Journal: The Journal of Biological Chemistry

Article Title: Systematic proteomics of endogenous human cohesin reveals an interaction with diverse splicing factors and RNA-binding proteins required for mitotic progression

doi: 10.1074/jbc.RA119.007832

Figure Lengend Snippet: Interaction of cohesin with splicing factors requires RAD21. A, nuclear extracts were prepared from the following: (i) HCT116 parental cells; (ii) HCT116 SMC1A-tagged cells; and (iii) HCT116 SMC1A-tagged, PDS5A KO cells. Dual-affinity purification was performed and separated by SDS-PAGE, and Western blotting was performed with the antibodies indicated. B, protein lysates were prepared from HCT116 cells and STAG2 mutant isogenic derivatives. Cohesin complexes were immunoprecipitated (IP) with SMC1A antibodies (or IgG control antibodies) and separated by SDS-PAGE, and Western blotting was performed with the antibodies indicated. C, HeLa cells were transfected with two independent STAG2 siRNAs (#1, s21089; #2, s21090) or scrambled siRNA and cultured for 72 h. Cells were then fixed, permeabilized, and double-stained with antibodies to SMC1A (cohesin) and HNRNPH (interacting splicing factor/RBP) for co-localization analysis. Cells were also stained with STAG2 antibodies to confirm depletion of STAG2. Microscopy and co-localization analysis were performed as in Fig. 3. DAPI, 4′,6-diamidino-2-phenylindole. Bar = 10 μm. D, HeLa cells were transfected with negative control siRNA (s4390843) or gene-specific siRNAs for SMC3 (s17426), STAG2 (s21090), RAD21 (s11726), STAG1 (s20074), sororin (s535461), NIPBL (s24588), WAPL (s22949), and PDS5B (s22912). Transfected cells were cultured for 3 days; protein lysates were prepared and separated by SDS-PAGE, and Western blotting was performed with the antibodies indicated to demonstrate depletion of the targeted proteins. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. E, HeLa cells were transfected with the siRNAs indicated and cultured for 3 days, and protein lysates were prepared. Cohesin complexes were then immunoprecipitated with SMC1A antibodies (or IgG control antibodies) and separated by SDS-PAGE, and Western blotting was performed with the antibodies indicated.

Article Snippet: Primary antibodies for immunoblotting were FLAG (M2) and tubulin (DM1α) from Sigma; sororin (ab192237) from Abcam; STAG2 (sc-81852), WAPL (sc-365189), HSPA8 (sc-7298), MAU2 (sc-243420), and NIPBL (sc-374625) from Santa Cruz Biotechnology; cyclin B1 (12231), cyclin E1 (20808), glyceraldehyde-3-phosphate dehydrogenase (2118), histone H3 (14269), and phospho-H3 (53348) from Cell Signaling Technologies; and ADAR1 (A303-884), DDX47 (A302-977), EFTUD2 (A300-957), HNRNPH (A300-511), PDCD11 (A303-804), PDS5A (A300-088), PDS5B (A300-538), PRPF31 (A303-919), RAD21 (A300-080), RBM10 (A301-006), RBM15 (A300-821), SF3B1 (A300-997), SF3B3 (A302-508), SMC1A (A300-055), SMC3 (A300-060), SNRNP200 (A303-453), and STAG1 (A302-579) from Bethyl Laboratories.

Techniques: Affinity Purification, SDS Page, Western Blot, Mutagenesis, Immunoprecipitation, Transfection, Cell Culture, Staining, Microscopy, Negative Control

Journal: Nature Communications

Article Title: Sororin locks the DNA-exit gate of cohesin to preserve sister-chromatid cohesion

doi: 10.1038/s41467-026-70484-2

Figure Lengend Snippet: A Schematic of cohesin-mediated sister-chromatid cohesion, illustrating Sororin’s interaction with Pds5 to antagonize Wapl. B Schematic representation of human Sororin variants, highlighting the CTR. C HeLa cells were transfected with an empty vector, H2B-GFP, or the indicated Sororin-GFP variants. Twenty-four hours post-transfection, cell lysates were immunoblotted for GFP and α-Tubulin ( n = 1). α-Tubulin from a parallel gel served as a loading control. Irrelevant lanes were excised from the gel image (indicated by a vertical line). Uncropped blots are available in the Source Data file. D Cells were transfected as in C . Twenty-four hours post-transfection, cells were treated with nocodazole for 3 h. Mitotic chromosome spreads were prepared and stained with DAPI and antibodies against GFP and ACA. The percentage of mitotic cells exhibiting predominantly arm-resolved or unresolved sister chromatids was quantified in at least 200 cells per condition across four independent experiments ( n = 4), except for parental cells and H2B-GFP-expressing cells, for which at least 50 cells per condition were analyzed in one experiment. Statistical significance was calculated with two-tailed, unpaired Student’s t -test. Error bars represent standard deviation (SD). Raw data are available in the Source Data file. E Cells were treated and analyzed as in D. Representative images are shown ( n = 1). Scale bar = 10 μm. F HeLa cells were transfected with H2B-GFP or the indicated H2B-Sororin-GFP variants. Twenty-four hours post-transfection, cell lysates were immunoblotted for GFP and GAPDH. GAPDH from a parallel gel served as a loading control. Blots are representative of three independent experiments. Uncropped blots are available in the Source Data file. G Cells were transfected as in F. Twenty-four hours post-transfection, cells were treated with nocodazole for 3 h. Mitotic chromosome spreads were stained with DAPI and antibodies against CENP-C. The percentage of mitotic cells with predominantly arm-resolved or unresolved sister chromatids was quantified from at least 300 cells per condition across three independent experiments ( n = 3). Statistical significance was calculated with two-tailed, unpaired Student’s t -test. Error bars represent SD. Raw data are available in the Source Data file. H Cells were treated and analyzed as in G . Representative images from three independent experiments are shown. Scale bar = 10 μm.

Article Snippet: The following rabbit polyclonal antibodies were used: GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), RAD21 (ab992, Abcam), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5A (A300-089A, Bethyl Laboratories), Pds5B (A300-537A and A300-538A, Bethyl Laboratories), SA1 (HPA035015, Sigma), SA2 (#5882, CST), Wapl (A300-268A, Bethyl Laboratories), Esco1 (29821-1-AP, Proteintech), Sororin (ab192237, Abcam), Sororin-pS145 (generated by immunization with the synthetic peptide LEMSKKVRRpSYSRLETLGSASTS, where pS indicates phosphoserine), and Flag-tag (A01868, GenScript).

Techniques: Transfection, Plasmid Preparation, Control, Staining, Expressing, Two Tailed Test, Standard Deviation

Journal: Nature Communications

Article Title: Sororin locks the DNA-exit gate of cohesin to preserve sister-chromatid cohesion

doi: 10.1038/s41467-026-70484-2

Figure Lengend Snippet: A HeLa cells stably expressing Sororin-GFP (W233A/E252K or F241A/F247A) were transfected with control siRNA, Sororin siRNA and/or Wapl siRNA. Forty-eight hours post-transfection, cell lysates were immunoblotted for Wapl, GFP, Sororin, and GAPDH. GAPDH from a parallel gel served as a loading control. Blots are representative of three independent experiments. Uncropped blots are available in the Source Data file. B The indicated stable cell lines were transfected as in A. Forty-eight hours post-transfection, cells were treated with nocodazole for 3 h. Mitotic chromosome spreads were stained with DAPI and antibodies against GFP and ACA. The percentages of mitotic cells exhibiting predominantly separated or unseparated sister chromatids were quantified from at least 300 cells per condition across three independent experiments ( n = 3). Statistical significance was calculated with two-tailed, unpaired Student’s t -test. Error bars represent SD. Raw data are available in the Source Data file. C Cells were treated and analyzed as in B . Representative images from three independent experiments are shown. Scale bars = 10 μm. D HeLa cells stably expressing inducible RAD21-SFB (Y3A/K50E) were treated with Dox and transfected with control siRNA, RAD21 siRNA, and/or Wapl siRNA. Sixty hours post-transfection, cell lysates were immunoblotted for Wapl, RAD21, and α-Tubulin. α-Tubulin from a parallel gel served as a loading control. Blots are representative of three independent experiments. Uncropped blots are available in the Source Data file. E Cells were treated as in D. Sixty hours post-transfection, cells were treated with nocodazole for 3 h. Mitotic chromosome spreads were stained with DAPI and antibodies against ACA. The percentage of mitotic cells exhibiting predominantly separated or unseparated sister chromatids was quantified from at least 300 cells per condition across three independent experiments ( n = 3). Statistical significance was calculated with two-tailed, unpaired Student’s t -test. Error bars represent SD. Raw data are available in the Source Data file. F Cells were treated and analyzed as in E . Representative images from three independent experiments are shown. Scale bars = 10 μm. G HeLa cells stably expressing SMC3-GFP (K1034E/1038E) were transfected with control siRNA, SMC3 siRNA, and/or Wapl siRNA. Seventy-two hours post-transfection, cell lysates were immunoblotted for GFP, SMC3, Wapl, and GAPDH. GAPDH from a parallel gel served as a loading control. Blots are representative of three independent experiments. Uncropped blots are available in the Source Data file. H Cells were transfected as in G . Seventy-two hours post-transfection, cells were treated with nocodazole for 3 h. Mitotic chromosome spreads were stained with DAPI and antibodies against ACA. The percentages of mitotic cells exhibiting fully separated, partially separated, or unseparated sister chromatids were quantified from at least 300 cells per condition across three independent experiments ( n = 3). Statistical significance was calculated with two-tailed, unpaired Student’s t -test. Error bars represent SD. Raw data are available in the Source Data file. I Cells were treated and analyzed as in H . Representative images from three independent experiments are shown. Scale bars = 10 μm. J Working model illustrating the function of Sororin’s CTR as a direct structural lock for cohesin’s DNA-exit gate by engaging the RAD21-SMC3 interface. Mutations in Sororin-CTR that disrupt binding to the RAD21-SMC3 interface cause Wapl-dependent opening of the DNA-exit gate and loss of sister-chromatid cohesion.

Article Snippet: The following rabbit polyclonal antibodies were used: GFP (A11122, Invitrogen), GAPDH (14C10, Cell Signaling Technology/CST), RAD21 (ab992, Abcam), SMC1 (A300-055A, Bethyl Laboratories), SMC3 (A300-060A, Bethyl Laboratories), Pds5A (A300-089A, Bethyl Laboratories), Pds5B (A300-537A and A300-538A, Bethyl Laboratories), SA1 (HPA035015, Sigma), SA2 (#5882, CST), Wapl (A300-268A, Bethyl Laboratories), Esco1 (29821-1-AP, Proteintech), Sororin (ab192237, Abcam), Sororin-pS145 (generated by immunization with the synthetic peptide LEMSKKVRRpSYSRLETLGSASTS, where pS indicates phosphoserine), and Flag-tag (A01868, GenScript).

Techniques: Stable Transfection, Expressing, Transfection, Control, Staining, Two Tailed Test, Binding Assay