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-Geminin (GMNN) , Proteintech , Cat# 10802-1-AP; RRID:AB_2110945.

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-Geminin (GMNN) , Proteintech , Cat# 10802-1-AP; RRID:AB_2110945.

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