paclitaxel  (Teva)

Journal: Journal of Cell Science

Article Title: Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling

doi: 10.1242/jcs.264494

Figure Lengend Snippet: Paclitaxel-induced cytoskeletal reorganisation around the nucleus in interphase. (A) Confocal micrographs of cells fixed after 16 h incubation in control medium or 5 nM paclitaxel. DNA was stained using Hoechst 33342 (cyan), and microtubules using α-tubulin immunofluorescence (magenta). MTOCs are marked with arrowheads. Scale bars: 20 μm. (B) STORM imaging of α-tubulin immunofluorescence in cells fixed after 16 h incubation in control medium or 5 nM paclitaxel. Lower panels show α-tubulin clusters generated with HDBSCAN analysis. Different colours distinguish individual α-tubulin clusters, representing individual microtubule filaments or filament bundles. Scale bars: 10 μm. (C) Violin plot comparing the diameter of α-tubulin clusters from B between control (blue) and paclitaxel-treated cells (red). The mean is shown in black with error bars showing the s.e.m. from three biological repeats ( n =3). The mean of each biological repeat is marked with a triangle and consists of >300 measurements from five or more cells. *** P =0.00012 (unpaired two-tailed t -test). (D,E) Confocal micrographs of control and paclitaxel-treated cells stained for DNA using Hoechst 33342 (cyan), microtubules using α-tubulin immunofluorescence (magenta), and either (D) F-actin using Alexa Fluor 488–phalloidin (green) or (E) vimentin using immunofluorescence (green). Scale bars: 20 μm. (F) Left, 2D slice of a reconstructed tomogram of the NE region in a paclitaxel-treated cell. Bundled vimentin filaments (green arrowheads) and microtubules (magenta arrowheads) are seen closely apposed to the NE (cyan arrowheads). Right, segmentation of the NE (cyan), microtubules (magenta) and vimentin filaments (green) from the tomogram. Scale bars: 100 nm. Images in A and D–F are representative of three biological repeats each with >50 cells.

Article Snippet: The extent of the cytoskeletal reorganisation correlated with the concentration of paclitaxel, as microtubules became increasingly bundled and the actin cytoskeleton more condensed with increasing concentrations (0 to 10 nM) of paclitaxel ( ).

Techniques: Incubation, Control, Staining, Immunofluorescence, Imaging, Generated, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling

doi: 10.1242/jcs.264494

Figure Lengend Snippet: Paclitaxel-induced cytoskeletal reorganisation around the nucleus in interphase. (A) Confocal micrographs of cells fixed after 16 h incubation in control medium or 5 nM paclitaxel. DNA was stained using Hoechst 33342 (cyan), and microtubules using α-tubulin immunofluorescence (magenta). MTOCs are marked with arrowheads. Scale bars: 20 μm. (B) STORM imaging of α-tubulin immunofluorescence in cells fixed after 16 h incubation in control medium or 5 nM paclitaxel. Lower panels show α-tubulin clusters generated with HDBSCAN analysis. Different colours distinguish individual α-tubulin clusters, representing individual microtubule filaments or filament bundles. Scale bars: 10 μm. (C) Violin plot comparing the diameter of α-tubulin clusters from B between control (blue) and paclitaxel-treated cells (red). The mean is shown in black with error bars showing the s.e.m. from three biological repeats ( n =3). The mean of each biological repeat is marked with a triangle and consists of >300 measurements from five or more cells. *** P =0.00012 (unpaired two-tailed t -test). (D,E) Confocal micrographs of control and paclitaxel-treated cells stained for DNA using Hoechst 33342 (cyan), microtubules using α-tubulin immunofluorescence (magenta), and either (D) F-actin using Alexa Fluor 488–phalloidin (green) or (E) vimentin using immunofluorescence (green). Scale bars: 20 μm. (F) Left, 2D slice of a reconstructed tomogram of the NE region in a paclitaxel-treated cell. Bundled vimentin filaments (green arrowheads) and microtubules (magenta arrowheads) are seen closely apposed to the NE (cyan arrowheads). Right, segmentation of the NE (cyan), microtubules (magenta) and vimentin filaments (green) from the tomogram. Scale bars: 100 nm. Images in A and D–F are representative of three biological repeats each with >50 cells.

Article Snippet: For paclitaxel treatment, paclitaxel (Merck PHL89806 ) resuspended in dimethyl sulfoxide (DMSO) to 10 mM was diluted in complete DMEM to final concentrations of 1, 5 or 10 nM as indicated.

Techniques: Incubation, Control, Staining, Immunofluorescence, Imaging, Generated, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling

doi: 10.1242/jcs.264494

Figure Lengend Snippet: Paclitaxel treatment results in nuclear deformation. (A) Nuclear solidity of Hoechst 33342-stained nuclei over 72 h in 0, 1, 5 or 10 nM paclitaxel. Error bars show the s.e.m. from three biological repeats ( n =3), each with more than 50 cells. (B) Example frames from a live-cell time-series used for the nuclear solidity analysis in A, showing Hoechst 33342-stained nuclei (cyan) and respective nuclear solidity measurements (yellow) taken every 2 h at 0–10 h after the addi­tion of 5 nM paclitaxel. Scale bars: 20 μm. (C) Quantification of nuclear solidity from cells cultured with complete medium (+serum) or serum-starved medium (−serum) in control conditions or after 16 h in 5 nM paclitaxel. The mean is shown in black with error bars showing the s.e.m. from three biological repeats ( n =3), each with at least 50 cells. The datapoint from each cell is marked with a dot colour-coded according to the biological replicate it came from, with the mean of each biological repeat marked with a triangle of the same colour. ** P =0.0075 (−serum); ** P =0.0011 (+serum) (unpaired two-tailed t -test for control versus paclitaxel). (D) Confocal micrographs of cells transiently transfected with GFP–LMNA to overexpress GFP–lamin A (green), fixed after 16 h incubation in control medium or 5 nM paclitaxel. Cells were stained for DNA using Hoechst 33342 (grey). Scale bars: 20 μm. (E) Quantification of nuclear solidity from wild-type cells, cells with lamin A/C knocked down (siLMNA), or cells with GFP–lamin A overexpressed, following 16 h incubation in control medium or 5 nM paclitaxel. For GFP–lamin A overexpression samples, only cells expressing GFP-lamin A were analysed. Error bars show s.e.m. from three biological repeats ( n =3), each with at least 30 cells. The datapoint from each cell is marked with a dot colour-coded according to the biological replicate it came from, with the mean of each biological repeat marked with a triangle of the same colour. * P =0.0311 (wild-type paclitaxel versus siLMNA paclitaxel), ** P =0.0099 (wild-type paclitaxel versus GFP–lamin A paclitaxel) (unpaired two-tailed t -test). (F) Violin plot comparing the ONM–INM distance between control and paclitaxel-treated cells, quantified from nuclear membrane segmentations from high-resolution tomograms using surface morphometrics . A total NE area of 1.64 μm 2 consisting of 1,236,316 datapoints over 23 tomograms from 6 cells was analysed for the control, and 3.97 μm 2 consisting of 3,781,706 datapoints over 41 tomograms from 9 cells was analysed for paclitaxel-treated cells. The black lines represent the modal values (21.0 nm for control; 10.5 nm and 25.5 nm for paclitaxel). (G) Example 2D slices of reconstructed tomograms of the NE in control and paclitaxel-treated cells. Segmentations of the ONM and INM that were used for the morphometrics analysis in F are shown in the lower panels, with the ONM coloured using a heatmap of the ONM–INM distance. Scale bars: 100 nm.

Article Snippet: For paclitaxel treatment, paclitaxel (Merck PHL89806 ) resuspended in dimethyl sulfoxide (DMSO) to 10 mM was diluted in complete DMEM to final concentrations of 1, 5 or 10 nM as indicated.

Techniques: Staining, Cell Culture, Control, Two Tailed Test, Transfection, Incubation, Over Expression, Expressing, Membrane

Journal: Journal of Cell Science

Article Title: Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling

doi: 10.1242/jcs.264494

Figure Lengend Snippet: Paclitaxel treatment results in aberrant organisation and decreased levels of lamin A/C and SUN2. (A) Confocal micrographs of cells fixed after 16 h incubation in control medium or 5 nM paclitaxel. DNA was stained using Hoechst 33342 (cyan), and lamin A/C (magenta) and lamin B1 (red) were stained using immunofluorescence. Areas of the lamina that are patchy or have lamin A/C or B1 missing are marked with arrowheads. Lower panels show magnified views of a patchy area of the lamina. Scale bars: 20 μm. (B) Western blots for lamin A/C and lamin B1 of whole-cell lysates following 16 h incubation in medium containing 0, 1, 5 or 10 nM paclitaxel. Cyclophilin B was used as a loading control. Lane 1 shows protein ladder (LD; 75 kDa for lamin A/C and lamin B1 panels, and 25 kDa for cyclophilin B panels). (C) Quantification of lamin A/C and lamin B1 protein levels from B. Each band was normalised to the corresponding cyclophilin B loading control. Error bars represent the s.d. from three biological repeats ( n =3) which are marked with triangles. ns, not significant; * P <0.05 (lamin A/C, 1 nM P =0.9059, 5 nM P =0.0452, 10 nM P =0.0128; lamin B1, 1 nM P =0.139, 5 nM P =0.0665, 10 nM P =0.0656) (one-sample t -test with null hypothesis mean=1). (D) Confocal micrographs of cells fixed after 16 h incubation in control medium or 5 nM paclitaxel. Cells were stained for lamin A/C (magenta) and SUN2 (green) using immunofluorescence. Scale bars: 20 μm. (E) Analysis of the colocalisation between lamin A/C and SUN1 or SUN2. Fluorescence colocalisation was quantified using Li's ICQ value , where more positive values represent better positive colocalisation. Error bars represent the s.e.m. from three biological repeats ( n =3), each with more than 30 cells. The datapoint from each cell is marked with a dot colour-coded according to the biological replicate it came from, with the mean of each biological repeat marked with a triangle of the same colour. ns, not significant (SUN2, P =0.8788); *** P =0.0002 (SUN1) (unpaired two-tailed t -test control versus paclitaxel). (F) Western blots for SUN1 and SUN2 of whole-cell lysates following 16 h incubation in medium containing 0, 1, 5 or 10 nM paclitaxel. Cyclophilin B was used as a loading control. Lane 1 shows protein ladder (LD; 98 kDa for SUN1 and SUN2 panels, and 28 kDa for cyclophilin B panels). (G) Quantification of SUN1 and SUN2 protein levels from F. Each band was normalised to the corresponding cyclophilin B loading control. Error bars represent the s.d. from three biological repeats ( n =3) which are marked with triangles. ns, not significant; * P <0.05; ** P <0.01 (SUN1, 1 nM P =0.9607, 5 nM P =0.2142, 10 nM P =0.1988; SUN2, 1 nM P =0.1794, 5 nM P =0.0133, 10 nM P =0.0042) (one-sample t -test with null hypothesis mean=1). Images in A are representative of three biological repeats, each with >50 cells.

Article Snippet: For paclitaxel treatment, paclitaxel (Merck PHL89806 ) resuspended in dimethyl sulfoxide (DMSO) to 10 mM was diluted in complete DMEM to final concentrations of 1, 5 or 10 nM as indicated.

Techniques: Incubation, Control, Staining, Immunofluorescence, Western Blot, Fluorescence, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling

doi: 10.1242/jcs.264494

Figure Lengend Snippet: The paclitaxel-induced decrease in lamin A/C levels occurs via SUN2. (A) Western blots for lamin A/C and SUN2 of whole cell lysates from cells cultured in complete medium (+serum) or serum-starved medium (−serum) and following 16 h incubation in 0, 1, 5 or 10 nM paclitaxel. Cyclophilin B was used as a loading control. Lane 1 shows protein ladder (LD; 75 kDa for lamin A/C panel, 20 kDa for cyclophilin B upper panel, 98 kDa for SUN2 panel and 28 kDa for cyclophilin B lower panel). (B,C) Quantification of lamin A/C (B) and SUN2 (C) protein levels from A. Each band was normalised to the corresponding cyclophilin B loading control. Error bars represent the s.d. from three biological repeats ( n =3) which are marked with triangles. ns, not significant; * P <0.05; ** P <0.01 (lamin A/C +Serum, 1 nM P =0.0504, 5 nM P =0.0301, 10 nM P =0.0092; lamin A/C −serum: 1 nM P =0.3119, 5 nM P =0.0472, 10 nM P =0.0272; SUN2 +serum, 1 nM P =0.1904, 5 nM P =0.0404, 10 nM P =0.0146; SUN2 −serum, 1 nM P =0.0849, 5 nM P =0.0182, 10 nM P =0.0015) (one-sample t -test with null hypothesis mean=1). (D) Western blots for lamin A/C of whole-cell lysates from wild-type or SUN2-knockdown (siSUN2) cells following 16 h incubation in 0, 1, 5 or 10 nM paclitaxel. Cyclophilin B was used as a loading control. Lane 1 shows protein ladder (LD; 62 kDa for lamin A/C panel, and 28 kDa for cyclophilin B panel). (E) Quantification of lamin A/C protein levels from D. Each band was normalised to the corresponding cyclophilin B loading control. Error bars represent the s.d. from three biological repeats ( n =3) which are marked with triangles. ns, not significant; * P <0.05; ** P <0.01 (wild type, 1 nM P =0.2779, 5 nM P =0.0185, 10 nM P =0.0032; siSUN2, 1 nM P =0.3984, 5 nM P =0.8031, 10 nM P =0.5198) (one-sample t -test with null hypothesis mean=1). (F) Top panel, western blot for polyubiquitin C following pull-down of SUN2 from whole cell lysates of control cells or cells treated with 5 nM paclitaxel for 16 h. Three biological repeats were used for each condition ( n =3). Bottom panel, to control for SUN2 protein levels, the same membrane was stripped and blotted for SUN2. (G) Quantification of polyubiquitin C levels from F, with each band normalised to SUN2. Error bars represent the standard deviation from three biological repeats ( n =3) which are marked with triangles. ** P =0.0014 (unpaired two-tailed t -test control versus paclitaxel).

Article Snippet: For paclitaxel treatment, paclitaxel (Merck PHL89806 ) resuspended in dimethyl sulfoxide (DMSO) to 10 mM was diluted in complete DMEM to final concentrations of 1, 5 or 10 nM as indicated.

Techniques: Western Blot, Cell Culture, Incubation, Control, Knockdown, Membrane, Standard Deviation, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling

doi: 10.1242/jcs.264494

Figure Lengend Snippet: Lamin A/C expression level affects cell sensitivity to and recovery from paclitaxel. (A) Cell confluency of wild-type (red), GFP–lamin A overexpression (green) and lamin A/C-knockdown (blue) cells over 48 h in 0, 1, 5 or 10 nM paclitaxel. Confluency was calculated from brightfield high-content live-cell images. Error bars represent the s.e.m. from five biological repeats. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001 (0 nM, GFP–lamin A versus wild type P =0.3186, siLMNA versus wild type P =0.6074; 1 nM, GFP–lamin A versus wild type P =0.0003, siLMNA versus wild type P =0.1662; 5 nM, GFP–lamin A versus wild type P =4.5×10 −5 , siLMNA versus wild type P =0.0267; 10 nM, GFP–lamin A versus wild type P =0.0207, siLMNA versus wild type P =0.0029) (one-way three-factor repeated measures ANOVA with Bonferroni post hoc test). (B) Quantification of cell viability of wild-type (red), GFP–lamin A overexpression (green) and lamin A/C-knockdown (blue) cells following 48 h incubation in 0, 1, 5 or 10 nM paclitaxel. Cell viability was calculated by subtracting the proportion of the total number of cells that were dead/apoptotic (identified by FLICA staining) from 1. Error bars represent s.e.m. from three biological repeats ( n =3) which are marked with triangles, each with at least 95 cells. ns, not significant; * P <0.05; ** P <0.01 (0 nM, siLMNA P =0.3244, GFP–lamin A P =0.6742; 1 nM, siLMNA P =0.2911, GFP–lamin A P =0.0107; 5 nM, siLMNA P =0.0325, GFP-lamin A P =0.0036; 10 nM, siLMNA P =0.8297, GFP-lamin A P =0.0428) (unpaired two-tailed t -test versus wild type). (C) Cell confluency of wild-type and lamin A/C knockdown cells in control medium (control), medium with the sustained presence of 5 nM paclitaxel (paclitaxel) or medium from which paclitaxel was removed after 24 h (recovery). Error bars represent the s.e.m. from three biological repeats.

Article Snippet: For paclitaxel treatment, paclitaxel (Merck PHL89806 ) resuspended in dimethyl sulfoxide (DMSO) to 10 mM was diluted in complete DMEM to final concentrations of 1, 5 or 10 nM as indicated.

Techniques: Expressing, Over Expression, Knockdown, Incubation, Staining, Two Tailed Test, Control