 24 hours. Data are averaged from three independent experiments, and each black dot indicates the value from the individual experiment. Error bars represent ± SEM. Statistics obtained through comparing between each knockout and control by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 3F-Source data. G-H. Quantification of the centrosomal signal intensity of IFT88 (G) or CEP19 (H) in control (sgGFP) and indicated knockout RPE cells serum starved for 24 hours. The data are combined from three independent experiments. Statistical significance was calculated from nested T test. The raw data, experimental conditions, detailed statistics are available in Figure 3G-H-Source Data. A.U., arbitrary units; n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Tube Formation Assay, Control, Knock-Out, Staining, Microscopy, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. Immunoblot (IB) analysis of expression of NCS1 (IB: NCS1) and α-tubulin (IB: Tub) in control (sgGFP) or indicated knockout RPE cells stably expressing either sgSafe (non-targeting) or sgNCS1. The cells were grown to confluence (without serum starvation), lysed and analyzed by immunoblot. Molecular weights (kDa) estimated from a protein marker are indicated. B-C. Cilium formation assay in control (sgGFP) and indicated knockout RPE cells stably expressing either sgSafe (non-targeting) or sgNCS1. The cells were serum starved for 24 (B) or 48 (C) hours. Data averaged from three independent experiments, and each black dot indicates the value from the individual experiment. Error bars represent ± SEM. Statistics obtained by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 4B-C-Source Data. D. Ciliary vesicle recruitment assay in control (sgGFP) or indicated knockout RPE cells stably expressing either sgSafe (control) or sgNCS1. Cells were grown to confluence (without serum starvation). Data are averaged from three independent experiments. Error bars represent ± SEM. Statistics obtained by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 4D-Source data. n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Western Blot, Expressing, Control, Knock-Out, Stable Transfection, Marker, Tube Formation Assay

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. Immunoblot (IB) analysis of the eluates from co-immunoprecipitation assay of NCS1 knockout RPE cells stably expressing wild-type or indicated point mutants of untagged NCS1 and N-terminally LAP (EGFP and S) tagged CEP89. The cell lysates were co-immunoprecipitated with GFP antibodies, resolved by SDS-PAGE and immunoblotted with indicated antibodies. Molecular weights (kDa) estimated from a protein marker are indicated. B. Box plots showing centrosomal signal intensity of NCS1. Control (sgGFP) or NCS1 knockout RPE cells stably expressing wild-type or indicated point mutants of NCS1were serum starved for 24 hours. Cells were fixed and stained with NCS1 antibody. Centrosomal signal intensity of NCS1 was measured from fluorescent images with the method described in Materials and Methods. A.U., arbitrary units. Data from a representative experiment are shown. The raw data and experimental conditions are available in -figure supplement 1B-Source data. C. Cilium formation assay in cells described in (B) serum starved for 24 hours. Data are averaged from three independent experiments, and each black dot indicates the value from the individual experiment. Error bars represent ± SEM. Statistics obtained through comparing between each mutant and wild-type NCS1 by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 5-figure supplement 1C-Source data. n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Knock-Out, Stable Transfection, Expressing, Immunoprecipitation, SDS Page, Marker, Control, Staining, Tube Formation Assay, Mutagenesis

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. A cartoon showing the method used to test the requirement of microtubules in ciliary vesicle recruitment and NCS1 localization. RPE cells were cultured in media containing 10% FBS for 72 hours, then serum starved (-FBS) for indicated times in the presence of nocodazole or dimethyl sulfoxide (DMSO). B. Immunofluorescence images taken via wide-filed microscopy. RPE cells were cultured as shown in (A), fixed, and stained with antibodies against α-Tubulin and FGFR1OP (FOP). Scale bar: 10 μm. C. The time course of cilium formation in cells treated with either DMSO (magenta) or nocodazole (blue). The cells were fixed at indicated time points, stained with α-ARL13B (to mark cilium) and α-CEP170 (to mark centriole), and imaged via wide-field microscopy. Data averaged from three independent experiments. Error bars represent ± SEM. Statistics obtained through comparing between DMSO and nocodazole treated cells at each time point by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 6C-Source data. D. The time course of ciliary vesicle recruitment in cells treated with either DMSO (magenta) or nocodazole (blue). The cells were fixed at indicated time points, stained with α-RAB34 (to mark ciliary vesicle) and α-CEP170 (to mark centriole), and imaged via wide-field microscopy. Data are averaged from three independent experiments. Error bars represent ± SEM. Statistics obtained through comparing between DMSO and nocodazole treated cells at each time point by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 6D-Source data. E. Box plots showing centrosomal signal intensity of NCS1 in RPE cells prepared using the method described in (A). The data from the representative experiment are shown. The raw data and experimental conditions are available in Figure 6E-Source data. F. Quantification of the centrosomal signal intensity of NCS1 in control or RAB34 knockout RPE cells serum starved for 24 hours. The data from the representative experiment are shown. The raw data and experimental conditions are available in Figure 6F-Source data. A.U., arbitrary units; n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Cell Culture, Immunofluorescence, Microscopy, Staining, Control, Knock-Out

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. Immunoblot (IB) analysis of the indicated tissue lysate from Ncs1 +/+ (7-week-old) or Ncs1 -/-(6-week-old) mice. 50 μg (for NCS1 detection) or 12 μg (for detection of other proteins) of each tissue lysate was loaded and analyzed by immunoblot with the indicated antibodies. Molecular weights (kDa) estimated from a protein marker are indicated.

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Western Blot, Marker

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A-E. Immunofluorescence images of indicated mouse tissues taken via spinning disk confocal microscopy. Tissue sections prepared from 8-week-old Ncs1 +/+ or Ncs1 -/-mice with the method described in materials and methods were stained for indicated markers. Arrowheads indicate NCS1 localization. The individual image is from a representative z-slice. Scale bar: 10 μm. P, photoreceptor cell layer; ONL, outer nuclear layer; OPL, outer plexiform layer.

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Immunofluorescence, Confocal Microscopy, Staining

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A-B. Box plots showing ciliary signal intensity of SSTR3 (A) or GPR161 (B) in isolated hippocampal neurons prepared from E18.5 Ncs1 +/+ or Ncs1 -/-mouse embryos. The cells were cultured in vitro for 7 days (DIV7), fixed, and stained with either SSTR3 or GPR161 together with α-ARL13B (to mark cilium) and α-FGFR1OP (to mark centriole), and imaged via wide-field microscopy. Data are averaged from five different neurons per genotype for SSTR3 and 3 Ncs1 +/+ and 4 Ncs1 -/-neurons for GPR161. Statistical significance was calculated from nested T test. The raw data, experimental conditions, detailed statistics are available in .

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Isolation, Cell Culture, In Vitro, Staining, Microscopy

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. A table showing viability of Ncs1 +/+, Ncs1 +/-, or Ncs1 -/-mice, generated by crossing Ncs1 +/-male and female, at P21. Detailed information of the mice and statistics are available in Figure 8A-Source Data. B-C. Body weight analysis of Ncs1 +/+, Ncs1 +/-, or Ncs1 -/-male (B) and female (C) mice. Raw data and detailed statistics are available from Figure 8B-C-Source Data. D. Measurements of the weights of inguinal fat (left) or epididymal fat (right) from 20-week-old Ncs1 +/+ or Ncs1 -/-male mice. Raw data and detailed statistics are available in Figure D-Source Data. E. Hematoxylin and Eosin (H&E) staining of the retina prepared from 50-week-old Ncs1 +/+ or Ncs1 -/-female mice. Scale bar: 50 μm. G, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; P, photoreceptor cell layer. Representative images from five Ncs1 +/+ or Ncs1 -/-mice are shown. Detailed information of the mice is available in Figure 8E-G-H-Source Data. F. Quantification of ONL/INL ration of the retina prepared from 50-week-old Ncs1 +/+ or Ncs1 -/-mice. 8 areas per mouse and 5 mice from each genotype were analyzed. Statistical significance was calculated from nested T test. The raw data, detailed information of the mice, and detailed statistics are available in Figure 8F-Source Data. G-H. H&E staining of the kidney (G) or Testis (H) prepared from 50-week-old Ncs1 +/+ or Ncs1 -/- female mice (G) or 20-week-old Ncs1 +/+ or Ncs1 -/-male mice. Scale bar: 1mm (G) and 100 μm (H). Representative images from five (G) or three (H) Ncs1 +/+ or Ncs1 -/-mice are shown. Detailed information of the mice is available in Figure 8E-G-H-Source Data. n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Generated, Staining

Journal: bioRxiv

Article Title: Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

doi: 10.1101/2023.01.06.523037

Figure Lengend Snippet: A. Immunoblot (IB) analysis of expression of NCS1, CEP89, EGFR, and RabGDI. The control (sgSafe) and indicated knockout cells were grown to confluence, lysed by nitrogen cavitation, and fractionated by differential centrifugation at 1,000, 15,000, and 100,000 x g. S: supernatant; P: pellet. Molecular weights (kDa) estimated from a protein marker are indicated. EGFR and RabGDI serves as representative markers for plasma membrane or cytoplasmic proteins, respectively. B. Immunoblot (IB) analysis of expression of NCS1 and α -tubulin in control (sgSafe) or or indicated RPE cells. Molecular weights (kDa) estimated from a protein marker are indicated. C. Immunofluorescence images taken via wide-filed microscopy in the cells described in (B) serum starved for 24 hours. Insets at the right panels are the enlarged images of the mother centriole. Scale bar: 10 µm. D. Box plots showing centrosomal signal intensity of NCS1 in cells described in (B) that were serum starved for 24 hours. A.U., arbitrary units. The data from a representative experiment are shown. The raw data and experimental conditions are available in Figure 5D-Source data. E. Cilium formation assay in the cells described in (B) serum starved for 24 hours. Data averaged from four independent experiments, and each black dot indicates the value from the individual experiment. Error bars represent ± SEM. Statistics obtained by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 5E-Source data. F. Ciliary vesicle recruitment assay in the cells described in (B) grown to confluence (without serum starvation). Data averaged from 5 independent experiments. Error bars represent ± SEM. Statistics obtained through comparing between each knockout and control by Welch’s t-test. The raw data, experimental conditions, and detailed statistics are available in Figure 5F-Source data. G. A cartoon depicting the model generated by our results. NCS1 is recruited to the distal appendage by CEP89 and captures ciliary vesicle via its myristoylation motif. n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: For co-immunoprecipitation with endogenous NCS1 shown in , the lysate was incubated with mouse monoclonal anti-NCS1 antibody (sc-376206, Santa Cruz) (1 μg of antibody per 4 mg of lysate) for 1 hour with end-over-end rotation.

Techniques: Western Blot, Expressing, Control, Knock-Out, Centrifugation, Marker, Clinical Proteomics, Membrane, Immunofluorescence, Microscopy, Tube Formation Assay, Generated