Journal: iScience

Article Title: Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

doi: 10.1016/j.isci.2021.103484

Figure Lengend Snippet:

Article Snippet: pcDNA3 HA-SUMO1 , , Addgene plasmid # 48966.

Techniques: Recombinant, Blocking Assay, cDNA Synthesis, Plasmid Preparation, Western Blot, Cell Culture, Mutagenesis, Software

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 1 Effects of varying doxorubicin doses on cellular outcomes in HCT116 cells with different p53 and p21 statuses. A Immunofluorescence images of HCT116 WT, p53−/−, and p21−/−cells treated with 100 nM or 2 μM doxorubicin for 36 h, showing nuclear morphology (DAPI, cyan) and actin cytoskeleton (phalloidin, red). Scale bar = 20 μm. B Quantification of multinucleated (left) and apoptotic cells (right) in WT, p53−/−, and p21−/−cells treated with 100 nM or 2 µM doxorubicin (n = 30 cells per line). Data are presented as mean ± SD, **p < 0.01, ****p < 0.0001. C Flow cytometry of cell cycle distribution (sub G1, G1, S, G2/M and > 4 N) over time (0, 24, 48, and 72 h) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin, with quantification of cells with > 4 N DNA content. Histograms (left) and quantitation (right) are shown. D, E Western blot analysis of p53, p21, and cleaved caspase-3 expression in WT, p53−/−, and p21−/−cells treated with 100 nM (D) or 2 µM (E) doxorubicin. β-actin was used as a loading control.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Flow Cytometry, Quantitation Assay, Western Blot, Expressing, Control

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 3 p21 is essential for maintaining genomic stability and normal mitotic progression. A Western blot analysis of p21 in HCT116 WT and p53−/−cells transduced with non-targeting (NT) or p21 shRNAs. β-actin was used as a loading control. B, C Fluorescence microscopy of nuclear morphology in HCT116 WT (B) and p53−/−(C) cells transduced with NT or p21-targeting shRNAs, treated with vehicle or 100 nM doxorubicin for 48 h. Nuclei and actin were stained with DAPI (cyan) and phalloidin (red), respectively. Scale bar = 20 µm. Quantitation of multinucleated cells is shown on the right (n = 50 cells per group). Data are mean ± SD, ***p < 0.001, ****p < 0.0001. D, E Flow cytometry analysis of cell cycle phases (Sub G1, G1, S, G2/M and > 4 N) in WT (D) and p53−/−(E) cells transduced with NT or p21 shRNA, treated with vehicle or 100 nM doxorubicin for 48 h, including quantification of cells with > 4 N DNA content. F Western blot showing p21 levels in HCT116 p21−/−cells transfected with empty vector (EV) or p21, with β-actin as a loading control. G Fluorescence microscopy of nuclear morphology (cyan) and actin (red) in HCT116 p21−/−cells transfected with EV or p21 after treatment with vehicle or 100 nM doxorubicin for 48 h. Scale bar = 10 μm. Quantitation of multinucleated cells (%) following 24 and 48 h of treatment is shown on the right (n = 50 cells per group). Data are mean ± SD, **p < 0.01.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Western Blot, Transduction, Control, Fluorescence, Microscopy, Staining, Quantitation Assay, Flow Cytometry, shRNA, Transfection, Plasmid Preparation

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 2 Role of p21 in high-dose doxorubicin-induced apoptosis. A Flow cytometry of cell cycle in HCT116 cells transduced with NT or p21 shRNA, treated with 2 µM doxorubicin for 48 h, showing subG1 cell population percentages. B Western blot analysis of Noxa and Mcl-1 expression in WT, p53−/−, and p21−/−cells treated with 2 µM doxorubicin. β-actin was used as a loading control.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Flow Cytometry, Transduction, shRNA, Western Blot, Expressing, Control

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 4 Mitotic defects in p21-deficient cells following low-dose doxorubicin treatment. A Live-cell imaging of HCT116 p21−/−cells expressing GFP-H2B, treated with 100 nM doxorubicin for 48 h. Images were captured every 15 min over 9 h. Scale bar = 5 μm. B, C Immunofluorescence showing the localization of Aurora B (B) and MKLP1 (C) in WT, p53−/−, and p21−/−cells synchronized to early anaphase by a thymidine-RO-3306 block. Hoechst 33342 (blue), α-tubulin (red), and Aurora B or MKLP1 (green) are shown. Quantification of cells with weak Aurora B (B) and MKLP1 (C) signals in early anaphase (n = 25 cells per group, repeated 4 times). Data are mean ± SD, ****p < 0.0001. D, E Immunofluorescence showing Aurora B (D) and MKLP1 (E) localization in WT and p53−/−cells transduced with NT or p21 shRNA.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Live Cell Imaging, Expressing, Blocking Assay, Transduction, shRNA

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 5 Impaired DNA damage repair in p21-deficient cells after low-dose doxorubicin treatment. A Western blot analysis of phosphorylated ATM (p-ATM), p-Chk1, Chk1, p-Chk2, and Chk2 in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for various time points. β-actin was used as a loading control. B Fluorescence microscopy of Lamin B1 (red) and γ-H2AX foci (green) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 48 h. Scale Bar = 20 μm. C Fluorescence microscopy showing nuclei (blue) and γ-H2AX foci (green) in HCT116 WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for 24 h and then released into fresh media for various time points. Scale bar = 20 μm. Quantification of nuclei with >10 γ-H2AX foci (n = 20 cells per group, repeated 4 times). Data are mean ± SD ****p < 0.0001.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Western Blot, Control, Fluorescence, Microscopy

Journal: Cell death discovery

Article Title: Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells.

doi: 10.1038/s41420-025-02416-w

Figure Lengend Snippet: Fig. 6 Activation of error-prone DNA repair pathways in p21-deficient cells treated with low-dose doxorubicin. A, C Immunofluorescence showing DNA-PKcs phosphorylation (A) or Mre11 (C) (red) in WT, p53−/−, and p21−/−cells treated with vehicle or 100 nM doxorubicin for 48 h. Nuclei were stained with Hoechst 33342 (cyan). Quantification of DNA-PKCs pS2056 (A) or Mre11 (C) signal intensity is shown (n = 16 cells per group, repeated 3 times). Scale bar = 10 μm. Data are mean ± SD **p < 0.01, ***p < 0.001, ****p < 0.0001. B, D Western blot of DNA- PKcs pS2056, DNA-PKcs (B) or Mre11 (D) in WT, p53−/−, and p21−/−cells treated with 100 nM doxorubicin for the indicated time points. β-actin was used as a loading control. E Fluorescence microscopy showing nuclear morphology (cyan) in WT, p53−/−, and p21−/−cells treated with vehicle, Mirin, or doxorubicin + Mirin for 48 h. Scale bar = 10 μm. F Quantification of multinucleated cells (%) following treatment as described in panel (E). data are mean ± SD, ****p < 0.0001 vs. vehicle, ####p < 0.0001 vs. Doxorubicin.

Article Snippet: The pcDNA3-HA-p21 plasmid (Addgene plasmid #78782; RRID) [24] was used for transfection.

Techniques: Activation Assay, Phospho-proteomics, Staining, Western Blot, Control, Fluorescence, Microscopy