Journal: Scientific Reports

Article Title: Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells

doi: 10.1038/s41598-019-48169-2

Figure Lengend Snippet: Selectivity Index of Danazol on several multidrug-resistant cancer cell lines.

Article Snippet: Parental cancer cell lines, including human cervical epithelioid carcinoma HeLaS3, human hepatoblastoma HepG2, and human non-small cell lung carcinoma NCI-H460, were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques:

Journal: Scientific Reports

Article Title: Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells

doi: 10.1038/s41598-019-48169-2

Figure Lengend Snippet: Cytotoxicity of natural and synthetic steroid hormones on HeLaS3 (parental) and KB/VIN (MDR) cancer cell lines.

Article Snippet: Parental cancer cell lines, including human cervical epithelioid carcinoma HeLaS3, human hepatoblastoma HepG2, and human non-small cell lung carcinoma NCI-H460, were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques:

Journal: Scientific Reports

Article Title: Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells

doi: 10.1038/s41598-019-48169-2

Figure Lengend Snippet: Results of ABCB1 gene expression analysis. HeLaS3 and KB/VIN were treated with danazol for 24, 48, and 72 hours. Total RNA was extracted and ABCB1 gene expression level of each sample was quantified by real-time PCR. The ABCB1 gene expression was significantly down-regulated by danazol treatment in KB/VIN cell line. Statistical differences were evaluated by ANOVA followed post hoc analysis (Tukey’s test). * Indicates p value < 0.05 compared with control group. Data presented as mean ± SE of at least three experiments, each in triplicate.

Article Snippet: Parental cancer cell lines, including human cervical epithelioid carcinoma HeLaS3, human hepatoblastoma HepG2, and human non-small cell lung carcinoma NCI-H460, were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control

Journal: Scientific Reports

Article Title: Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells

doi: 10.1038/s41598-019-48169-2

Figure Lengend Snippet: Results of cell cycle analysis in HeLaS3 and MDR KB/VIN cells. HeLaS3 and KB/VIN were treated with culture medium ( a ), DMSO ( b ), paclitaxel ( c ; positive control), danazol ( d ), β-estradiol ( e ), and deoxycorticosterone ( f ) for 24, 48, and 72 hours. DNA contents and cell cycle distribution of each sample were determined by PI solution (X-axis PE). Danazol, β-estradiol, and deoxycorticosterone arrested KB/VIN cell at G2/M phase and caused apoptosis (increased sub G1) in a time-dependent manner.

Article Snippet: Parental cancer cell lines, including human cervical epithelioid carcinoma HeLaS3, human hepatoblastoma HepG2, and human non-small cell lung carcinoma NCI-H460, were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Cell Cycle Assay, Positive Control

Journal: Scientific Reports

Article Title: Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells

doi: 10.1038/s41598-019-48169-2

Figure Lengend Snippet: Results of caspase activity and ROS levels detection assay in HeLaS3 and MDR KB/VIN cells. HeLaS3 and KB/VIN cells were treated with or without danazol for 72 hours. The activities of caspase-8 ( a ) and caspase-9 ( b ) were evaluated by Cell Meter™ Caspase Activity Apoptosis Assay Kits. Danazol activated caspase-8 in KB/VIN cells while the level remained the same in HeLaS3 cells compared with respective control group. ( c ) The treatment of danazol increased the ROS level in both HeLaS3 and KB/VIN cell lines. Menadione, a ROS inducer, was used as a positive control. Statistical differences were evaluated by the ANOVA followed post hoc analysis (Tukey’s test). * indicates p value < 0.05 compared with control group. Data presented as mean ± SE of at least two experiments, each in duplicate.

Article Snippet: Parental cancer cell lines, including human cervical epithelioid carcinoma HeLaS3, human hepatoblastoma HepG2, and human non-small cell lung carcinoma NCI-H460, were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Activity Assay, Detection Assay, Apoptosis Assay, Control, Positive Control

Journal: Scientific Reports

Article Title: Danazol mediates collateral sensitivity via STAT3/Myc related pathway in multidrug-resistant cancer cells

doi: 10.1038/s41598-019-48169-2

Figure Lengend Snippet: MYC signaling mediates danazol-induced G2/M arrest in MDR KB/VIN cells. HeLaS3 and KB/VIN were treated with or without danazol for 72 hours. The whole-treated cells were extracted and analyzed by ELISA using antibodies specific to human ( a ) STAT3-pY705, ( b ) cMYC, ( c ) CDC25, ( d ) CDK1, and ( e ) p21. The results showed danazol reduced the concentration of MYC (c-Myc) and its upstream protein STAT3-pY705 in MDR KB/VIN cells. In addition, decreases in downstream signaling proteins, CDC25 and CDK1, were shown. Each experiment was performed in duplicate. Student’s t-test, *p < 0.05 compared with cells treated with medium only control.

Article Snippet: Parental cancer cell lines, including human cervical epithelioid carcinoma HeLaS3, human hepatoblastoma HepG2, and human non-small cell lung carcinoma NCI-H460, were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Control

Journal: The FASEB Journal

Article Title: DeSUMOylating isopeptidase 1 participates in the faithful chromosome segregation and vincristine sensitivity

doi: 10.1096/fj.202401560RR

Figure Lengend Snippet: Acceleration of M‐phase progression due to DESI1 knockdown. (A) HeLa S3 cells underwent treatment with DESI1‐targeting (siDESI1) or non‐targeting (siCtrl) siRNAs for 48 h, followed by the analysis of whole‐cell lysates via Western blot analysis. (B–D) HeLa S3 cells were treated with siCtrl or siDESI1 #1 for 48 h and 6 μM RO‐3306 during the final 20 h. Subsequently, cells were cultured with fresh medium for 45 min without RO‐3306, and the fixed cells were immunostained using an anti‐α‐tubulin antibody and 1 μM Hoechst 33342. M‐phase progression was evaluated based on the morphologies of DNA and microtubules. In (B), a schematic depiction of the experimental method is shown. In (C), representative images are displayed. M‐phase cells were divided into two groups: before (P/PM/M, pink arrows) and after (A/T/Cyto, blue arrows) the anaphase onset. Scale bar, 20 μm. In (D), the ratios of each group ( n > 188 per condition) and mitotic index ( n > 1002 per condition), a ratio of the number of M‐phase cells to the total number of cells, are shown as the mean ± SD calculated from three independent experiments. p ‐values were determined using the Student's t ‐test. (E) HeLa S3/FLAG‐DESI1 WT and HeLa S3/FLAG‐DESI1 C108S cells were treated with siCtrl or siDESI1 #3 for 28 h. Then, expression of FLAG‐DESI1 WT or FLAG‐DESI1 C108S was induced by treatment with 0.4 μg/mL or 0.85 μg/mL doxycycline (Dox), respectively, for an additional 20 h. Whole‐cell lysates were analyzed by Western blot analysis. (F, G) HeLa S3/FLAG‐DESI1 WT and HeLa S3/FLAG‐DESI1 C108S cells were treated with siCtrl or siDESI1 #3 for 48 h and with 6 μM RO‐3306 during the final 20 h in the presence of Dox (0.4 μg/mL for WT; 0.85 μg/mL for C108S). Subsequently, cells were cultured in fresh medium for 45 min without RO‐3306. M‐phase cells were examined for mitotic subphases as described in (C). In (F), a schematic depiction of the experimental method is shown. M‐phase progression ( n > 190 per condition) and mitotic index ( n > 1002 per condition) were evaluated as described in (D). The results are shown in (G) as the mean ± SD calculated from three independent experiments. p ‐values were determined using Tukey's multiple comparisons test.

Article Snippet: Human cervical cancer HeLa S3, endometrioid cancer HEC‐1‐A (Japanese Collection of Research Bioresources, Osaka, Japan), and Lenti‐X 293T cells (Clontech Laboratories, Mountain View, CA, USA) were cultured in Dulbecco's modified Eagle medium, supplemented with 5% (HeLa S3, Lenti‐X 293T) or 10% (HEC‐1‐A) fetal bovine serum (FBS), 20 mM HEPES‐NaOH (pH 7.4), and 2 mM of L (+)‐glutamine at 37°C in a 5% CO 2 environment.

Techniques: Knockdown, Western Blot, Cell Culture, Expressing

Journal: The FASEB Journal

Article Title: DeSUMOylating isopeptidase 1 participates in the faithful chromosome segregation and vincristine sensitivity

doi: 10.1096/fj.202401560RR

Figure Lengend Snippet: M‐phase progression is delayed by DESI1 overexpression. (A) HeLa S3/FLAG‐DESI1 WT and HeLa S3/FLAG‐DESI1 C108S cells were treated with 2 μg/mL and 4.3 μg/mL Dox for 20 h, respectively. Whole‐cell lysates were then analyzed using Western blot analysis. (B, C) HeLa S3/FLAG‐DESI1 WT and HeLa S3/FLAG‐DESI1 C108S cells were cultured with Dox at the concentrations described in (A) in the presence of 6 μM RO‐3306 for 20 h. After a 75‐min release from RO‐3306, cells were fixed and stained for α‐tubulin and DNA. A schematic depiction of the experimental method is shown in (B). M‐phase progression was evaluated as described in Figure . The percentages of each group ( n > 329 per condition) and the mitotic index ( n > 1009 per condition) are shown as the mean ± SD calculated from three independent experiments. p ‐values were determined using the Student's t‐ test.

Article Snippet: Human cervical cancer HeLa S3, endometrioid cancer HEC‐1‐A (Japanese Collection of Research Bioresources, Osaka, Japan), and Lenti‐X 293T cells (Clontech Laboratories, Mountain View, CA, USA) were cultured in Dulbecco's modified Eagle medium, supplemented with 5% (HeLa S3, Lenti‐X 293T) or 10% (HEC‐1‐A) fetal bovine serum (FBS), 20 mM HEPES‐NaOH (pH 7.4), and 2 mM of L (+)‐glutamine at 37°C in a 5% CO 2 environment.

Techniques: Over Expression, Western Blot, Cell Culture, Staining

Journal: The FASEB Journal

Article Title: DeSUMOylating isopeptidase 1 participates in the faithful chromosome segregation and vincristine sensitivity

doi: 10.1096/fj.202401560RR

Figure Lengend Snippet: Delay in M‐phase progression due to SENP1 knockdown. (A) HeLa S3 cells were treated with either siCtrl or SENP1‐targeting siRNA for 48 h, and whole‐cell lysates were analyzed using Western blot analysis. (B–D) HeLa S3 cells were treated with siCtrl or siSENP1 for 48 h and cultured with 6 μM RO‐3306 during the final 20 h. The cells were then cultured without RO‐3306 for an additional 75 min. M‐phase progression was evaluated as described in Figure . A schematic depiction of the experimental method is shown in (B). Representative images are displayed in (C). DNA (red), microtubules (green). Scale bar, 20 μm. The results are shown as the mean ± SD calculated from three independent experiments (left graph, n > 269; right graph, n > 1044 per condition). p ‐values were determined using the Student's t‐ test. (E) HeLa S3/FLAG‐SENP1 cells were treated with 0–4 μg/mL Dox for 24 h, and whole‐cell lysates were analyzed using Western blot analysis. (F) HeLa S3/FLAG‐SENP1 cells were simultaneously incubated with 2 μg/mL Dox and 6 μM RO‐3306 for 20 h. The cells were then cultured without RO‐3306 for 60 min. M‐phase progression was evaluated as described in Figure . The results are shown as the mean ± SD calculated from three independent experiments (left graph, n > 362; right graph, n > 1005 per condition). p ‐values were determined using the Student's t‐ test.

Article Snippet: Human cervical cancer HeLa S3, endometrioid cancer HEC‐1‐A (Japanese Collection of Research Bioresources, Osaka, Japan), and Lenti‐X 293T cells (Clontech Laboratories, Mountain View, CA, USA) were cultured in Dulbecco's modified Eagle medium, supplemented with 5% (HeLa S3, Lenti‐X 293T) or 10% (HEC‐1‐A) fetal bovine serum (FBS), 20 mM HEPES‐NaOH (pH 7.4), and 2 mM of L (+)‐glutamine at 37°C in a 5% CO 2 environment.

Techniques: Knockdown, Western Blot, Cell Culture, Incubation

Journal: The FASEB Journal

Article Title: DeSUMOylating isopeptidase 1 participates in the faithful chromosome segregation and vincristine sensitivity

doi: 10.1096/fj.202401560RR

Figure Lengend Snippet: DESI1 is essential for accurate chromosome segregation. (A–D) HeLa S3 cells underwent treatment with siCtrl or siDESI1 #1 for 48 h and 6 μM RO‐3306 for the final 20 h. Following release from RO‐3306, cells were observed under time‐lapse analysis in the presence of 0.1 μM Hoechst 33342 for 180 min at 5‐min intervals. In (A), a series of representative images are displayed. Arrows point to individual cells. In (B), the length of mitotic subphases (red, P/PM; yellow, M; green, A/T) for each cell is depicted ( n = 40). In (C), the proportions of each subphase at the specified time points are graphed. The dashed black lines represent the percentages of mitotic cells. In (D), the time spans from M‐phase entry to furrow compression, prophase/prometaphase, and metaphase are presented as the mean ± SD ( n = 40). p ‐values were calculated using Welch's t ‐test. (E) Cells with DESI1 knockdown were synchronized as outlined in Figure . Images of representative cells exhibiting chromosome bridge and lagging chromosome are displayed, and their percentages are graphed as the mean ± SD calculated from three independent experiments ( n = 50 per condition). p ‐values were calculated using the Student's t ‐test. Scale bar, 10 μm. (F) DESI1 was depleted from HeLa S3/FLAG‐DESI1 WT and HeLa S3/FLAG‐DESI1 C108S cells and re‐expressed via Dox treatment (WT, 0.4 μg/mL; C108S, 0.85 μg/mL). The cells were synchronized as outlined in Figure . The mean values ± SD were calculated from three independent experiments ( n ≥ 50 per condition). p ‐values were calculated using Tukey's multiple comparisons test. (G, H) HeLa S3 cells underwent treatment with siCtrl or siDESI1 #1 for 48 h, and during the final 24 h cells were synchronized by treatment with 7.5 μM STLC. Subsequently, cells were fixed with 100% methanol. A schematic representation of the experimental method is displayed in (G). In (H), the mitotic index is graphed as the mean ± SD calculated from three independent experiments ( n > 500 per condition). p ‐values were calculated using Student's t‐ test. (I, J) HeLa S3 cells were treated with siCtrl or siDESI1 #1 for 24 h and then monitored by time‐lapse analysis at 20‐min intervals for 24 h in the presence of 50 ng/mL nocodazole and 0.1 μM Hoechst 33342. In (I), representative images of mitotic arrest, slippage, and abnormal cytokinesis are displayed. In (J), the duration of M phase of individual cell is shown on the left ( n = 40). Red, green, and black asterisks indicate mitotic slippage, abnormal cytokinesis, and cell death, respectively. On the right, the percentage of cells exhibiting slippage, abnormal cytokinesis, and cell death are shown as the mean ± SD calculated from three independent experiments ( n = 40). p ‐values were calculated using Student's t‐ test.

Article Snippet: Human cervical cancer HeLa S3, endometrioid cancer HEC‐1‐A (Japanese Collection of Research Bioresources, Osaka, Japan), and Lenti‐X 293T cells (Clontech Laboratories, Mountain View, CA, USA) were cultured in Dulbecco's modified Eagle medium, supplemented with 5% (HeLa S3, Lenti‐X 293T) or 10% (HEC‐1‐A) fetal bovine serum (FBS), 20 mM HEPES‐NaOH (pH 7.4), and 2 mM of L (+)‐glutamine at 37°C in a 5% CO 2 environment.

Techniques: Knockdown

Journal: The FASEB Journal

Article Title: DeSUMOylating isopeptidase 1 participates in the faithful chromosome segregation and vincristine sensitivity

doi: 10.1096/fj.202401560RR

Figure Lengend Snippet: Reduction of Aurora B localization on chromosomes by DESI1 knockdown. (A, B) HeLa S3/FLAG‐DESI1 WT and HeLa S3/FLAG‐DESI1 C108S cells were treated with siCtrl or siDESI1 #3 for 48 h in the presence of Dox (WT, 0.4 μg/mL; C108S, 0.85 μg/mL). In the final 20 h, cells were treated with 6 μM RO‐3306. Following a 30‐min release from RO‐3306, cells were fixed with 4% formaldehyde in PBS (−) and stained for α‐tubulin (red), Aurora B (green), and DNA (blue). Representative images are displayed in (A). Scale bar, 10 μm. The fluorescence intensity of Aurora B on chromosomes was quantified using ImageJ, and the results are shown as the mean ± SD in (B) ( n ≥ 30 per condition). p ‐values were calculated using the Games‐Howell multiple comparison test. (C) HeLa S3 cells were treated with siCtrl or siDESI1 #1 for 48 h and cultured in the presence of 4 mM thymidine for the final 24 h. After thymidine removal, cells were cultured for 9 h and then treated with 6 μM RO‐3306 for an additional 10 h. Following the removal of RO‐3306, cells were treated with 5 μM STLC for 1 h. Subsequently, mitotic cells were collected and analyzed by Western blot analysis. (D) HeLa S3 cells were treated with siRNA and thymidine as outlined in (C). After thymidine removal, G2 and M‐phase cells were obtained after 9‐h culture without any drug (G2) and 16‐h culture with 5 μM STLC (M), respectively, and subjected to Western blot analysis. (E, F) HeLa S3 cells were treated with siCtrl or siDESI1 #1 for 48 h and 6 μM RO‐3306 during the final 20 h. After a 5‐min culture without RO‐3306, cells were treated with 40 μM MG‐132 for an additional 25 min, fixed using 4% formaldehyde in PBS (−), and stained for α‐tubulin (red), Aurora B (green), and DNA (blue). Representative images are displayed in (E). Scale bar, 20 μm. The fluorescence intensity of Aurora B on chromosomes was quantified, and the results are shown as the mean ± SD ( n ≥ 35 per condition). p ‐values were calculated using Tukey's multiple comparisons test.

Article Snippet: Human cervical cancer HeLa S3, endometrioid cancer HEC‐1‐A (Japanese Collection of Research Bioresources, Osaka, Japan), and Lenti‐X 293T cells (Clontech Laboratories, Mountain View, CA, USA) were cultured in Dulbecco's modified Eagle medium, supplemented with 5% (HeLa S3, Lenti‐X 293T) or 10% (HEC‐1‐A) fetal bovine serum (FBS), 20 mM HEPES‐NaOH (pH 7.4), and 2 mM of L (+)‐glutamine at 37°C in a 5% CO 2 environment.

Techniques: Knockdown, Staining, Fluorescence, Comparison, Cell Culture, Western Blot

Journal: The FASEB Journal

Article Title: DeSUMOylating isopeptidase 1 participates in the faithful chromosome segregation and vincristine sensitivity

doi: 10.1096/fj.202401560RR

Figure Lengend Snippet: The reduction in mRNA levels of FoxM1 target genes upon DESI1 knockdown. (A, B) HeLa S3 cells were transfected with siCtrl or siFoxM1. After a 6‐h incubation, cells were treated with 5 μM STLC for 16 h. The mitotic cells were harvested via mitotic shake‐off, and the mRNA levels of Aurora B, cyclin B1, and CENP‐F were analyzed by real‐time PCR. In (A), the fold changes in mRNA levels are expressed relative to siCtrl, and the results are displayed as the mean ± SD calculated from three independent experiments. p ‐values were calculated using Welch's t‐ test. Knockdown was confirmed by Western blot analysis in (B). (C–E) HeLa S3/Strep‐HA‐FoxM1/FLAG‐DESI1 cells (C), HeLa S3/Strep‐HA‐FoxM1 cells (D), and HeLa S3/Strep‐HA‐DESI1 cells (E) were treated with 4 μg/mL Dox for 48 h and 5 μM STLC during the last 16 h. The mitotic cells were harvested via mitotic shake‐off and solubilized with RIPA buffer. The lysates were subjected to the pull‐down assay with Strep‐Tactin resin, and co‐precipitated protein was detected by Western blot analysis. (F) HeLa S3/Strep‐HA‐FoxM1 cells were transfected with siCtrl or siDESI1 (#1) and cultured in medium containing 4 μg/mL Dox for 22 h. During the last 16 h, 5 μM STLC was added. The mitotic cells were harvested via mitotic shake‐off, and the mRNA levels of Aurora B were analyzed by real‐time PCR. The fold changes in mRNA levels are expressed relative to siCtrl (Dox−), and the results are displayed as the mean ± SD calculated from three independent experiments. p ‐values were calculated using Games‐Howell multiple comparison test. (G) The cells were treated with siDESI1 (#1), and mRNA was obtained as outlined in (A). The mRNA levels of CENP‐F were analyzed by real‐time PCR. (H) HeLa S3 cells were transfected with siCtrl or siDESI1 #1. After a 24‐h incubation, cells were cultured in a medium containing 4 mM thymidine for 24 h, followed by an additional 9‐h culture without thymidine. The mRNA levels of Aurora B and cyclin B1 were analyzed by real‐time PCR. The fold changes in mRNA levels are expressed relative to siCtrl, and the results are displayed as the mean ± SD calculated from three independent experiments. p ‐values were calculated using Welch's t‐ test. (I, J) HeLa S3/Strep‐HA‐FoxM1 cells were transfected with siCtrl or siDESI1 #1, and after a 6‐h culture, the medium was replaced with a fresh one, followed by a 22‐h culture. The cells were then incubated with 4 μg/mL Dox and 6 μM RO‐3306 for an additional 20 h. After a 45‐min release from RO‐3306, the cells were fixed and immunofluorescence staining was performed as described in Figure , In (I), representative images are displayed. Scale bar, 10 μm. The blue arrows point to cells that have progressed beyond the anaphase. In (J), M‐phase progression was evaluated as described in Figure . M‐phase cells were divided into four groups: prophase/prometaphase (P/PM), metaphase (M), anaphase/telophase (A/T), and cytokinesis (Cyto). The results are shown as the mean ± SD calculated from three independent experiments (left graph, n > 272; right graph, n > 1002 per condition). p ‐values were determined using the Tukey's multiple comparisons test.

Article Snippet: Human cervical cancer HeLa S3, endometrioid cancer HEC‐1‐A (Japanese Collection of Research Bioresources, Osaka, Japan), and Lenti‐X 293T cells (Clontech Laboratories, Mountain View, CA, USA) were cultured in Dulbecco's modified Eagle medium, supplemented with 5% (HeLa S3, Lenti‐X 293T) or 10% (HEC‐1‐A) fetal bovine serum (FBS), 20 mM HEPES‐NaOH (pH 7.4), and 2 mM of L (+)‐glutamine at 37°C in a 5% CO 2 environment.

Techniques: Knockdown, Transfection, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Pull Down Assay, Cell Culture, Comparison, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: DeSUMOylating isopeptidase 1 participates in the faithful chromosome segregation and vincristine sensitivity

doi: 10.1096/fj.202401560RR

Figure Lengend Snippet: DESI1 knockdown results in decreased sensitivity to vincristine. (A–E) HeLa S3 cells were transfected with siCtrl or siDESI1 #1. Following a 24‐h culture, cells were exposed to 1.5 nM vincristine (VCR) for a period of 4 days. In (A), representative images are displayed. Scale bar, 20 μm. In (B), cells were immunostained for γ‐tubulin, and the number of centrosomes was counted. The graph depicts the percentage of cells with more than three centrosomes as the mean ± SD calculated from three independent experiments ( n > 204). p ‐values were calculated using Tukey's multiple comparisons test. In (C), cells were fixed and stained for α‐tubulin (green) and DNA (red) over a period of 1–4 days. Representative images are displayed. Scale bar, 20 μm. White arrowheads point to cells initiating chromosome segregation. In (D), the mitotic index is displayed, and mitotic cells were categorized into two groups: before (P/PM/M) and after (A/T/Cyto) the onset of anaphase. The ratios of the mitotic index ( n > 1004 per condition) and each group ( n > 208 per condition) are displayed as the mean ± SD calculated from three independent experiments. p ‐values were calculated using the Student's t‐ test. The statistical significance between siCtrl‐treated cells and siDESI1 (#1)‐treated cells for the ratio of cells in A/T/Cyto are as follows: p = .012 after a 1‐day culture; p = .025 after a 2‐day culture. In (E), the representative images of cells with normal metaphase (a), cytokinesis (b), and abnormal cells with multipolar spindle (c), multipolar cytokinesis (d), and two spindles (e) are shown. Scale bar, 20 μm. (F) HeLa S3 cells were treated with siDESI1 #1. After 24 h, cells were incubated with 1.5 nM VCR, 5 μM proTAME, or their combination for a duration of 3 days, and a CCK‐8 assay was performed. The absorbance ratio is expressed relative to control cells (siCtrl, DMSO), and the results are displayed as the mean ± SD calculated from five independent experiments. p ‐values were calculated using the Student's t‐ test or Welch's t‐ test.

Article Snippet: Human cervical cancer HeLa S3, endometrioid cancer HEC‐1‐A (Japanese Collection of Research Bioresources, Osaka, Japan), and Lenti‐X 293T cells (Clontech Laboratories, Mountain View, CA, USA) were cultured in Dulbecco's modified Eagle medium, supplemented with 5% (HeLa S3, Lenti‐X 293T) or 10% (HEC‐1‐A) fetal bovine serum (FBS), 20 mM HEPES‐NaOH (pH 7.4), and 2 mM of L (+)‐glutamine at 37°C in a 5% CO 2 environment.

Techniques: Knockdown, Transfection, Staining, Incubation, CCK-8 Assay, Control

Journal: Cell Metabolism

Article Title: Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response

doi: 10.1016/j.cmet.2022.10.011

Figure Lengend Snippet:

Article Snippet: human cervical cancer S3 (HeLa S3) , ATCC , CRL-4000; RRID: CVCL 4388.

Techniques: Recombinant, Reverse Transcription, Staining, Clone Assay, Mutagenesis, SYBR Green Assay, DNA Extraction, Enzyme-linked Immunosorbent Assay, RNA Sequencing, CRISPR, Knock-Out, Quantitative RT-PCR, Software