Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2021 Nov 25;49(21):12284–12305. doi: 10.1093/nar/gkab1120

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

PMC Copyright notice

Figure 5. — Increased DNA damage and impaired DDR in Hnrnpr^tm1a/tm1a motoneurons. (A) Western blot analysis of subcellular fractions from control motoneurons (0 Gy) or motoneurons subjected to γ-irradiation (9 Gy). Motoneurons were fractioned into cytosolic (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions. Fractions were probed with the indicated antibodies. (B) Western blot analysis of chromatin fractions of NSC-34 cells incubated with Benzonase (Bn) or the indicated amounts of RNase A and separated into supernatant and pellet by centrifugation. (C) Immunoprecipitation of γ-H2AX from nuclear fractions of control (0 Gy) or irradiated (9 Gy) NSC-34 cells. Proteins were analyzed by western blot using antibodies against γ-H2AX or hnRNP R. In, input; IP, immunoprecipitation. (D) Representative images of alkaline comet assays performed on Hnrnpr⁺^/+ and Hnrnpr^tm1a/tm1a motoneurons cultured for 6 DIV under control conditions (0 Gy) and after exposure to γ-irradiation (9 Gy). (E) Quantification of comet mean tail moments. Data are mean ± SD (n = 3 independent experiments; N = 71 nuclei for Hnrnpr⁺^/+ (0 Gy), N = 55 nuclei for Hnrnpr⁺^/+ (9 Gy), N = 82 nuclei for Hnrnpr^tm1a/tm1a (0 Gy) and N = 76 nuclei for Hnrnpr^tm1a/tm1a (9 Gy)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P ≤ 0.01. (F) Representative images of γ-H2AX immunofluorescence staining of Hnrnpr⁺^/+ and Hnrnpr^tm1a/tm1a motoneurons under non-irradiated conditions (0 Gy) or after exposure to γ-irradiation (9 Gy) followed by the indicated recovery time. Scale bar: 5 μm. (G) Quantification of nuclear γ-H2AX immunostaining shown in (F). Data are mean ± SD (n = 4 independent experiments; N = 38 nuclei for Hnrnpr⁺^/+ (0 Gy), N = 40 nuclei for Hnrnpr⁺^/+ (9 Gy, 3 min recovery), N = 40 nuclei for Hnrnpr⁺^/+ (9 Gy, 10 min recovery), N = 41 nuclei for Hnrnpr⁺^/+ (9 Gy, 30 min recovery), N = 40 nuclei for Hnrnpr⁺^/+ (9 Gy, 24 h recovery), N = 42 nuclei for Hnrnpr^tm1a/tm1a (0 Gy), N = 39 nuclei for Hnrnpr^tm1a/tm1a (9 Gy, 3 min recovery), N = 41 nuclei for Hnrnpr^tm1a/tm1a (9 Gy, 10 min recovery), N = 40 nuclei for Hnrnpr^tm1a/tm1a (9 Gy, 30 min recovery) and N = 39 nuclei for Hnrnpr^tm1a/tm1a (9 Gy, 24 h recovery)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P ≤ 0.01, ***P ≤ 0.001. (H) Western blot analysis of hnRNP R levels in cultured motoneurons transduced with control or shRNA against both hnRNP R isoforms (shRNA-FL+ΔN). Gapdh was used as loading control. (I) Representative images of alkaline comet assay performed on motoneurons transduced with control and shRNA-FL+ΔN under non-irradiated conditions (0 Gy) or after exposure to γ-irradiation (9 Gy). (J) Quantification of comet mean tail moments. Data are mean ± SD (n = 3 independent experiments; N = 32 nuclei for Control (0 Gy), N = 29 nuclei for Control (9 Gy), N = 37 nuclei for shRNA-FL+ΔN (0 Gy) and N = 30 nuclei for shRNA-FL+ΔN (0 Gy)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; **P ≤ 0.01. (K) Representative images of γ-H2AX immunofluorescence staining of control and shRNA-FL+ΔN-transduced motoneurons under non-irradiated conditions (0 Gy) or after exposure to γ-irradiation (9 Gy) followed by the indicated recovery time. EGFP was used as a marker to identify transduced cells. Scale bar: 5 μm. (L) Quantification of nuclear γ-H2AX immunostaining in (K). Data are mean ± SD (n = 3 independent experiments; N = 34 nuclei for control (0 Gy), N = 32 nuclei for control (9 Gy, 10 min recovery), N = 22 nuclei for control (9 Gy, 24 h recovery), N = 21 nuclei for shRNA-FL+ΔN (0 Gy), N = 33 nuclei for shRNA-FL+ΔN (9 Gy, 10 min recovery), N = 36 nuclei for shRNA-FL+ΔN (9 Gy, 24 h recovery)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; ***P ≤ 0.001. (M) Representative images of γ-H2AX immunofluorescence staining of motoneurons transduced with control, shRNA-FL+ΔN, shRNA-FL+ΔN; ΔN(OE) or shRNA-FL+ΔN; FL(OE) constructs under non-irradiated conditions (0 Gy) or after exposure to γ-irradiation (9 Gy) followed by 10 min recovery. EGFP was used as a marker to identify cells transduced with the control or shRNA-FL+ΔN construct. The rescue constructs shRNA-FL+ΔN; ΔN(OE) and shRNA-FL+ΔN; FL(OE) expressed EGFP-tagged hnRNP R-ΔN or hnRNP R-FL, respectively, in addition to an shRNA targeting both endogenous hnRNP R isoforms. Scale bar: 5 μm. (N) Quantification of nuclear γ-H2AX immunostaining in (M). Data are mean ± SD (n = 3 independent experiments; N = 25 nuclei for control (0 Gy), N = 25 nuclei for control (9 Gy), N = 25 nuclei for shRNA-FL+ΔN (0 Gy), N = 25 nuclei for shRNA-FL+ΔN (9 Gy), N = 25 nuclei for shRNA-FL+ΔN; ΔN(OE) (0 Gy), N = 25 nuclei for shRNA-FL+ΔN; ΔN(OE) (9 Gy), N = 25 nuclei for shRNA-FL+ΔN; FL(OE) (0 Gy) and N = 25 nuclei for shRNA-FL+ΔN; FL(OE) (9 Gy)). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; ***P ≤ 0.001.