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

Journal: Oncogene

Article Title: Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress

doi: 10.1038/s41388-021-02105-9

Figure Lengend Snippet: A HT1080 cells were treated with DMSO or TH287(10 μM) for 24 h and then subjected to cell cycle distribution analysis by flow cytometry. The proportions of G2/M-phase cells were statistically analyzed. B Left, representative immunofluorescence imaging of phosphorylated Histone H3 (pH3) in HT1080 cells treated with DMSO or TH287 (10 μM) for 24 h. Scale bar, 5 μm. Right, representative cell-cycle profiles of HT1080 cells treated with DMSO or TH287 (10 μM) for 24 h assessed by pH3 (mitotic cells) and PI flow cytometric analysis (FACS). 2N DNA content indicates cells in G1 phase. 4 N DNA content indicates cells in either G2 or M phase. The mitotic index (percentage of pH3-positve cells) for each group was shown. C Flow cytometric analysis of intracellular ROS levels measured by DCFH-DA and dihydroethidium (DHE). HT1080 cells were treated with DMSO or TH287(10 μM) for 24 h. D Flow cytometric analysis of superoxide measured by dihydroethidium (DHE). HT1080 cells were treated with DMSO or TH287(10 μM) for 24 h. E Flow cytometric analysis of intracellular ROS levels measured by DCFH-DA. HT1080 were treated with TH287 (10 μM) alone or in combination with MitoQ (1 μM) for 24 h. F MitoSOX Red was used to assess mitochondrial superoxide levels. HT1080 cells were treated with DMSO or TH287(10 μM) for 24 h. Mitochondrial superoxide was measured using MitoSOX Red, detected by flow cytometric analysis. G HT1080 cells were pretreated with DMSO or UCN-01 (300 nM) for 4 h before addition of TH287 (10 μM) treatment for 24 h, and subjected to cell cycle distribution analysis by flow cytometry. The proportions of G2/M-phase cells were statistically analyzed. H Left, representative immunofluorescence imaging of pH3 in HT1080 cells treated with DMSO or UCN-01 (300 nM) for 4 h before addition of TH287 (10 μM) treatment for an additional 24 h. Scale bar, 5 μm. Right, representative cell-cycle profiles of HT1080 cells treated with DMSO or UCN-01 (300 nM) for 4 h before addition of TH287 (10 μM) treatment for an additional 24 h assessed by pH3/PI FACS. The mitotic index (percentage of pH3-positve cells) for each group was shown. I Flow cytometric analysis of intracellular ROS levels measured by DCFH-DA. HT1080 cells were treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. J Flow cytometric analysis of superoxide measured by dihydroethidium (DHE). HT1080 cells were treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. K HT1080 cells were treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. Mitochondrial superoxide was detected by flow cytometric analysis after staining with MitoSOX Red. L HT1080 cells were treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. Mitochondrial superoxide, indicated by MitoSOX Red, was detected using fluorescence microscope. Scale bar, 5 μm. Data shown were representative of three independent experiments and data presented in bars as mean ± S.D. The statistical differences between the two groups were analyzed by two-sided unpaired Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: TH287 (Selleck Chemicals, Houston, TX, USA) and UCN-01 (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Flow Cytometry, Immunofluorescence, Imaging, Staining, Fluorescence, Microscopy

Journal: Oncogene

Article Title: Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress

doi: 10.1038/s41388-021-02105-9

Figure Lengend Snippet: A Representative fluorescence imaging of mitochondria stained with MitoTracker Red in HT1080 cells treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. Scale bar, 5 μm. B HT1080 and U2OS cells were treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. Cells were stained with MitoTracker Red and analyzed using a flow cytometer. C HT1080 and U2OS cells were treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. Cells were stained with nonylacridine orange (NAO) and analyzed using a flow cytometer. D Representative fluorescence imaging of mitochondria stained with MitoTracker Red in HT1080 cells treated with paclitaxel (50 nM) alone or in combination with UCN-01 (300 nM) for 24 h. Scale bar, 5 μm. E HT1080 cells were treated with paclitaxel (50 nM) alone or in combination with UCN-01 (300 nM) for 24 h. Cells were stained with MitoTracker Red and analyzed using a flow cytometer. F HT1080 cells were treated with paclitaxel (50 nM) alone or in combination with UCN-01 (300 nM) for 24 h. Cells were stained with nonylacridine orange (NAO) and analyzed using a flow cytometer. G HT1080 cells were treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. Mitochondria fractions were isolated with the Cell Mitochondria Isolation Kit. The intact mitochondria were isolated from 20 million treated cells and mitochondrial proteins were quantified using BCA Protein Assay. Equal amounts of proteins were loaded in each lane. The mitochondrial nature of the isolated proteins was confirmed with Western blotting analysis using antibodies against various mitochondrial components. H HT1080 cells were treated with paclitaxel (50 nM) alone or in combination with UCN-01 (300 nM) for 24 h. Western blotting analysis using antibodies against various mitochondrial components were treated as described in G . I Mitochondrial DNA (mtDNA) copy number measured by quantitative PCR. The relative amounts of Cytochrome Oxidase I (COX I) in total DNA were determined as described in Materials and methods. Data shown were representative of three independent experiments and data presented in bars as mean ± S.D. The statistical differences between the two groups were analyzed by two-sided unpaired Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: TH287 (Selleck Chemicals, Houston, TX, USA) and UCN-01 (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Fluorescence, Imaging, Staining, Flow Cytometry, Isolation, Bicinchoninic Acid Protein Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress

doi: 10.1038/s41388-021-02105-9

Figure Lengend Snippet: A Transmission electron micrographs of HT1080 cells treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. There are more mitochondria and Mitochondrial vacuolation are apparent in TH287-treated cells. Magnifications: high power lens, ×4000, Scale bar, 2.0 μM. N, Nucleus. M, Mitochondria. REF, Rough endoplasmic reticulum. Go, Golgi apparatus. ASS, Autolysosome. Boxes represent the areas seen in the high-power-magnification images. Scale bar, 0.5 μM. Quantitative analysis of number of mitochondria in HT1080 cells treated with TH287 and/or UCN-01. B HT1080 cells treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h were stained with JC-1 according to Materials and methods and analyzed using a flow cytometer. Red fluorescence represents the mitochondrial aggregate form of JC-1 (JC-1 polymers), which indicates the intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1 (JC-1 monomers), which indicates the dissipation of mitochondrial transmembrane potential. Ratios of JC-1 monomer to JC-1 polymer (red/green fluorescence) was calculated. C HT1080 cells were transfected with non-targeted control siRNA or CHK1-directed siRNA for 48 h. Subsequently, cells were treated with 10 μM TH287 for 24 h and then were treated as described in B . Kinetic profile of ECAR and OCR in HT1080 cells was measured in real-time, under basal conditions and in response to mitochondrial drugs oligomycin, FCCP, Rotenone & antimycin A for OCR ( D ), and in response to glucose, oligomycin and 2-DG for ECAR ( E). Indices of mitochondrial respiratory function, calculated from OCR profile: basal OCR, ATP-linked OCR and maximal OCR (see also Materials and Methods section). Indices of glycolytic pathway activation, calculated from ECAR profile: glycolysis, glycolytic capacity and glycolytic reserve (see also Materials and Methods section). F and G Kinetic profile of ECAR and OCR in HT1080 cells transfected with with non-targeted control siRNA or CHK1-directed siRNA was measured as described in D and E . Data shown were representative of three independent experiments and data presented in bars as mean ± S.D. The statistical differences between the two groups were analyzed by two-sided unpaired Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: TH287 (Selleck Chemicals, Houston, TX, USA) and UCN-01 (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Transmission Assay, Staining, Flow Cytometry, Fluorescence, Membrane, Polymer, Transfection, Control, Activation Assay

Journal: Oncogene

Article Title: Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress

doi: 10.1038/s41388-021-02105-9

Figure Lengend Snippet: A Flow cytometry analysis and quantification of mitochondrial mass in HT1080 cells pretreated with CCCP (12.5 μM) for 48 h, followed by treatment with TH287 (10 μM) for 24 h. B Western blotting analysis of OXPHOS complex and COXIV protein levels in HT1080 cells pretreated with CCCP (12.5 μM) for 48 h before TH287 treatment for 24 h. C HT1080 cells pretreated with CCCP (12.5 μM) for 48 h before TH287 treatment for 24 h and were then subjected to cell cycle distribution analysis by flow cytometry. D Flow cytometric analysis of intracellular ROS levels measured by DCFH-DA. HT1080 were pretreated with CCCP (12.5 μM) for 48 h before TH287 treatment for 24 h. E Flow cytometric analysis of Mitochondrial superoxide levels by MitoSOX Red in HT1080 cells pretreated with CCCP (12.5 μM) for 48 h, followed by treatment with TH287 (10 μM) for 24 h. F Flow cytometry analysis and quantification of mitochondrial mass in HT1080 cells pretreated with CCCP (12.5 μM) for 48 h, followed by treatment with paclitaxel (50 nM) for 24 h. G Flow cytometric analysis of Mitochondrial superoxide levels by MitoSOX Red in HT1080 cells pretreated with CCCP (12.5 μM) for 48 h, followed by treatment with paclitaxel (50 nM) for 24 h. Data shown were representative of three independent experiments and data presented in bars as mean ± S.D. The statistical differences between the two groups were analyzed by two-sided unpaired Student’s t test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: TH287 (Selleck Chemicals, Houston, TX, USA) and UCN-01 (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Flow Cytometry, Western Blot

Journal: Oncogene

Article Title: Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress

doi: 10.1038/s41388-021-02105-9

Figure Lengend Snippet: A Western blotting analysis of PGC-1α protein levels in HT1080 cells treated with TH287 (10 μM) alone or TH287 in combination with UCN-01 (300 nM) for 24 h. B HT1080 cells were transfected with non-targeted control siRNA or PGC-1α-directed siRNA for 48 h. Western blotting analysis the RNAi efficiency of PGC-1α in HT1080 cells. C HT1080 cells were transfected with non-targeted control siRNA or PGC-1α-directed siRNA for 48 h. Subsequently, cells were treated with 10 μM TH287 for 24 h, and then subjected to cell cycle distribution analysis by flow cytometry. The proportions of G2/M-phase cells were statistically analyzed. D HT1080 cells were transfected with non-targeted control siRNA or PGC-1α-directed siRNA for 48 h. Subsequently, cells were treated with 10 μM TH287 for 24 h and mitotic index was assessed by pH3/PI FACS. The mitotic indices were statistically analyzed. E HT1080 cells were transfected with non-targeted control siRNA or PGC-1α-directed siRNA for 48 h. Flow cytometric analysis of intracellular ROS levels measured by DCFH-DA in HT1080-siSCR and HT1080-siPGC-1α cells treated with TH287 (10 μM) for 24 h. F HT1080 cells were transfected with non-targeted control siRNA or PGC-1α-directed siRNA for 48 h. Flow cytometric analysis of Mitochondrial superoxide levels by MitoSOX Red in HT1080-siSCR and HT1080-siPGC-1α cells treated with TH287 (10 μM) for 24 h. G HT1080 cells were transfected with non-targeted control siRNA or siPGC-1α-directed siRNA for 48 h. Subsequently, cells were treated with 10 μM TH287 for 24 h and were stained with MitoTracker Red and analyzed using a flow cytometer. H HT1080 cells were transfected with non-targeted control siRNA or siPGC-1α-directed siRNA for 48 h. Subsequently, cells were treated with 10 μM TH287 for 24 h and Mitochondria fractions were isolated. The mitochondrial nature of the isolated proteins was confirmed with Western blotting analysis using antibodies against various mitochondrial components. Data shown were representative of three independent experiments and data presented in bars as mean ± S.D. The statistical differences between the two groups were analyzed by two-sided unpaired Student’s t test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: TH287 (Selleck Chemicals, Houston, TX, USA) and UCN-01 (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot, Transfection, Control, Flow Cytometry, Staining, Isolation

Journal: Oncogene

Article Title: Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress

doi: 10.1038/s41388-021-02105-9

Figure Lengend Snippet: A HT1080 cells were treated with DMSO or TH287 (10 μM) for 6 h and then subjected to cell cycle distribution analysis by flow cytometry. The proportions of G2/M-phase cells were statistically analyzed. B HT1080 cells were treated with DMSO or TH287 (10 μM) for 6 h and subjected to pH3/PI FACS. The mitotic indices were statistically analyzed. C Flow cytometric analysis of intracellular ROS levels measured by DCFH-DA. HT1080 cells were treated with TH287 (10 μM) for 6 h. D Flow cytometric analysis of Mitochondrial superoxide levels by MitoSOX Red in HT1080 cells treated with TH287 (10 μM) for 6 h. E Mitochondrial mass measured by MitoTracker Red staining. HT1080 cells were treated with TH287 (10 μM) for 6 h. F HT1080 cells were treated with DMSO or TH287 (10 μM) for 12 h and then subjected to cell cycle distribution analysis by flow cytometry. The proportions of G2/M-phase cells were statistically analyzed. G HT1080 cells were treated with DMSO or TH287 (10 μM) for 12 h and subjected to pH3/PI FACS. The mitotic indices were statistically analyzed. H Flow cytometric analysis of intracellular ROS levels measured by DCFH-DA. HT1080 cells were treated with TH287 (10 μM) for 12 h. I Flow cytometric analysis of Mitochondrial superoxide levels by MitoSOX Red in HT1080 cells treated with TH287 (10 μM) for 12 h. J Mitochondrial mass measured by MitoTracker Red staining. HT1080 cells were treated with TH287 (10 μM) for 12 h. K Flow cytometry analysis and quantification of mitochondrial mass in HT1080 cells pretreated with TH287 (10 μM) for 12 h, followed by treatment with UCN-01 (300 nM) for 24 h. L Flow cytometric analysis of intracellular ROS levels measured by DCFH-DA. HT1080 cells were pretreated with TH287 (10 μM) for 12 h, followed by treatment with UCN-01 (300 nM) for 24 h. M Flow cytometric analysis of Mitochondrial superoxide levels by MitoSOX Red in HT1080 cells pretreated with TH287 (10 μM) for 12 h, followed by treatment with UCN-01 (300 nM) for 24 h. N Western blotting analysis of p-AKT, total AKT, p-ERK, total ERK, CyclinD and p21 protein levels in HT1080 cells treated with UCN-01 (300 nM) for 24 h and Quantification of EdU-positive cells after 300 nM UCN-01 treatment for 24 h. O Western blotting analysis of p-AKT, total AKT, p-ERK, total ERK, CyclinD and p21 protein levels in HT1080 cells treated with CCCP (12.5 μM) for 48 h and Quantification of EdU-positive cells after 12.5 μM CCCP treatment for 48 h. P Western blotting analysis of p-AKT, total AKT, p-ERK, total ERK, CyclinD and p21 protein levels in HT1080 cells transfected with non-targeted control siRNA or PGC-1α-directed siRNA for 48 h and Quantification of EdU-positive cells after the depletion of PGC-1α by RNAi for 48 h. Data shown were representative of three independent experiments and data presented in bars as mean ± S.D. The statistical differences between the two groups were analyzed by two-sided unpaired Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns stand for no significant.

Article Snippet: TH287 (Selleck Chemicals, Houston, TX, USA) and UCN-01 (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Flow Cytometry, Staining, Western Blot, Transfection, Control

Journal: Oncogene

Article Title: Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress

doi: 10.1038/s41388-021-02105-9

Figure Lengend Snippet: A Western blotting analysis of γ-H2AX protein levels in HT1080 cells treated with TH287 (10 μM) alone or TH287 in combination with UCN-01 (300 nM) for 24 h. B Western blotting analysis of γ-H2AX protein levels in HT1080 cells treated with TH287 (10 μM) alone or TH287 in combination with NAC (10 mM) for 24 h. C Western blotting analysis of γ-H2AX protein levels in HT1080 cells treated with TH287 (10 μM) alone or TH287 in combination with MitoQ (1 μM) for 24 h. D The amount of DNA strand breaks was quantified by measuring the amount of tail-DNA using the neutral comet assay as described in methods. HT1080 cells treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h. Representative pictures were shown on the left. Values represent average ± S.D. from two independent experiments (≥100 comets per experiment). Scale bar, 25 μm. E Scatterplots of apoptotic cells. Cells were treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM) for 24 h, and then harvested for analysis of apoptosis using Annexin V PE Apoptosis Detection Kit I. F Clonogenic assay of HT1080 cells treated with TH287 (10 μM) alone or in combination with UCN-01 (300 nM). G Clonogenic assay of HT1080 cells treated with TH287 (10 μM) alone or in combination with MitoQ (1 μM). H Western blotting analysis of p-CHK1, total CHK1 and γ-H2AX protein levels in HT1080 cells pretreated with CCCP (12.5 μM) for 48 h before TH287 (10 μM) treatment for 24 h. I HT1080 cells were transfected with non-targeted control siRNA or siPGC-1α-directed siRNA for 48 h. Western blotting analysis of γ-H2AX protein levels in HT1080-siSCR and HT1080-siPGC-1α cells treated with TH287 (10 μM) for 24 h. Data shown were representative of three independent experiments and data presented in bars as mean ± S.D. The statistical differences between the two groups were analyzed by two-sided unpaired Student’s t test. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns stand for no significant.

Article Snippet: TH287 (Selleck Chemicals, Houston, TX, USA) and UCN-01 (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Western Blot, Neutral Comet Assay, Clonogenic Assay, Transfection, Control

Journal: Oncogene

Article Title: Disrupted mitochondrial homeostasis coupled with mitotic arrest generates antineoplastic oxidative stress

doi: 10.1038/s41388-021-02105-9

Figure Lengend Snippet: A , B , C Growth curves and final weights of tumors from transplanted HT1080 cells in nude mice. Mice were randomized into one of four groups; vehicle only ( n = 6), 6 mg/kg TH287 only ( n = 6), 3 mg/kg UCN-01 only ( n = 6) or 6 mg/kg TH287 plus 3 mg/kg UCN-01 ( n = 6). Final weights were taken at day 16. D Representative IHC images showing the γ-H2AX. Scale bar, 20 μm. E Quantification of the relative intensity of IHC staining of γ-H2AX was performed using ImageJ software. F Representative IHC images showing the NDUFB8 and Cytochrome C. Scale bar, 20 μm. G Quantification of the relative intensity of IHC staining of NDUFB8 and Cytochrome C was performed using ImageJ software. H A schematic model. When cell cycle is decelerated, via the inhibition of CHK1, pre-depletion of mitochondria, or blocking mitochondrial biogenesis by PGC-1α depletion, so that M-arrest is spared or attenuated, mitochondria will be more likely to stay homeostatic, thus generating less ROS and rendering cancer cells less responsive to the therapeutic agents. Data shown were representative of three independent experiments and data presented in bars as mean ± S.D. The statistical differences between the two groups were analyzed by two-sided unpaired Student’s t test and ANOVA was used to compare significant differences among multiple experimental groups. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: TH287 (Selleck Chemicals, Houston, TX, USA) and UCN-01 (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Immunohistochemistry, Software, Inhibition, Blocking Assay