Journal: bioRxiv

Article Title: The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination

doi: 10.1101/2025.09.30.678750

Figure Lengend Snippet: A) Schematic of active P-TEFb release from 7SK-snRNP to promote RNA polymerase II (RNAPII) pause release in response to stress and growth stimuli. B) Protein levels of HEXIM1, LARP7 and Tubulin (loading control) in U2OS cells 48 h after siRNA transfection. nonT = non-targeting control siRNA. C) Approach for siRNA depletion or small molecule inhibitor treatment prior to DNA fibre labelling in presence of camptothecin (CPT, 10 μM) or hydroxyurea (HU, 200 μM). D) Replication fork speeds (IdU label) after CPT or HU treatment in presence of HEXIM1 (+) or nonT (-) siRNA. Data from 2 repeats. E) Replication fork speeds (IdU label) after CPT or HU treatment in presence of LARP7 (+) or nonT (-) siRNA. Data from 2-3 repeats. F) DRB inhibits P-TEFb and p38 MAPK inhibitor (P38i) inhibits 7SK-snRNP/P-TEFb dissociation. G) Replication fork speeds (IdU label) after CPT or HU treatment +/- p38i. Data from 3 repeats. H) Replication fork speeds (IdU label) after CPT or HU treatment +/- 100 μM DRB. Data from 2 repeats. I) Nascent chromatin capture proteomics in HeLa S3 cells shows limited enrichment or depletion of HEXIM1, LARP7 and MEPCE at ongoing (nascent/mature chromatin), stalled (3 mM HU, 30 min/untreated) or collapsed (1 μM CPT, 40 min + roscovinite/roscovitine) replication forks compared to PCNA or RAD51. Roscovitine was included to prevent new origin firing. Scatter graphs show aggregates and medians (black points) of independent repeats with overall median (line). Kruskal-Wallis with multiple comparisons test, **** p ≤ 0.0001; ns: not significant.

Article Snippet: The membrane was then incubated overnight at 4 °C in mouse anti-DNA-RNA hybrid antibody (S9.6 hybridoma growth medium, ATCC HB-8730, 1:1,000) or mouse anti-dsDNA antibody (Abcam ab27156, 1:100,000) in sterile 5 % BSA/TBST, before being washed in TBST and incubated in goat anti-mouse HRP (Cell Signaling 7074, 1:5,000) 5 % milk/TBST for 1 hour at room temperature.

Techniques: Control, Transfection

Journal: bioRxiv

Article Title: The 7SK small nuclear ribonucleoprotein links the cell responses to transcription and replication stress by promoting replication fork reversal and homologous recombination

doi: 10.1101/2025.09.30.678750

Figure Lengend Snippet: A) Protein levels of CDK9 in soluble extract (SE) and nuclear pellet (NP) fractions after 2 h treatment with 10 μM CPT or 200 μM HU +/- HEXIM1 siRNA (48 h). B) Protein levels of CDK9 in SE and nuclear NP fractions +/- LARP7 siRNA as in A. C) Relative levels of SE (7SK-snRNP bound) versus NP (chromatin-bound) P-TEFb after treatment +/- HEXIM1 siRNA as in A. n=3. D) Relative levels of SE versus NP P-TEFb after treatment +/- LARP7 siRNA as in B. n=3. E) Representative images of nascent RNA labelling with 5-ethynyluridine (EU). DNA was detected using DAPI. Bars: 50 μm. F) Nuclear EU intensities after treatment with 10 μM CPT and EU for 20 or 50 min. n=1. G) Nuclear EU intensities after treatment with 10 μM CPT or 200 μM HU (2 h) +/- HEXIM1 siRNA. n=3. H) Slot blots of genomic DNA stained with S9.6 antibody (RNA:DNA hybrids) and double-stranded DNA (dsDNA; loading control) after treatment with 10 μM CPT or 200 μM HU (20 min) +/- HEXIM1 siRNA. RNase H treatment was used as control. I) RNA:DNA hybrid quantification as in E. n=4. J) Replication fork speeds (IdU label) after CPT or HU treatment overexpression of turboGFP-RNase H1 (+) or eGFP vector control (-). Data from 2 repeats. K) Approach for siRNA depletion and 10 μM CPT or 200 μM HU treatment prior to immunostaining. L) Percentages of cells with >10 γH2AX foci after 2 h CPT or HU +/- HEXIM1 siRNA. n=2-3. M) Percentages of cells with >9 53BP1 foci after 2 h CPT or HU +/- HEXIM1 siRNA. n=3. Scatter blots show aggregates and medians (black points) of independent repeats with overall median (line). ANOVA or Kruskal-Wallis with multiple comparisons test, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: not significant.

Article Snippet: The membrane was then incubated overnight at 4 °C in mouse anti-DNA-RNA hybrid antibody (S9.6 hybridoma growth medium, ATCC HB-8730, 1:1,000) or mouse anti-dsDNA antibody (Abcam ab27156, 1:100,000) in sterile 5 % BSA/TBST, before being washed in TBST and incubated in goat anti-mouse HRP (Cell Signaling 7074, 1:5,000) 5 % milk/TBST for 1 hour at room temperature.

Techniques: Staining, Control, Over Expression, Plasmid Preparation, Immunostaining