ccna2  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Aberrant CDK4/6-driven cell-cycle reentry drives neuronal loss and defines a therapeutic target in C9orf72 ALS/FTD

doi: 10.1016/j.isci.2025.114596

Figure Lengend Snippet: Post-mitotic iPSC-derived motor neurons from C9orf72 carriers aberrantly enter cell-cycle (A) Representative images of control and C9orf72 motor neuron cultures. Scale bars, 100 μm. (B–C) mRNA levels of Ki67 and GMNN at 1-, 1.5-, and 2-month-old control and C9orf72 iPSC-derived motor neurons. (D) Representative western blot images of GMNN and actin and quantification of protein levels from 2-month-old control and C9orf72 iPSC-derived motor neurons. (E–F) Flow cytometry of propidium iodide-stained 2-month-old iPSC-derived motor neurons from control (lines: 35L5, 35L11, and 37L20) and C9orf72 (lines: 16L14, 40L3, and 42L11). (G) Percentage of neurons in S-phase from controls and C9orf72 neurons. (H and I) mRNA levels of CCNA2 and CCNB2 at 1-, 1.5-, and 2-month-old control and C9orf72 iPSC-derived motor neurons. (J and K) mRNA levels of CDK2 and CDK4 at 1-, 1.5-, and 2-month-old control and C9orf72 iPSC-derived motor neurons. (L) Representative western blot images of CCNA2 and GAPDH and quantification of protein levels from 2-month-old control and C9orf72 iPSC-derived motor neurons. (M) Representative western blot images of CDK4 and actin and quantification of protein levels from 2-month-old control and C9orf72 iPSC-derived motor neurons. Data are presented as mean ± SEM (B–D, G–M). Data presented in (B–D) is from 3 control and 3 C9orf72 iPSC iPSC-derived neuron cultures from 3 independent differentiation experiments. Two-tailed t test with Welch’s correction was applied. ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Data presented in (G) is from 3 control and 3 C9orf72 iPSC-derived neuron cultures. Two-tailed t test with Welch’s correction was applied. ns, not significant, ∗ p < 0.05. Data presented in (H–K) 3 control and 3 C9orf72 iPSC-derived neuron cultures from 3 independent differentiation experiments. Two-tailed t test with Welch’s correction was applied. ns, not significant, ∗ p < 0.05 and ∗∗ p < 0.01. Data presented in (L and M) is from 3 control and 3 C9orf72 iPSC-derived neuron cultures from 3 independent differentiation experiments. Two-tailed t test with Welch’s correction was applied. ∗ p < 0.05. In cases where the loading control was located above the target protein on the original membrane, the band was repositioned below for consistency and clarity.

Article Snippet: Rabbit anti-Cyclin A2 (CCNA2) , Cell Signaling , Cat# 67955; RRID:AB_2909603.

Techniques: Derivative Assay, Control, Western Blot, Flow Cytometry, Staining, Two Tailed Test, Membrane

Journal: iScience

Article Title: Aberrant CDK4/6-driven cell-cycle reentry drives neuronal loss and defines a therapeutic target in C9orf72 ALS/FTD

doi: 10.1016/j.isci.2025.114596

Figure Lengend Snippet: Poly (GR) induces an increase in cyclins and CDKs levels (A) Schematic representation of the CRISPR/Cas9 strategy to generate C9orf72 homozygous and heterozygous lines from a healthy control line. (B) Generation of C9orf72 heterozygous and homozygous knockout iPSC lines by CRISPR Cas9. (C) Representative western blot image of C9orf72 protein levels in homozygous (line 3) and heterozygous (line 8) knockout lines by CRISPR Cas9. (D–G) mRNA levels of Ki67, GMNN, CDK4, and CCNA2 at 1-, 1.5-, and 2-month-old control and C9orf72 iPSC-derived motor neurons. (H) Representative immunostaining images of iPSC-derived motor neurons cultures treated with 1 and 2 μM of poly (GR). Scale bars, 200 μm. (I and J) Quantification of protein levels of CCND1 and CDK4 in 2-month-old control iPSC-derived motor neurons treated with 1 and 2 μM of poly (GR). (K) Representative immunostaining images of iPSC-derived motor neurons cultures treated with 1 and 2 μM of poly (GP). Scale bars, 200 μm. (L and M) Quantification of protein levels of CCND1 and CDK4 in 2-month-old control iPSC-derived motor neurons treated with 1 and 2 μM of poly (GR). (N) Representative western blot image of control iPSC-derived neurons treated with Poly (GR) and poly (GP). (O and P) Quantification of protein levels of CDK4 in 2-month-old control iPSC-derived motor neurons treated with 1 and 2 μM of poly (GR) and poly (GP). Data are presented as mean ± SEM (D–G, I and J, L and M, O and P). Data presented in (D–G) is from 3 independent differentiation experiments of a control iPSC line (parental line) and one C9orf72 heterozygous and one homozygous knockout line. Two-tailed t test with Welch’s correction was applied. ns, not significant. Data in (I and J, L and M) is from 3 control iPSC lines treated with DPRs from 3 independent differentiation experiments. Two-tailed t test with Welch’s correction was applied. ns, not significant, ∗∗ p < 0.01. Data in (O and P) is from 3 control iPSC lines treated with DPRs from 2 independent differentiation experiments. Two-tailed t test with Welch’s correction was applied. ns, not significant, ∗∗ p < 0.01. In cases where the loading control was located above the target protein on the original membrane, the band was repositioned below for consistency and clarity.

Article Snippet: Rabbit anti-Cyclin A2 (CCNA2) , Cell Signaling , Cat# 67955; RRID:AB_2909603.

Techniques: CRISPR, Control, Knock-Out, Western Blot, Derivative Assay, Immunostaining, Two Tailed Test, Membrane

Journal: iScience

Article Title: Aberrant CDK4/6-driven cell-cycle reentry drives neuronal loss and defines a therapeutic target in C9orf72 ALS/FTD

doi: 10.1016/j.isci.2025.114596

Figure Lengend Snippet: CDK4/6 inhibitor Palbociclib (PD33002291) prevents cell-cycle reentry in iPSC-derived motor neuron from C9orf72 carriers (A and B) Western blot and quantification of protein levels of phosphorylated RB and topoisomerase II α (TopoII) in 2-month-old iPSC-derived motor neurons from C9orf72 carriers treated with Palbociclib 1 and 5 μM. (C–E) mRNA levels of Ki67 and CCNA2 and CDK1 in 2-month-old C9orf72 iPSC-derived motor neurons treated with Palbociclib 5 μM. (F) Flow cytometry of propidium iodide-stained 2-month-old iPSC-derived motor neurons from controls, C9orf72 and C9orf72 neurons treated with Palbociclib 5 μM. (G) Quantification of the percentage of C9orf72 iPSC-derived motor neurons in S-phase. Data are presented as mean ± SEM (A–E, G). Data presented in (A and B) is from iPSC-derived neurons from 3 C9orf72 and 3 C9orf72 treated with Palbociclib 1 and 5 μM from 2 differentiation experiments, one-way ANOVA with Newman-Keuls post hoc test was applied ∗ p < 0.05.∗∗ p < 0.01 and ∗∗∗ p < 0.001. Data presented in (C–E) is from iPSC-derived neurons from 3 C9orf72 and 3 C9orf72 neurons with Palbociclib 5 μM from 2 differentiation experiments. Two-tailed t test with Welch’s correction was applied ∗ p < 0.05 and ∗∗ p < 0.01. Data in (G) is from iPSC-derived neurons from 3 controls, 3 C9orf72 , and 3 C9orf72 treated with Palbociclib from 1 differentiation experiment, one-way ANOVA with Newman-Keuls post hoc test was applied ∗∗ p < 0.01. See also .

Article Snippet: Rabbit anti-Cyclin A2 (CCNA2) , Cell Signaling , Cat# 67955; RRID:AB_2909603.

Techniques: Derivative Assay, Western Blot, Flow Cytometry, Staining, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Super enhancer-driven LINC01013 mediates hypoxia-induced mitochondrial dysfunction by HSPA9 to determine pulmonary arterial smooth muscle cell fate

doi: 10.1007/s00018-025-06071-3

Figure Lengend Snippet: Silencing of LINC01013 inhibited hypoxia-induced hPASMC proliferation and inflammation. A , B hPASMC proliferation was determined by CCK8 and EdU incorporation assays ( n = 6). EdU (red), DAPI (blue), scale bar, 50 μm. C Western blotting analysis of PCNA, cyclin A and cyclin D in hPASMCs ( n = 6). D RT‒qPCR analysis showed the mRNA levels of TNF-α and IL-6 after LINC01013 knockdown, β-actin was used as a internal reference gene ( n = 6). E Western blotting analysis of TNF-α and IL-6 in hPASMCs. ( n = 6). All values are presented as the mean ± SEM. Statistical analysis was performed with one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. Nor, normoxia; Hyp, hypoxia; NC, negative control; si, siRNA

Article Snippet: The antibody against CEBPB (PB9171, BA0670, 1:500, Boster, Wuhan, China), H3K27ac (A7253, 1:500, ABclonal, Wuhan, China), H3K4me1 (A2355, 1:500, Wuhan, China), PCNA (A00125, 1:500, Boster, Wuhan, China), Cyclin A (PB0515, 1:500, Boster, Wuhan, China), Cyclin D (BM4272, 1:500, Boster, Wuhan, China), IL-6 (AF7236, 1:500, Beyotime, Shanghai, China), TNF-α (AF8208, 1:500, Beyotime, Shanghai, China), PKM2 (4053, 1:1000, Cell Signaling, MA, US), HK II (66974-1-Ig, 1:1000, Proteintech, IL, USA), PDH (2784, 1:1000, Cell Signaling, MA, US), HSPA9 (14887-1-AP, 1:5000, Proteintech, IL, USA), VDAC1 (10866-1-AP, 1:5000, Proteintech, IL, USA), and β-actin (TA-09, 1:1000, ZSGB‐BIO, Beijing, China) was incubated at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, and proteins were visualized with enhanced chemiluminescence reagents.

Techniques: Western Blot, Knockdown, Negative Control

Journal: Cancer Biology & Therapy

Article Title: Interaction between ZMIZ2 and AR promotes prostate cancer proliferation in vitro and in vivo

doi: 10.1080/15384047.2025.2604936

Figure Lengend Snippet: To uncover the molecular regulatory network underlying the functions of ZMIZ2 and AR, RNA-seq analysis was meticulously performed to identify the common downstream target genes of these two factors. (a) RNA-seq analysis of differentially expressed genes after ZMIZ2 silencing or AR silencing. (b) Venn diagram analysis of genes commonly upregulated by ZMIZ2 and AR. (c) KEGG analysis of genes commonly upregulated by ZMIZ2 and AR. (d) GO analysis of genes commonly upregulated by ZMIZ2 and AR. (e) Heatmap of the expression levels of cell cycle-related genes after ZMIZ2 silencing or AR silencing. (f–g) A flow cytometry assay was employed to determine the cell cycle distribution. (h) QPCR was utilized to assess the mRNA transcriptional levels of cell cycle-related genes. (i) Western blot analysis was carried out to detect the protein expression levels of CDK1, CCNA2, and CCNE2 in each sample group. Significant differences are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001; ns indicates not significant; n = 3.

Article Snippet: The ZMIZ2 antibody (Novus Biologicals, LLC, Centennial, CO, USA), AR antibody (Santa Cruz Biotechnology, Dallas, TX, USA), CDK1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), CCNA2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), CCNE2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) were added and incubated overnight at 4 °C and then incubated with HRP-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 30 min.

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Western Blot

Journal: Cancer Biology & Therapy

Article Title: Interaction between ZMIZ2 and AR promotes prostate cancer proliferation in vitro and in vivo

doi: 10.1080/15384047.2025.2604936

Figure Lengend Snippet: Depletion of ZMIZ2 expression is associated with reduced AR enrichment on the promoters of downstream target genes, accompanied by a concurrent decrease in H3K27ac levels. (a) Flow chart of the ChIP experiment. (b) The binding sites of AR on the promoters of CDK1, CCNA2, and CCNE2. (c–h) ChIP analysis of AR enrichment on the CDK1, CCNA2, and CCNE2 promoters and H3K27ac levels. Significant differences are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001; ns indicates not significant; n = 3.

Article Snippet: The ZMIZ2 antibody (Novus Biologicals, LLC, Centennial, CO, USA), AR antibody (Santa Cruz Biotechnology, Dallas, TX, USA), CDK1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), CCNA2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), CCNE2 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) were added and incubated overnight at 4 °C and then incubated with HRP-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 30 min.

Techniques: Expressing, Binding Assay