Fig 2: STN1 depletion does not affect cohesin levels or mitotic timing.A, Western blot analysis of total SMC and acetylated SMC3 (Ac-SMC3) in HeLa cells, as indicated. B, representative images of SMC3 levels in pre-extracted cells. DAPI is shown in blue and SMC3 in red. The scale bar represents 20 µm. C, Western blot of chromatin-bound cohesin subunits. Histone H3 was used as the loading control. D, HeLa cells stably expressing H2B-RFP were imaged in 5-min intervals for 3 h. The scale bar represents 20 µm. E, dot plot of the time between nuclear envelope breakdown until cytokinesis in individual cells. Black lines and numbers below the graph indicate the average time in minutes to complete mitosis. (*p < 0.05 and **p < 0.01).

Fig 3: CST associates with the cohesin complex.A, co-IP of nuclease-treated lysates from HEK293T cells expressing Flag-tagged CTC1, Flag-tagged STN1, or the full CST complex. The asterisk indicates background band. B, representative images of proximity ligation assay (PLA) performed in HeLa cells with antibodies to STN1 or SMC3 alone or in combination. Red represents PLA foci; blue represents DAPI. The scale bar represents 5 µm. C, violin plot of PLA foci per nucleus. Results are representative of four independent, biological experiments. Bold dashed line represents the median, and dashed lines represent the first and third quartiles. (****p < 0.0001). co-IP, coimmunoprecipitation; CST, CTC1-STN1-TEN1.

Fig 4: CST deficiency results in SCC loss.A, Western blot of STN1 knockdown and MAD2 levels in HeLa cells. Actinin was used as the loading control. B, example images of metaphase spreads with normal chromosomes or SCC loss. The scale bar represents 5 µm. C, fold increase in cohesion loss after metaphase spread analysis. n = 3 independent, biological replicates. D, representative images of chromosome FISH from cells isolated by mitotic shake-off. Red represents centromere 6 probe; blue represents DAPI. The scale bar represents 5 µm. E, fold increase in nuclei with >4 chromosome 6 foci. n = 4 independent, biological replicates. F, knockdown of CTC1, STN1, or TEN1 by siRNA in HeLa cells. siNT was used as the nontarget control and actinin as the loading control. G, graph of SCC loss after metaphase spread analysis, as indicated. n = 3 independent, biological replicates. (*p < 0.05, **p < 0.01, and ***p < 0.001). CST, CTC1-STN1-TEN1; SCC, sister chromatid cohesion; shNT, nontargeting shRNA; shSTN1, shRNA knockdown of STN1; shSTN1+Flag-STN1, shSTN1-7 cells plus shRNA-resistant Flag-STN1.

Fig 5: Purified CST–Pola-primase and stoichiometry of the subunits.a, Silver-stained SDS-PAGE of one of the HEK cell CST–Pola-primase preparations used in this study (HEK CST-PP), recombinant human CST overexpressed in insect cells (Insect CST), and recombinant human Pola-primase overexpressed in insect cells (Insect PP). M, marker proteins. The HEK cell and insect cell STN1 proteins have slightly different mobilities due to a Myc tag on the former. b, Western blot with antibodies to STN1 and to epitope tags on the HEK cell CTC1 and TEN1 subunits (thus no signal for CTC1 or TEN1 in the Insect CST). The HEK cell-expressed CTC1 consistently runs as a doublet. Amount of protein loaded (pmoles) is based on direct determination of protein concentration for Insect CST but is calculated from the STN1 western blots for HEK CST-PP. HEK FLAG/HA CST-PP indicates double immunopurification via FLAG and HA tags. c and d, Quantification of STN1 from the western blot gives (c) the phosphorimager counts of STN1 per µL for the HEK cell preparation and (d) the counts of STN1 per pmol for purified insect cell CST; the calculation below the graphs gives the concentration of STN1 in the HEK cell CST–Pola-primase preparation. e, Western blot with antibodies to the two POLA subunits. The endogenous POLA1 from HEK cells consistently runs as a doublet, likely due to processing between lys 123 and lys 124 producing a stable 165 kDa species39; in contrast, the POLA1 in the Pola-primase overexpressed in insect cells is mostly a single species with a minor smaller species. Amount of protein loaded (pmoles) is based on direct determination of protein concentration for Insect PP but is calculated from the POLA western blots for HEK CST-PP. f, g, Quantification of POLA1 and POLA2 from the western blot gives (f) the counts of POLA1 and POLA2 per µL of the HEK cell preparation and (g) the counts per pmol for the purified insect cell Pola-primase; the calculation below the graphs gives the concentration of POLA in the HEK cell CST–Pola-primase preparation. Dividing the concentration of POLA from f and g by the concentration of CST from c and d gives the stoichiometry of 0.21 POLA per CST in the HEK cell preparation. These results were consistent between two biological replicates of the entire set of experiments. For gel source data, see Supplementary Fig. 1 Source Data