Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 1. IPO13, respectively, is an import receptor for nCLU and export receptor for KU70. (a–d) HeLa cells were subjected to CLSM 16 h post-transfection to co-express either DsRed2 or DsRed2-IPO13 with GFP-nCLU, Scale bar = 10 µM (a) or GFP-KU70, Scale bar = 10 µM (c) and treated ± 125 µM H2O2 for 1 h prior to imaging live. Quantitative analysis of GFP-nCLU (b) or GFP-KU70 (d) localisation was carried out using the ImageJ software on images, such as those in (a,c), to determine the nuclear-to-cytoplasmic-fluorescence ratio (Fn/c), as described in Materials and Methods. Values represent the mean ± SEM (n > 50 cells) from a single typical experiment from a series of 2 (b) or 3 (d) similar experiments. (e–i) HeLa cells were subjected to CLSM 72 h post-transfection with non-targeting or IPO13 siRNA. (e) Total cell extracts were probed by Western blotting using rabbit-anti-IPO13 (Protein Tech), with mouse-anti-actin (Abcam, Cambridge, UK) as a control and imaged using the ChemiDoc Gel Imaging System (Biorad, Hercules, CA, USA). At 16 h post-transfection, cells were transfected with either GFP-nCLU, Scale bar = 10 µM (h) or GFP-KU70, Scale bar = 10 µM (h) and treated with H2O2 as per (a,c) above. Quantitative analysis of GFP-nCLU (g) or GFP-KU70 (i) localisation was carried out as in (b,d).Values represent the mean ± SEM (n > 31 cells) from a single typical experiment from a series of 2 (g) or 3 (i) similar experiments. (j) HeLa

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Transfection, Imaging, Software, Western Blot, Control

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 2. IPO13 efficiently traffics nCLU into the nucleus under oxidative stress, but IPO13-mediated nuclear export of KU70 is inhibited, as confirmed by fluorescence recovery after photo bleaching (FRAP) analysis. (a) CLSM images of HeLa cells transfected to co-express either mCherry or mCherry and IPO13 (expressed separately from the same plasmid using an IRES translation initiation site, pIRES) with GFP-nCLU and treated ± 125 µM H2O2 for 1 h, Scale bar = 10 µM. Cells were imaged prior to photobleaching (Pre) in the indicated nuclear region (dotted outline in yellow) and then monitored every 20 s for 8 min. (b) Digitised images, such as those in (a), were analysed to determine the fractional recovery of nuclear fluorescence (Frec(Fn-b)). Results shown are for a single representa- tive cell under each condition. Curves, such as those generated in (b), were used to determine the maximal recovery of nuclear fluorescence (c) and the initial rate of recovery, up to 100 s post-bleaching (Frec (Fn-b)/s−1); (d) Results represent the mean ± SEM (n > 20), typical results from 3 separate experiments. p-values represent statistical differences as determined by Student’s t-test. (e) CLSM images of HeLa cells transfected to co-express either DsRed2 or DsRed2-IPO13 with GFP-KU70 and treated ± 125 µM H2O2 for 1 h, Scale bar = 10 µM. Cells were imaged live prior to photo bleaching (Pre) in the indicated cytoplasmic region (dotted outline in blue) and then monitored every 20 s for 8 min. (f) Digitised images, such as those in (e), were analysed to determine the fractional change of nuclear fluorescence (Frec(Fn-b)). The more negative a value in this assay, the more nuclear export is occurring; therefore, when there is less loss of nuclear fluorescence, this is indicative of less export and vice versa. Results shown are for a single representative cell under each condition. Curves such as those generated in (b), were used to determine the maximal loss of nuclear fluorescence (g) and the initial rate of export, up to 100 s post-bleaching (Frec (Fn-b)/s−1); (h) Results represent the mean ± SEM (n > 20), typical results from 3 separate experiments. p-values represent statistical differences as determined by Student’s t-test.

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Transfection, Plasmid Preparation, Generated

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 3. IPO13 plays a significant role in stress-induced DNA damage and repair, in part through effects on nCLU. (a) Fluorescence images of comets produced by in-gel neutral comet assay (single- cell electrophoresis) from HeLa cells ectopically expressing either GFP or GFP-tagged IPO13 after treatment without or with 50 µM H2O2 for 1 h or treatment followed by 2 h recovery in fresh media. H denotes the comet head and T denotes the comet tail (middle top panel). (b) Tail DNA content (%) was quantified using the OpenComet plugin for ImageJ to determine the percentage of DNA in the comet tail (mean ± SEM, n > 100 comets per sample). Results represent a single typical experiment from a series of 3 independent experiments. (c) Fluorescent images of comets produced as in (a) from HeLa cells transfected with either non-targeting (NT) or IPO13 siRNA after treatment as in (a) with 125 µM of H2O2. (d) Tail DNA content of pictures, such as those in (c), was quantified as in (b). (e) Fluorescent images of comets produced as in (a) from HeLa cells transfected with either NT or IPO13 siRNA that ectopically expressed either GFP or GFP-nCLU after treatment as in (a) with 125 µM H2O2. (f) Tail DNA content from images, such as those in (e), was quantified as in (b), with results representing a single typical experiment from 2 independent experiments (mean ± SEM, n > 200 comets per sample).

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Fluorescence, Produced, Neutral Comet Assay, Electrophoresis, Expressing, Transfection

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 4. IPO13 contributes to nCLU-induced cell death and apoptosis. (a,b) Flow cytometric analysis for cell death in IPO13+/+ and IPO13−/−ESC transfected with GFP or GFP-nCLU and treated with H2O2 for 1 h prior to FACS analysis for percentage of cell death (PI-positive cells) within the GFP- or GFP-nCLU-transfected cell populations. (a) Representative plots of untreated and 600 µM H2O2- treated IPO13+/+ and IPO13−/−ESCs, gated to include the GFP positive populations. (b) Pooled data (n = 6 independent experiments) for % of GFP- or GFP-nCLU-expressing cells that are PI-positive (mean ± SEM) under increasing concentrations of H2O2 treatment as indicated. p-values represent statistical differences as determined by two-way ANOVA using Prism 7. (c,d) Flow cytometric analysis for apoptosis (Annexin V and/or PI staining) in IPO13+/+ and IPO13−/−ESCs treated with or without 12 µM Camptothecin (CTH) for 6 h. (c) Representative dot plots for untreated and CTH-treated conditions are typical of three independent assays. In each panel, the upper left quadrant (Q1) shows only PI-positive cells, which are necrotic. The upper right quadrant (Q2) shows cells positive for both PI and Annexin V cells. The bottom right quadrant (Q3) shows cells positive for Annexin V only, and the bottom left quadrant (Q4) shows unstained cells. (d) Pooled data (n = 3 independent experiments) for % of untreated or CTH-treated IPO13+/+ and IPO13−/−ESCs positive for Annexin V staining (Q2 + Q3) (mean ± SEM).

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Transfection, Expressing, Staining

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 5. Stress disrupts the localisation of nuclear transport machinery components but not IPO13. (a,b) HeLa cells were treated ± 125 µM H2O2 for 1 h or ± 43 ◦C for 1 h prior to staining with mouse-anti-Ran (BD Biosciences) and counter-staining with DAPI, Scale bar = 10 µM. Quantitative analysis of Ran localisation (b) was carried out using the ImageJ software on images, such as those in (a), to determine the nuclear-to-cytoplasmic-fluorescence ratio (Fn/c) of Ran, as described in Materials and Methods. Values represent the mean ± SEM (n > 50 cells) from a single typical experiment from a series of 2 similar experiments. (c,d) Typical CLSM images of HeLa cells, 16 h post-transfection to express GFP or GFP-IMPα (c) and treated ± 125 µM H2O2 for 1 h prior to imaging live, Scale bar = 10 µM. Quantitative analysis of GFP or GFP- IMPα (d) was carried out as in (b). Values represent the mean ± SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (e,f) Typical line fluorescence intensity histograms of GFP-IMPα (e) were measured across the nuclear envelope as indicated by the yellow line on the corresponding cell images (c). (f) Quantitative analysis of GFP-IMPα was carried out using the ImageJ software on images, such as those in (c), to determine the nuclear-envelope-to-nuclear ratio (Fne/n), as described in Materials and Methods. Values represent the mean ± SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (g,h) Typical CLSM images of HeLa cells, 16 h post-transfection to express GFP or GFP-IPOβ1 (c) and treated ± 125 µM H2O2 for 1 h prior to imaging live, Scale bar = 10 µM. Quantitative analysis of GFP or GFP-IPOβ1 (d) was carried out as in (b). Values represent the mean + SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (i,j) Typical line fluorescence intensity histograms of GFP-IPOβ1 (i) were measured across the nuclear envelope, as indicated by the yellow line on the corresponding cell images (g). (j) Quantitative analysis of GFP-IPOβ1 was carried out as in (f). Values represent the mean ± SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (k,l) Typical CLSM images of HeLa cells, 16 h post-transfection to express GFP or GFP-IPO7 (k) and treated ± 125 µM H2O2 for 1 h prior to imaging live, Scale bar = 10 µM. Quantitative analysis of GFP or GFP-IPO7 (l) was carried out as in (b). Values represent the mean ± SEM (n > 30 cells) from a single typical experiment from a series of 3 similar experiments. (m,n) Typical line fluorescence intensity histograms of GFP-IPO7 (m) were measured across the nuclear envelope, as indicated by the yellow line on the corresponding cell images (k). (n) Quantitative analysis of GFP-IPO7 was carried out as in (f). Values

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Staining, Software, Transfection, Imaging, Fluorescence

Journal: Cells

Article Title: The Nuclear Transporter Importin 13 Can Regulate Stress-Induced Cell Death through the Clusterin/KU70 Axis.

doi: 10.3390/cells12020279

Figure Lengend Snippet: Figure 6. IPO13, unlike IPO7, continues to traffic into the nucleus under H2O2-induced oxida- tive stress. (a) CLSM images of HeLa cells transfected to express GFP-IPO7 or GFP-IPO13 and treated ± 125 µM H2O2 for 1 h. Cells were imaged prior to photobleaching (Pre) in the indicated nuclear region (dotted outline in yellow) and then monitored every 20 s for 8 min. (b) Digitised images, such as those in (a) were analysed to determine the fractional recovery of nuclear fluores- cence (Frec(Fn-b)), Scale bar = 10 µM. Results shown are for a single representative cell under each condition. Curves, such as those generated in (b), were used to determine the maximal recovery of nuclear fluorescence (c) and the time post-bleaching to reach half-maximal recovery (t1/2). (d) Results represent the mean ± SEM (n = 20); typical results from 2 separate experiments. p-values indicate statistical differences as determined by Student’s t-test.

Article Snippet: At 16 h post-transfection, cells were treated with 125 μM H2O2 for 1 h, after which an additional glutaraldehyde–protein crosslinking step was performed (see Materials and Methods) before lysis and IP using GFP-Trap beads (Chromotek). (n) Input or IP samples were probed by Western Blotting using rabbit-anti-IPO13 (Protein Tech) or mouse-antiGFP (Roche) antibodies. (o) Densitometric analysis was performed on images, such as those in (a), for binding of IPO13 to GFP-KU70 under H2O2-treated conditions and untreated conditions with and without co-transfection of mCherry-nCLU.

Techniques: Transfection, Generated