sk es 1 cells  (ATCC)

Journal: bioRxiv

Article Title: Condensin and topoisomerases cooperate to relieve topological stress at stalled replication forks

doi: 10.1101/2025.08.06.668895

Figure Lengend Snippet: a Condensin binds nascent DNA. HeLa-S3 cells were labelled for 20 minutes with 10 μM EdU and chased for 60 minutes with thymidine. Proteins associated to nascent DNA before and after the thymidine chase were analyzed by iPOND-MS as described . b Condensin I and II are dispensable for normal fork progression. HeLa-S3 cells depleted for condensin I (siCAPG) or condensin II (siCAPG2) for 48 hours with siRNAs were sequentially labeled with IdU and CldU for 15 min before DNA fiber spreading. The length distribution of CldU tracks length is shown for four independent experiments. Box and whiskers indicate 25 th -75 th and 10 th -90 th percentiles, respectively. Median length is indicated. c The ATR-CHK1 pathway is functional in the absence of condensin II. HeLa-S3 cells were transfected with siCtrl, siCAPG and siCAPG2 for 48 hours. Cells were then treated with 4 mM HU for 2 hours and the activation of CHK1 was detected with an anti-pCHK1 (S345) antibody. CAPG and CAPG2 depletion was verified by Western blotting. Total CHK1 and tubulin were used as loading controls. d Condensin II promotes fork restart. HeLa-S3 cells were transfected with siCtrl or siCAPG2 for 48 hours and were treated with 4 mM HU for 3 hours after a 30 minutes IdU pulse. IdU and CldU tracks were analyzed by DNA fiber spreading 30 minutes after HU removal and CldU addition. Red and green signals are indicative of fork restart. Red only tracks correspond to stalled forks and green tracks to new origin firing (n = 3). e Condensin II is required for fork slowing after exposure to a low dose of HU. U2OS cells were transfected with siCtrl and siCAPG2 for 48 hours. Cells were first labelled for 30 minutes with IdU, and CldU was then added for 30 minutes in the presence of 50 μM HU. The length of IdU and CldU tracks was determined by DNA fiber spreading and was plotted as the ratio of CldU to IdU (n=3). Box and whiskers indicate median, 25th–75th and 10th–90th percentiles. f Condensin II promotes the resection of nascent DNA at HU-arrested forks. HeLa-S3 cells were transfected with siCtrl and siCAPG2 for 48 hours and were sequentially labeled for 15 minutes with IdU and CldU. Cells were either collected immediately or treated for 2 hours with 4 mM HU before DNA fiber analysis. The ratio of CldU to IdU track length was plotted for 4 independent experiments. Box and whiskers indicate median, 25th–75th and 10th–90th percentiles.

Article Snippet: Human cervical adenocarcinoma HeLa S3 cells (ATCC, CCL2-2) and osteocarcinoma U2OS cells (ATCC, HTB-86) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or McCoy’s 5a medium, respectively, supplemented with 10% fetal calf serum (FCS) and 100 U/mL penicillin/streptomycin (PS).

Techniques: Labeling, Functional Assay, Transfection, Activation Assay, Western Blot

Journal: bioRxiv

Article Title: Condensin and topoisomerases cooperate to relieve topological stress at stalled replication forks

doi: 10.1101/2025.08.06.668895

Figure Lengend Snippet: a Analysis of the frequency of reversed forks in control and condensin II-depleted cells by electron microscopy (EM). Control (shCtrl) or CAPG2-depleted (shCAPG2) HeLa-S3 cells were treated for 72 hours with 10 μM/ml doxycycline and then for 2 hours with 4 mM HU +/- 50 μM mirin before EM analysis. Electron micrographs of representative replication intermediates in shCAPG2 cells are shown. b The frequency of reversed forks in shCtrl and shCAPG2 cells is shown for the indicated conditions. Means and the number of analyzed forks are indicated (n=2). RI # indicates the number of analyzed replication intermediates. c Mechanism of fork reversal. See main text for details. d Reversed arms are shorter in the absence of condensin II. The length of reversed arms (in nt) was plotted for the indicated samples. Median values are indicated (n=2). RI # indicates the number of analyzed replication intermediates. P-values: Mann-Whitney rank sum test. e CAPG2 depletion restores fork restart in SMARCAL1-deficient cells. HeLa-S3 cells were transfected with siCtrl, siSMARCAL1, siCAPG2 or co-transfected with siSMARCAL1 and siCAPG2 for 48 hours. Cells were treated with 4 mM HU for 3 hours after a 30 minutes IdU pulse and fork restart was monitored 30 min after HU removal and CldU addition, as indicated in (n=3). f Defective fork slowdown in SMARCAL1-deficient cells is rescued by CAPG2 depletion. U2OS cells were transfected with siCtrl, siSMARCAL1, siCAPG2 or co-transfected with siSMARCAL1 and siCAPG2 for 48 hours. Cells were labeled for 30 minutes with IdU and 30 minutes with CldU in the presence of 50 μM HU and processed for DNA fiber spreading. The ratio of CldU to IdU track length is shown for 3 independent experiments. Box and whiskers correspond to 25th–75th and 10th–90th percentiles. Median ratio is indicated. g CAPG2 depletion restores fork resection in SMARCAL1-depleted cells. HeLa-S3 cells were transfected with siCtrl, siSMARCAL1, siCAPG2 or co-transfected with siSMARCAL1 and siCAPG2 for 48 hours. Cells were sequentially labeled for 15 minutes with IdU and CldU, and were either collected immediately or treated for 2 hours with 4 mM HU before DNA fiber analysis. The ratio of CldU to IdU track length was plotted for four independent experiments. Box and whiskers indicate median, 25th–75th and 10th–90th percentiles.

Article Snippet: Human cervical adenocarcinoma HeLa S3 cells (ATCC, CCL2-2) and osteocarcinoma U2OS cells (ATCC, HTB-86) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or McCoy’s 5a medium, respectively, supplemented with 10% fetal calf serum (FCS) and 100 U/mL penicillin/streptomycin (PS).

Techniques: Control, Electron Microscopy, MANN-WHITNEY, Transfection, Labeling

Journal: bioRxiv

Article Title: Condensin and topoisomerases cooperate to relieve topological stress at stalled replication forks

doi: 10.1101/2025.08.06.668895

Figure Lengend Snippet: a Condensin II acts with TOP2A to promote fork slowing. U2OS cells were transfected for 48 hours with siCtrl, siCAPG2, siTOP2A, or co-transfected with siTOP2A and siCAPG2. Cells were labeled for 30 minutes with IdU and 30 minutes with CldU in the presence of 50 μM HU and processed for DNA fiber spreading. The ratio of CldU to IdU track length is shown for three independent experiments. Box and whiskers correspond to median, 25th–75th and 10th–90th percentiles. b Condensin II acts with TOP2A to promote fork resection. HeLa-S3 cells were transfected with siCtrl, siCAPG2, siTOP2A, or co-transfected with siTOP2A and siCAPG2 for 48 hours. Cells were sequentially labeled for 15 minutes with IdU and CldU, and were either collected immediately or treated for 2 hours with 4 mM HU before DNA fiber analysis. The ratio of CldU to IdU track length was plotted for four independent experiments. Box and whiskers indicate median, 25th–75th and 10th–90th percentiles. c TOP1 depletion restores fork slowing in CAPG2-deficient cells. U2OS cells were transfected with siCtrl, siCAPG2, siTOP1, or co-transfected with siTOP1 and siCAPG2 for 48 hours. Cells were labeled and analyzed by DNA fiber spreading as indicated in panel a (n=4). d TOP1 depletion restores fork resection in CAPG2-deficient cells. HeLa-S3 cells were transfected with siCtrl, siCAPG2, siTOP1, or co-transfected with siTOP1 and siCAPG2 for 48 hours. Cells were labeled and analyzed by DNA fiber spreading as indicated in panel b (n=3). e TOP1 depletion restores fork resection in SMARCAL1-deficient cells. HeLa-S3 cells were transfected with siCtrl, siSMARCAL1, siTOP1, or co-transfected with siSMARCAL1 and siTOP1 for 48 h. Cells were labeled and analyzed by DNA fiber spreading as indicated in panel b (n=3). f Depletion of TOP2A does not restore fork resection in SMARCAL1-deficient cells. HeLa-S3 cells were transfected with siCtrl, siSMARCAL1, siTOP2A, or co-transfected with siSMARCAL1 and siTOP2A for 48 h. Cells were labeled and analyzed by DNA fiber spreading as indicated in panel b (n=3).

Article Snippet: Human cervical adenocarcinoma HeLa S3 cells (ATCC, CCL2-2) and osteocarcinoma U2OS cells (ATCC, HTB-86) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or McCoy’s 5a medium, respectively, supplemented with 10% fetal calf serum (FCS) and 100 U/mL penicillin/streptomycin (PS).

Techniques: Transfection, Labeling