Journal: Nature Communications

Article Title: Transiently formed nucleus-to-cilium microtubule arrays mediate senescence initiation in a KIFC3-dependent manner

doi: 10.1038/s41467-024-52363-w

Figure Lengend Snippet: a Western blot detecting KIFC3 and senescence markers in RCTE cells with or without IR exposure. Three experiments were repeated independently with similar results. Immunofluorescent images showing KIFC3 (green) and polyglutamylated sinc-MTs (labeled with GT335 antibody, red) ( b ) and relative intensity of KIFC3 ( c ) in RCTE cells with or without IR exposure. CENTRIN2 (cyan) labels the ciliary base . n = 50 cells. Scale bar, 10 μm. SIM series section ( d ) and 3D surface-rendering reconstruction ( e ). Scale bar, 10 μm. Localization of KIFC3 (green) along polyglutamylated sinc-MTs (red) in RCTE cells exposed to IR. f Immunofluorescence images showing KIFC3 and sinc-MTs (labeled with α-tubulin) in control or siTTLL5 RCTE cells after IR exposure. CENTRIN2 labels the ciliary base. Scale bar, 10 μm. Western blot of senescence markers ( g ), quantitation of relative protein levels of senescence markers ( h ), SA-β-gal staining ( i ), relative mRNA level of SASP genes ( j ) in control or shKIFC3 RCTE cells at day 10 after IR exposure. Scale bar, 50 μm. All results from n = 3 independent experiments. Data are the mean ± SEM. Statistical significance was determined using one-way ANOVA. Three experiments were repeated independently with similar results ( d – f ). Source data are provided as a file.

Article Snippet: Sub-cloning templates of human CENEXIN1 and KIFC3 were purchased from Addgene (#73334) and DNASU(#HsCD00442644), and ODF2(iso6) was kindly provided by Dr. Kyung Lee (NIH/NCI).

Techniques: Western Blot, Labeling, Immunofluorescence, Control, Quantitation Assay, Staining

Journal: Nature Communications

Article Title: Transiently formed nucleus-to-cilium microtubule arrays mediate senescence initiation in a KIFC3-dependent manner

doi: 10.1038/s41467-024-52363-w

Figure Lengend Snippet: a Immunoprecipitation showing no interaction between V5-tagged KIFC3 and Myc-tagged FBF1 when overexpressed in 293 T cells. b , c Immunoprecipitation of endogenous CENEXIN1 with Myc-tagged FBF1 in IR-treated RCTE cells. d V5-tagged KIFC3 immunoprecipitates with EGFP-tagged CENEXIN1 when overexpressed in 293 T cells. e , f Endogenous CENEXIN1 immunoprecipitates with V5-tagged KIFC3 in control or IR-treated RCTE cells. g Immunofluorescence images showing PML-NBs translocation of CENEXIN1 (green) in IR-treated RCTE cells. PML (red) labels the PML-NBs. Scale bar, 10 μm. h 3D surface-rendering reconstruction of SIM section images showing the spatial relationship among CENEXIN1 (green), KIFC3 (red), and EB1 (cyan) labeled plus-ends of sinc-MTs at ciliary base in IR-treated RCTE cells. Scale bar, 10 μm. Three experiments were repeated independently with similar results ( a – h ). Source data are provided as a file.

Article Snippet: Sub-cloning templates of human CENEXIN1 and KIFC3 were purchased from Addgene (#73334) and DNASU(#HsCD00442644), and ODF2(iso6) was kindly provided by Dr. Kyung Lee (NIH/NCI).

Techniques: Immunoprecipitation, Control, Immunofluorescence, Translocation Assay, Labeling

Journal: Nature Communications

Article Title: Transiently formed nucleus-to-cilium microtubule arrays mediate senescence initiation in a KIFC3-dependent manner

doi: 10.1038/s41467-024-52363-w

Figure Lengend Snippet: a Diagram of WT and KIFC3 variants used in experiments. b Endogenous CENEXIN1 immunoprecipitates with V5-tagged WT or KIFC3 DN in RCTE cells, asterisk labels KIFC3 ∆N . c Immunofluorescent images showing localization of NeonGreen(NG)-tagged KIFC3 truncation variants (green) in RCTE cells. γ-tubulin (red) labels the ciliary base. Scale bar, 10 μm. Immunofluorescence images showing the effect of re-expression of KIFC3 or KIFC3 DN on CENEXIN1 and FBF1 translocation. CENEXIN1 (green), FBF1 (red) and PML (cyan) were immunostained by antibodies, respectively ( d ) and quantitation of PML-NBs ( e ) in shKIFC3 RCTE cells with or without IR exposure ( n = 40 cells). Scale bar, 10 μm. Immunofluorescence images showing the changes of CENEXIN1 (red) and PML (cyan) in control or over-expression NG-tagged KIFC3 DN RCTE cells with or without IR treatment ( f ) and quantitation of PML-NBs numbers per cell ( n = 40 cells) ( g ). Localization of KIFC3 DN was shown by NG direct fluorescence (green). Scale bar, 10 μm. h SA-β-gal staining of IR-treated shKIFC3 RCTE cells re-expressing KIFC3 or KIFC3 DN . Scale bar, 100 μm. i Immunofluorescence images showing the impact on the PML-NBs translocation of CENEXIN1 (green) and FBF1 (red) after re-expressing CEP170C-tagged KIFC3 in IR-treated shKIFC3 RCTE cells. γ-tubulin (cyan) labels the ciliary base. YFP direct fluorescence shown CEP170C-tagged KIFC3. Scale bar, 10 μm. All results from n = 3 independent experiments. Data are the mean ± SEM. Statistical significance was determined using one-way ANOVA. Three experiments were repeated independently with similar results ( b , c , h , i ). Source data are provided as a file.

Article Snippet: Sub-cloning templates of human CENEXIN1 and KIFC3 were purchased from Addgene (#73334) and DNASU(#HsCD00442644), and ODF2(iso6) was kindly provided by Dr. Kyung Lee (NIH/NCI).

Techniques: Immunofluorescence, Expressing, Translocation Assay, Quantitation Assay, Control, Over Expression, Fluorescence, Staining

Journal: Nature Communications

Article Title: Transiently formed nucleus-to-cilium microtubule arrays mediate senescence initiation in a KIFC3-dependent manner

doi: 10.1038/s41467-024-52363-w

Figure Lengend Snippet: a – d SA-β-gal staining ( a ), quantitation of SA-β-gal-positive cells ( n = 3 independent experiments, 6-8fields per experiment, 200 cells per field) ( b ) in control or siCENEXIN1 RCTE cells with or without IR exposure. Western blot of senescence markers ( c ), and relative mRNA level of SASP genes ( d ) in control or shCENEXIN1 RCTE cells with or without IR exposure. For IR treatment, cells were collected at day 10 after irradiation. Scale bar, 100 μm. SA-β-gal staining ( e ) and quantitation of SA-β-gal-positive cells ( n = 3 independent experiments, 6-8fields per experiment, 200–300 cells per field) ( f ), in control or CENEXIN1 -/- RCTE cells re-expressing CENEXIN1 or ODF2 (iso6) at day 10 after IR exposure. Scale bar, 100 μm. SA-β-gal staining ( g ), quantitation of SA-β-gal-positive cells ( n = 3 independent experiments, 3-4fields per experiment, 200–500 cells per field) ( h ), and relative mRNA level of SASP genes ( i ) in IL-1β-treated (3 ng/ml for 5 days) control or shCENEXIN1 RCTE cells. Scale bar, 50 μm. j Proposed working model: Exposure to irreparable stresses triggers the reorganization of microtubules (MTs), leading to the nucleation of sinc-MTs in the proximity of the nuclear envelop towards the ciliary base. Concurrently, the minus-end-directed kinesin KIFC3 is recruited to the ciliary base. It subsequently facilitates the transportation of the CENEXIN1-FBF1 cargo complex along the sinc-MTs, directing it towards the nucleus. This process initiates cellular senescence in stressed human cells. All results from n = 3 independent experiments. Data are the mean ± SEM. Statistical significance was determined using one-way ANOVA. Three experiments were repeated independently with similar results ( c ). Source data are provided as a file.

Article Snippet: Sub-cloning templates of human CENEXIN1 and KIFC3 were purchased from Addgene (#73334) and DNASU(#HsCD00442644), and ODF2(iso6) was kindly provided by Dr. Kyung Lee (NIH/NCI).

Techniques: Staining, Quantitation Assay, Control, Western Blot, Irradiation, Expressing