Journal: bioRxiv

Article Title: Genome-wide DNA supercoiling arises from transcription and SMC activity and mediates transcriptional negative feedback

doi: 10.64898/2026.03.26.714539

Figure Lengend Snippet: a. Western blot showing AID depletions of condensin I subunit CAP-H and condensin II subunit CAP-H2, in HCT116 cells synchronized at prometaphase. b. Flow cytometry gating to sort cells at G1 phase and S phase. c. Flow cytometry gating to validate cell synchronization at prometaphase by nocodazole treatment, and at the G2/M checkpoint by RO-3306 treatment before drug release into early prophase. d. Pulsed-field gel electrophoresis (PFGE) of extracted genomic DNA from human cells with or without gamma irradiation treatment. Extracted gDNA were treated with nuclease S1 to convert single-stranded DNA breaks into double-stranded DNA breaks before PFGE analysis.

Article Snippet: LF-PVDF membranes were then blocked by 5% nonfat milk for 1 h and incubated overnight at 4 °C with primary antibody against CAP-H (Proteintech, 11515-1-AP, 1:1000 dilution) or CAP-H2 (Proteintech, 26172-1-AP, 1:1000 dilution).

Techniques: Western Blot, Flow Cytometry, Pulsed-Field Gel, Electrophoresis, Irradiation

Journal: bioRxiv

Article Title: Genome-wide DNA supercoiling arises from transcription and SMC activity and mediates transcriptional negative feedback

doi: 10.64898/2026.03.26.714539

Figure Lengend Snippet: a. Example regions of genome-wide supercoiling distribution in prometaphase cells under conditions of untreated, condensin I subunit CAP-H AID depletion, and condensin II subunit CAP-H2 AID depletion (left). Changes in genome-wide supercoiling under condensin AID depletion conditions. Dotted lines indicate general reduction in supercoiling levels. Bin size: 100 kb (right). b. Average supercoiling level across the genome in HCT116 cells and GM12878 cells. ATMP density is determined by the ratio between pull-down ATMP-adducted DNA and input DNA, and compared between living cells with DNA breaks (IR: gamma Irradiation) and with intact genome (No IR). Significance was calculated by two-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). c. Average supercoiling level across the genome in HCT116 cells at different cell-cycle stages. ATMP density is determined by the ratio between pull-down ATMP-adducted DNA and input DNA, and compared between living cells with DNA breaks (IR: gamma Irradiation) and with intact genomic DNA (No IR). Significance was calculated by two-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). d. Average supercoiling level across the genome in HCT116 cells at prometaphase, under conditions of untreated, condensin I AID depletion, and condensin II AID depletion. Significance was calculated by one-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). e. Average supercoiling level across the genome in HCT116 cells at prometaphase, with and without gamma irradiation, under conditions of untreated, condensin I AID depletion, and condensin II AID depletion. Significance was calculated by two-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). f. Average supercoiling level across the genome in asynchronous HCT116 cells, with and without gamma irradiation, under conditions of untreated, condensin I AID depletion, and condensin II AID depletion. Significance was calculated by two-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: LF-PVDF membranes were then blocked by 5% nonfat milk for 1 h and incubated overnight at 4 °C with primary antibody against CAP-H (Proteintech, 11515-1-AP, 1:1000 dilution) or CAP-H2 (Proteintech, 26172-1-AP, 1:1000 dilution).

Techniques: Genome Wide, Irradiation

Journal: bioRxiv

Article Title: Genome-wide DNA supercoiling arises from transcription and SMC activity and mediates transcriptional negative feedback

doi: 10.64898/2026.03.26.714539

Figure Lengend Snippet: a. Western blot showing AID depletions of condensin I subunit CAP-H and condensin II subunit CAP-H2, in HCT116 cells synchronized at prometaphase. b. Flow cytometry gating to sort cells at G1 phase and S phase. c. Flow cytometry gating to validate cell synchronization at prometaphase by nocodazole treatment, and at the G2/M checkpoint by RO-3306 treatment before drug release into early prophase. d. Pulsed-field gel electrophoresis (PFGE) of extracted genomic DNA from human cells with or without gamma irradiation treatment. Extracted gDNA were treated with nuclease S1 to convert single-stranded DNA breaks into double-stranded DNA breaks before PFGE analysis.

Article Snippet: LF-PVDF membranes were then blocked by 5% nonfat milk for 1 h and incubated overnight at 4 °C with primary antibody against CAP-H (Proteintech, 11515-1-AP, 1:1000 dilution) or CAP-H2 (Proteintech, 26172-1-AP, 1:1000 dilution).

Techniques: Western Blot, Flow Cytometry, Pulsed-Field Gel, Electrophoresis, Irradiation

Journal: bioRxiv

Article Title: Genome-wide DNA supercoiling arises from transcription and SMC activity and mediates transcriptional negative feedback

doi: 10.64898/2026.03.26.714539

Figure Lengend Snippet: a. Example regions of genome-wide supercoiling distribution in prometaphase cells under conditions of untreated, condensin I subunit CAP-H AID depletion, and condensin II subunit CAP-H2 AID depletion (left). Changes in genome-wide supercoiling under condensin AID depletion conditions. Dotted lines indicate general reduction in supercoiling levels. Bin size: 100 kb (right). b. Average supercoiling level across the genome in HCT116 cells and GM12878 cells. ATMP density is determined by the ratio between pull-down ATMP-adducted DNA and input DNA, and compared between living cells with DNA breaks (IR: gamma Irradiation) and with intact genome (No IR). Significance was calculated by two-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). c. Average supercoiling level across the genome in HCT116 cells at different cell-cycle stages. ATMP density is determined by the ratio between pull-down ATMP-adducted DNA and input DNA, and compared between living cells with DNA breaks (IR: gamma Irradiation) and with intact genomic DNA (No IR). Significance was calculated by two-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). d. Average supercoiling level across the genome in HCT116 cells at prometaphase, under conditions of untreated, condensin I AID depletion, and condensin II AID depletion. Significance was calculated by one-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). e. Average supercoiling level across the genome in HCT116 cells at prometaphase, with and without gamma irradiation, under conditions of untreated, condensin I AID depletion, and condensin II AID depletion. Significance was calculated by two-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). f. Average supercoiling level across the genome in asynchronous HCT116 cells, with and without gamma irradiation, under conditions of untreated, condensin I AID depletion, and condensin II AID depletion. Significance was calculated by two-way ANOVA analysis based on two replicates per condition (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Article Snippet: LF-PVDF membranes were then blocked by 5% nonfat milk for 1 h and incubated overnight at 4 °C with primary antibody against CAP-H (Proteintech, 11515-1-AP, 1:1000 dilution) or CAP-H2 (Proteintech, 26172-1-AP, 1:1000 dilution).

Techniques: Genome Wide, Irradiation