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. 2024 May 2;121(19):e2318438121. doi: 10.1073/pnas.2318438121

Fig. 6.

Fig. 6.

C-circles and C-rich ssDNAs are generated through C-strand priming and subsequent strand displacement. (A) Illustration depicting C-strand synthesis priming by CST-PP (CTC1/STN1/TEN1-Primase-Polymerase alpha)-complex and the strand displacement of Okazaki fragments by PCNA and DNA polymerase delta (POLD) during DNA replication on the lagging strand. (B) C-circle assay for U2OS cells expressing shRNAs targeting STN1, POLD3, or nontargeting (NT) control. Cells were treated with Mirin (50 μM, 48 h) DNA amount: Left—100 ng, Right—30 ng. (C) Quantification of the C-circle assay in (B) as the relative amount of C-circle assay products (mean ± SD; unpaired t test). (D) 4SET assay for SaoS2 expressing shRNAs targeting STN1, or nontargeting (NT) control. Cells were treated with Mirin (50 μM, 48 h). Samples were fractionated into soluble fraction or whole DNA. G-rich probe was used to detect C-rich telomeric sequences, and C-rich probe was used to detect G-rich telomeric sequences. C-rich single-stranded DNAs are indicated by an arrow. DNA amount (Saos2 whole: 200 ng, soluble: 60 ng). (E) Quantification of the 4SET assay in (D); as the relative amount of C-rich ssDNA (whole). (mean ± SD; unpaired t test). (F) 4SET assay for U2OS cells expressing shRNAs targeting POLD3, or nontargeting (NT) control. Samples were fractionated into soluble fraction or whole DNA. G-rich probe was used to detect C-rich telomeric sequences, and C-rich probe was used to detect G-rich telomeric sequences. C-rich single-stranded DNAs are indicated by an arrow. DNA amount (whole: 300 ng, soluble: 100 ng). (G) 4SET assay for U2OS cells expressing shRNAs targeting STN1, POLD3, or nontargeting (NT) control after transfection of siRNAs targeting DNA2 or control (siLuc). Samples were fractionated into soluble fraction or whole DNA. G-rich probe was used to detect C-rich telomeric sequences, and C-rich probe was used to detect G-rich telomeric sequences. C-rich single-stranded DNAs are indicated by an arrow. DNA amount (whole: 210 ng, soluble: 70 ng).