Fig. 6: The interaction between the CC domain of ITSN1-S and the NT domain of NDH II directly suppressed DNA replication and nascent DNA synthesis by inhibiting R-loops resolution in breast cancer cells. | Cell Death & Disease

Fig. 6: The interaction between the CC domain of ITSN1-S and the NT domain of NDH II directly suppressed DNA replication and nascent DNA synthesis by inhibiting R-loops resolution in breast cancer cells.

From: Endocytic protein intersectin1-S shuttles into nucleus to suppress the DNA replication in breast cancer

Fig. 6

A Several exogenous different domain structure fragments of NDH II or ITSN1-S were transfected into HEK-293T cells, respectively. Expression of exogenous fragments was monitored by anti-flag antibody in Western blot analysis. B Cellular extracts of the above cells were applied, and IP was performed by an anti-flag M2 affinity gel. Expression of ITSN1-S or NDH II was determined by Western blot analysis. C 3×flag-vector, 3×flag-ITSN1-S, and 3×flag-ITSN1-S-△CC were transfected into shITSN1-S#2/MDA-MB-231 cells and tested with anti-flag and anti-ITSN1-S antibodies by Western blot. β-actin was used as a loading control. D–F Proliferation ability was examined by ATP/viability assay (D), SRB assay (E), and EdU incorporation assays (F) in shITSN1-S-3×flag-ITSN1-S-WT/MDA-MB-231 and shITSN1-S-3×flag-ITSN1-S-△CC/MDA-MB-231 cells. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t-test, *P < 0.05). Scale bars, 25 μm. G Quantification by qPCR of nascent DNA abundance of indicated cells. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t-test, ***P < 0.001). H 3×flag-vector, 3×flag-ITSN1-S, and 3×flag-ITSN1-S-△CC were transfected into KOITSN1/MDA-MB-231 cells and tested with anti-flag and anti-ITSN1-S antibodies by Western blot. β-actin was used as a loading control. I Proliferation ability was examined by EdU incorporation assay in KOITSN1–3×flag-ITSN1-S-WT/MDA-MB-231 and KOITSN1–3×flag-ITSN1-S-△CC/MDA-MB-231 cells. Scale bars, 25 μm. Quantitative results were analyzed in the right panel. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t-test, *P < 0.05). J Quantification by qPCR of nascent DNA abundance in KOITSN1–3×flag-ITSN1-S-WT/MDA-MB-231 and KOITSN1–3×flag-ITSN1-S-△CC/MDA-MB-231 cells. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t-test, **P < 0.01). K R-loops were detected by immunofluorescence with the anti-RNA/DNA hybrid antibody S9.6 in KOITSN1–3×flag-ITSN1-S-WT/MDA-MB-231 and KOITSN1–3×flag-ITSN1-S-△CC/MDA-MB-231 cells. Pretreatment with RNase H for 12 h was used as a negative control. Scale bars, 25 μm. Quantitative results were analyzed in the right panel (two-tailed Student’s t-test, *P < 0.05).

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