Fig. 1: Irreparable stress induces transient assembly of sinc-MTs that promote cellular senescence in human cells. | Nature Communications

Fig. 1: Irreparable stress induces transient assembly of sinc-MTs that promote cellular senescence in human cells.

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

Fig. 1

a Immunofluorescent images of sinc-MTs in IR-treated RCTE cells. α-tubulin (green) labels MTs. CENTRIN2 (red) labels the ciliary base. Scale bar, 10 μm. b Stacked z-steps deconvolution image of RCTE cells exposed to IR on day2 using α-tubulin (green) labeling MTs. Scale bar, 10 μm. c Quantification of sinc-MTs-positive cells, calculated as the ratio of positive cells to the total number of cells (n = 20 fields, 10–20 cells per field). d Immunofluorescence showing sinc-MTs and the cilium in IR-treated RCTE cells. α-tubulin (green) labels MTs. ARL13B (red) labels primary cilium. Scale bar, 10 μm. e Confocal z-stack of IR-treated RCTE cells, with arrows pointing at sinc-MTs (α-tubulin, green) near the nuclear envelope (NUP153, red). Scale bar, 5 μm. f Immunofluorescence of sinc-MTs plus-end binding protein EB1 (green) near the ciliary base (CENTRIN2, red) in IR-treated RCTE cells. Scale bar, 10 μm. g Confocal z-stack images in IR-treated RCTE cells using antibodies against α-tubulin (blue), CAMSAP2 (red), and EB1 (green). Arrows indicate the minus- and plus-ends of sinc-MT filaments. Scale bar, 5 μm. h Localization PML (red) and FBF1 (green) in IR-treated RCTE cells with or without colchicine treatment. Arrows indicate the ciliary base. Scale bar, 10 μm. i Western blot of CDK5RAP2 and senescence markers in control or siCDK5RAP2 RCTE cells 10 days post-IR. j Immunofluorescence of sinc-MTs (α-tubulin, green) in IR-treated control or siCDK5RAP2 RCTE cells. CENTRIN2 (red) labels the ciliary base. Scale bar, 10 μm. k Immunostaining of PML-NBs (red) and FBF1 (green) in IR-treated control or siCDK5RAP2 RCTE cells. Scale bar, 10 μm. Relative mRNA level of SASP genes (l), SA-β-gal staining (m), quantitation of the percentage of SA-β-gal-positive cells (n = 3 independent experiments, 6-8fields per experiment, 100–200 cells per field) (n) in control or siCDK5RAP2 RCTE cells 10 days post-IR. Scale bar, 50 μm. All results from n = 3 independent experiments. Data are the mean ± SEM. One-way ANOVA was used analyzing (c, l, n). Three experiments were repeated independently with similar results (a, b, d–k). Source data are provided as a Source Data file.

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