Journal: Molecular Cell

Article Title: The ASC-1 Complex Disassembles Collided Ribosomes

doi: 10.1016/j.molcel.2020.06.006

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-ASCC3 , Bethyl , Cat. #A304-015A.

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Sequencing, Mutagenesis, Software, Cloning

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC2 recruits ASCC3 to stalled forks in a manner dependent upon ASCC2’s ubiquitin binding activity. ( A ) Western blot analyses of U2OS WT, ASCC2-KO, and ASCC3-KO cells. Immunoblotting was performed with anti-ASCC2, anti-ASCC3, and anti-γ-tubulin antibodies. The γ-tubulin blot was used as a loading control in this and subsequent figures except for where the α-tubulin blot was used as a loading control. The asterisk (*) indicates a non-specific band. ( B ) Representative images of endogenous ASCC2-EdU PLA foci formation in U2OS WT and ASCC2-KO that were either treated without or with 4 mM HU for 4 h. Nuclei were stained with DAPI in blue in this and subsequent figures. Scale bars in this and subsequent figures: 5 μm. ( C ) Quantification of endogenous ASCC2-EdU PLA foci formation from (B). The PLA experiments were performed independently twice with reproducible data in this, 1F, 1G, and 1H panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 492-506 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( D ) Quantification of PLA foci formation in U2OS treated with 4 mM HU for 4 h. PLA assays were performed in several conditions as indicated. This PLA experiment was performed once. A total of 492-515 cells per condition were analyzed. **** P <0.0001. ( E ) Representative images of endogenous ASCC3-EdU PLA foci formation in no HU- or HU-treated U2OS WT and ASCC3-KO cells. ( F ) Quantification of endogenous ASCC3-EdU PLA foci formation from (E). A total of 1389-1396 cells per condition were analyzed. **** P <0.0001. ( G ) Quantification of Myc-tagged ASCC2-EdU PLA foci formation in no HU- or HU-treated U2OS ASCC2-KO cells expressing various Myc-ASCC2 alleles as indicated. A total of 603-617 cells per condition were analyzed. **** P <0.0001. ( H ) Quantification of endogenous ASCC3-EdU PLA foci formation in no HU- or HU-treated U2OS ASCC2-KO cells stably expressing various Myc-ASCC2 alleles as indicated. A total of 806-816 cells per condition were analyzed. **** P <0.0001. ( I ) Western blot analyses of U2OS ASCC2-KO cells stably expressing various Myc-ASCC2 alleles as indicated. Immunoblotting was performed with anti-ASCC3 and anti-γ-tubulin antibodies.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Ubiquitin Proteomics, Binding Assay, Activity Assay, Western Blot, Control, Staining, MANN-WHITNEY, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC2 interacts with polyubiquitylated PCNA and is recruited by PCNA ubiquitylated at K164 to stalled forks. ( A ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS cells transfected with siControl or siRNA against SHPRH, HLTF or RFWD3. The PLA experiments were performed twice independently with reproducible data in this, 2D, and 2E panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 805-846 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( B ) Western blot analyses of U2OS WT and SHPRH-KO cells. Immunoblotting was performed with anti-SHPRH, anti-ASCC2, anti-ASCC3, and anti-γ-tubulin antibodies. ( C ) Representative images of ASCC2-EdU PLA foci in HU-treated U2OS WT and SHPRH-KO cells. ( D ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS WT and SHPRH-KO cells from (C). U2OS ASCC2-KO cells were included as a control. A total of 817-822 cells per condition were analyzed. **** P <0.0001. ( E ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS SHPRH-KO cells expressing various Myc-SHPRH alleles as indicated. A total of 827-832 cells per condition were analyzed. **** P <0.001. ( F ) Representative images of ASCC2-EdU PLA foci in no HU- or HU-treated RPE1 parental (WT), A1 (PCNA-K164R), and B1 (PCNA-K164R) cells. ( G ) Quantification of ASCC2-EdU PLA foci formation in RPE1 WT, A1, B1, A1 revertant (A1.R, PCNA-K164K), and B1 revertant (B1.R, K164K) cells that were treated with or without HU. This PLA experiment was done once. A total of 525-540 cells per condition were analyzed. **** P <0.0001. ( H ) Coimmunoprecipitation with anti-HA antibody in UV-irradiated, ATRi-treated and USP1-depleted HEK293T cells expressing the vector alone, HA-tagged ASCC2, or HA-tagged ASCC2 carrying LLL-AAA mutations. Immunoblotting was performed with anti-Ubiquityl-PCNA (K164), anti-PCNA, and anti-HA antibodies.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Transfection, MANN-WHITNEY, Western Blot, Control, Expressing, Irradiation, Plasmid Preparation

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 unwinds DNA to promote ssDNA-RPA accumulation upon replication stress. ( A ) Quantification of the percentage of cells with ≥ 10 BrdU foci in U2OS WT and ASCC3-KO cells that were pulse-labeled with BrdU for 20 min prior to their treatment with or without 4 mM HU for 4 h. A total of 802-825 cells per condition were scored in blind. SDs from three independent experiments are indicated in this and subsequent panels. *** P <0.001. ( B ) Quantification of the percentage of cells with ≥ 30 BrdU foci in U2OS WT and ASCC3-KO cells that were labeled with BrdU for 20 h prior to their treatment with or without 4 mM HU for 4 h. A total of 800-849 cells per condition were scored in blind. *** P <0.001. ( C ) Quantification of the percentage of cells with ≥ 10 BrdU foci in U2OS ASCC3-KO cells expressing various Myc-ASCC3 alleles as indicated. These cells were pulse-labeled with BrdU for 20 min prior to their treatment with HU. A total of 601-641 cells per condition were scored in blind. *** P <0.001. ( D ) Quantification of the percentage of cells with ≥ 30 BrdU foci in U2OS ASCC3-KO cells expressing various Myc-ASCC3 alleles as indicated. These cells were labeled with BrdU for 20 h prior to their treatment with HU. A total of 502-521 cells per condition were scored in blind. * P <0.05; ** P <0.01. ( E ) Quantification of EdU+ cells with ≥ 30 RPA32 foci in U2OS WT and ASCC3-KO cells that were pulse-labeled with EdU for 10 min prior to treatment with or without 4 mM HU for 6 h. A total of 800-873 cells per condition were scored in blind. *** P <0.001. ( F ) Quantification of the percentage of EdU+ cells with ≥ 30 RPA32 foci in U2OS ASCC3-KO cells expressing various Myc-ASCC3 alleles as indicated. A total of 801-856 cells per condition were scored in blind. *** P <0.001. ( G ) Quantification of the percentage of cells with ≥ 10 BrdU foci in U2OS WT and TRIP4-KO cells that were pulse-labeled with BrdU for 20 min prior to their treatment with or without HU. A total of 601-650 cells per condition were scored in blind. ** P <0.01. ( H ) Quantification of the percentage of EdU+ cells with ≥ 30 RPA32 foci in U2OS WT and TRIP4-KO cells that were pulse-labeled with EdU for 10 min prior to their treatment with or without HU. A total of 742-977 cells per condition were scored in blind. ** P <0.01. ( I ) Schematic diagram of in vitro RPA binding assays. ( J ) Western blot analyses of recombinant RPA70 recovered from streptavidin pulldown of biotinylated ssDNA from indicated reactions. Immunoblotting was done with an anti-RPA70 antibody. ( K ) Quantification of recombinant RPA70 from (J). * P <0.05.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Labeling, Expressing, In Vitro, Binding Assay, Western Blot, Recombinant

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 interacts with RPA and this interaction is further induced by replication stress. ( A ) Anti-Myc coIPs in HEK293T cells transfected with eGFP-ASCC3 together with either the vector alone, Myc-RPA32, or Myc-RPA70. Immunoblotting was done with anti-Myc and anti-GFP antibodies. ( B ) Anti-Myc coIPs in HEK293T cells transfected with HA-RPA70 together with either the vector alone or Myc-ASCC3. Immunoblotting was done with anti-Myc and anti-HA antibodies. ( C ) Anti-Myc coIPs in HEK293T cells transfected with HA-RPA32 together with the vector alone or Myc-ASCC3. Immunoblotting was done with anti-Myc and anti-HA antibodies. ( D ) Coimmunoprecipitations (coIPs) with IgG, anti-ASCC2, or anti-ASCC3 antibodies in HCT116 cells. Immunoblotting was done with anti-ASCC2, anti-ASCC3, anti-RPA70, and anti-RPA32 antibodies. ( E ) Anti-RPA32 coIPs in HCT116 cells. IgG was used as a control. Immunoblotting was performed with anti-RPA32 and anti-ASCC3 antibodies. ( F ) Anti-HA coIPs in no HU- or HU-treated HEK293T cells expressing the vector alone or HA-RPA70. Immunoblotting was done with anti-HA, anti-ASCC2, and anti-ASCC3 antibodies. ( G ) Anti-HA coIPs in HEK293T cells expressing the vector alone or HA-RPA70 in the presence or absence of DNase I. Immunoblotting was done with anti-HA and anti-ASCC3 antibodies. ( H ) Representative images of ASCC3-RPA32 PLA foci formation in no HU- or HU-treated U2OS WT and ASCC3-KO cells. ( I ) Quantification of ASCC3-RPA32 PLA foci formation from (H). The PLA experiments were performed twice independently with reproducible data. Data from one representative experiment are shown as scatter plot graphs with the mean indicated. A total of 402-418 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test. **** P <0.0001.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 promotes fork reversal upon replication stress. ( A ) Quantification of the percentage of U2OS WT and ASCC3-KO cells with ≥ 10 SMARCAL1 foci. Cells were treated with or without 4 mM HU for 2 h. A total of 501-531 cells per condition were scored in blind. SDs from three independent experiments are indicated in this and 6B panels. *** P <0.001. ( B ) Quantification of the percentage of cells with ≥ 10 SMARCAL1 foci in U2OS ASCC3-KO cells expressing the vector alone, Myc-ASCC3, or Myc-ASCC3-G1354D prior to treatment with HU. A total of 501-537 cells per condition were scored in blind. ** P <0.01. ( C ) Representative images of SMARCAL1-EdU PLA foci formation in no HU- or HU-treated U2OS WT and ASCC3-KO cells. ( D ) Quantification of SMARCAL1-EdU PLA foci formation from (C). The PLA experiments were performed twice independently with reproducible data in this and 5E panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 768-793 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( E ) Quantification of SMARCAL1-EdU PLA foci formation in HU-treated U2OS ASCC3-KO cells expressing the vector alone, Myc-ASCC3, or Myc-ASCC3-G1354D. A total of 612-635 cells per condition were analyzed. **** P <0.0001. ( F ) Representative images of DNA fibers from U2OS WT or ASCC3-KO cells treated with or without HU. ( G ) Quantification of the CldU/IdU ratio from (F). DNA fiber experiments were performed three times independently with reproducible data. A total of 388-418 fibers per condition were analyzed. **** P <0.0001. ( H ) Quantification of the CldU/IdU ratio for HU-treated U2OS ASCC3-KO cells expressing the vector alone, Myc-ASCC3, or Myc-ASCC3-G1354D. DNA fiber experiments were performed twice independently with reproducible data. A total of 333-357 fiber per condition were analyzed. **** P <0.0001. ( I ) Western blot analyses of U2OS WT and ASCC3-KO cells transfected with indicated siRNAs. Immunoblotting was performed with anti-BRCA1 and anti-γ-tubulin antibodies. ( J ) Western blot analysis of U2OS WT and ASCC3-KO cells transfected with indicated siRNA. Immunoblotting was performed with anti-BRCA2 and anti-γ-tubulin antibodies. ( K ) Representative images of DNA fibers from U2OS WT or ASCC3-KO cells transfected with siControl, siBRCA1, or siBRCA2. ( L ) Quantification of the CldU/IdU ratio from (K). DNA fiber experiments were performed twice independently with reproducible data. A total of 400-407 fibers per condition were analyzed. **** P <0.0001.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Expressing, Plasmid Preparation, MANN-WHITNEY, Western Blot, Transfection

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 antagonizes RAD51-mediated recombination. ( A ) Quantification of the CldU/IdU ratio in HU-treated U2OS WT and ASCC3-KO cells expressing control siRNA or siRNA against PRIMPOL (siPRIMPOL). DNA fiber experiments were performed twice independently with reproducible data in this, 6B, and 6C panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this, 6B, and 6C panels. A total of 407-421 fibers per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( B ) Quantification of the CldU/IdU ratio in U2OS WT and ASCC3-KO cells that were treated with HU in the presence or absence of REV1 inhibitor JH-RE-06 (REV1i). A total of 413-414 fibers per condition were analyzed. **** P <0.0001. ( C ) Quantification of the CldU/IdU ratio in HU-treated U2OS WT and ASCC3-KO cells expressing control siRNA or two independent siRNA against RAD51 as indicated. A total of 407-415 fibers per condition were analyzed. **** P <0.0001. ( D ) Quantification of EdU+ cells with ≥ 20 RAD51 foci in U2OS WT and ASCC3-KO cells that were pulse-labeled with EdU for 10 min prior to treatment with or without 4 mM HU for 6 h. A total of 801-853 cells per condition were scored in blind. SDs from three independent experiments are indicated in this, and 6E-6G panels. *** P <0.001. ( E ) Quantification of EdU+ cells with ≥ 20 RAD51 foci in U2OS ASCC3-KO cells expressing various Myc-ASCC3 alleles as indicated. A total of 802-827 cells per condition were scored in blind. ** P <0.01; *** P <0.001. ( F ) Quantification of cells with ≥ 10 IR-induced RAD51 foci in U2OS WT and ASCC3-KO cells that were treated with 10 Gy IR prior to fixation. A total of 503-554 cells per condition were scored in blind. ** P <0.01. ( G ) Quantification of the percentage of cells with restoration of GFP expression following HR-mediated repair of I-SceI-induced DSBs. ** P <0.01. ( H ) Schematic diagram of strand exchange assays. ( I ) In vitro strand exchange assays. Top panel: Recombinant RAD51 (270 nM) was premixed with 1µM of 3’-ssDNA tail for 5 min, followed by further incubation with 350 nM of labeled duplex DNA and increasing concentration (150 nM, 300 nM, 600 nM) of ASCC3 HR WT-TRIP4 or ASCC3 HR -DD-AA-TRIP4 for 15 min. * indicates the 32 P-labeled DNA end. Bottom panel: Quantification of strand exchange efficiency (%) relative to RAD51 alone reaction from top panel. SDs from four independent experiments are shown. * P <0.05; **** P <0.0001.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Expressing, Control, MANN-WHITNEY, Labeling, In Vitro, Recombinant, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 promotes efficient ATR activation and genomic stability. ( A ) Western blot analyses of U2OS and HCT116 WT and ASCC3-KO cells treated with no HU or HU (4 mM) for various times as indicated. Immunoblotting was done with antibodies against RPA32-pS33, RPA32, CHK1-pS345, CHK1, and α-tubulin. ( B ) Western blot analyses. U2OS WT and ASCC3-KO cells were either untreated or treated with UV (30 J/m 2 ), camptothecin (CPT) (1 μM CPT for 1 h) or aphidicolin (APH) (0.4 μM for 24 h). Immunoblotting was done with antibodies against RPA32-pS33, RPA32, CHK1-pS345, CHK1, and α-tubulin. ( C ) Schematic diagram of experimental setup. APH: aphidicolin; RO-3306: CDK1 inhibitor; RAD52i: RAD52 inhibitor AICAR. ( D ) Quantification of the average number of EdU foci in both U2OS WT and ASCC3-KO prometaphase cells treated with or without RAD52i (20 μM). A total of 188-207 prometaphase cells per condition were scored in blind. SDs from three independent experiments are shown in this and subsequent panels. *** P <0.001. ( E ) Quantification of the percentage of metaphase chromosomes with gaps and breaks in both U2OS WT and ASCC3-KO following exposure to aphidicolin in S phase. A total of 20 metaphase cells (with a total of 905-975 metaphase chromosomes) per condition were scored in blind. *** P <0.001. ( F ) Quantification of the average number of micronuclei in both U2OS WT and ASCC3-KO G1 daughter cells following exposure to aphidicolin in previous S phase. A total of 508-553 cells per condition were scored in blind. *** P <0.001. ( G ) Quantification of the percentage of both U2OS WT and ASCC3-KO G1 daughter cells with ≥ 5 53BP1 nuclear bodies following exposure to aphidicolin in previous S phase. A total of 510-517 cells per condition were scored in blind. *** P <0.001. ( H ) Model for control of replication stress responses by ASCC3. See the text for details.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Activation Assay, Western Blot, Control

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Venn diagram of overlaps between 5´ TOP motif mRNAs ( n = 97) and transcripts with significantly increased A-site pauses in the first 5 codons in ZNF598 RING -expressing hiPSCi ( n = 704), and comparison of 5´ UTR lengths and TPM levels from RNA-Seq in both groups ( p -value from two-sided Wilcoxon test). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. b , Comparison of ribosome recruitment scores for 5´ UTRs quantified by direct analysis of ribosome targeting (DART) for histone mRNAs with start site pauses ( n = 29), other mRNAs with start site pauses ( n = 351), other well-translated mRNAs ( n = 1340), and other detectable mRNAs ( n = 3791). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× the interquartile range. c , Density heatmaps of 50-80 nt ribosome footprints according to length and 5’ end position around CDS start (left) and stop sites (right) for a second biological replicate. d , Density heatmaps of 50-80 nt ribosome footprints according to length and 3’ end position around CDS start (left) and stop sites (right) for a second biological replicate. e , Metagene profiles of 50-80 nt ribosome footprints around CDS start and stop sites in hiPSCi ( n = 2 biological replicates). f , Polysome profiles and immunoblot analysis of uS10 and eS10 in sucrose gradient fractions from control HEK293i or after a short (2.5 min) treatment with 2 µg/ml homoharringtonine (HAR) ( n = 1 biological replicate). g , Metagene profiles of 50-80 nt ribosome footprints around CDS start and stop sites in ZNF598-depleted hiPSCi treated with GCN2 inhibitor (GCN2i, A-92, 1.25 µM ; n = 2 biological replicates). h , Polysome profiles (from Fig. ) and immunoblot analysis of uS3 and uS5 in sucrose gradient fractions from control hiPSCi, after short (2.5 min) or long (2 hours) treatment with 2 µg/ml homoharringtonine, or after treatment with 0.05 mg/l anisomycin (ANS) for 15 minutes ( n = 1 biological replicate). Membranes from Fig. were stripped and re-probed with antibodies against uS3 and uS5. i , Global protein synthesis measurements by OPP labeling in control hiPSCi and/or after treatment with 6.25, 12.5, and 25 µM 4EGI-1 for 1 day. Median fluorescence intensity was quantified by flow cytometry ( > 10,000 cells/ analysis) and normalized to the average value in controls ( n = 2 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Comparison, RNA Sequencing, Western Blot, Control, Labeling, Fluorescence, Flow Cytometry

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Schematic model of the consequences of ZNF598 depletion or ZNF598 RING expression in inducible hiPS cells. b , Polysome profiles (top) and immunoblot analysis of ZNF598 and uS5 in polysome gradient fractions (bottom) of inducible hiPS cells expressing sgControl, a ZNF598 sgRNA or ZNF598 RING ( n = 1 biological replicate). c , Metagene profiles of ribosomal A-site occupancy from monosome footprints around CDS start and stop sites ( n = 2 biological replicates). d , Volcano plot of differential ribosome pause sites upon ZNF598 RING expression in inducible hiPS cells (two-tailed Fisher’s exact test with Benjamini–Hochberg correction, adjusted P ≤ 0.01). e , Nucleotide (top) and amino acid (bottom) motif analysis of significantly increased pause sites in well-translated mRNAs (>0.5 footprints per codon in all samples; n = 3,421) in ZNF598 RING -expressing inducible hiPS cells. f , Volcano plot of differential ribosome pausing analysis upon ZNF598 knockdown (sgZNF598) in inducible hiPS cells as in d . g , Nucleotide (top) and amino acid (bottom) motif analysis of significantly increased pause sites in well-translated mRNAs (>0.5 footprints per codon in all samples; n = 2,463) in sgZNF598 inducible hiPS cells. h , i , GO term enrichment analysis of genes with significantly increased pause sites (one-tailed Fisher’s exact test with Benjamini–Hochberg correction, adjusted P ≤ 0.01) within the first five codons in ZNF598 RING -expressing inducible hiPS cells ( h ) and throughout the ORF in sgZNF598 inducible hiPS cells ( i ) filtered for TPM > 1 in RNA-seq from inducible hiPS cells. j , Distribution of monosome footprints (in reads per million (rpm)) along the H1-5 (left) and H3C2 (right) mRNA in control and ZNF598 RING -expressing inducible hiPS cells ( n = 2 biological replicates). Significant differential pauses are indicated with red arrows. k , Representative histograms of cell-cycle analysis in inducible hiPS cells by DNA staining with EdU followed by flow cytometry. l , Changes in the fraction of cells in different cell-cycle phases calculated by flow cytometry analysis after EdU staining ( n = 3 biological replicates; >10,000 cells per analysis; P values from an unpaired two-tailed t -test).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Western Blot, Two Tailed Test, Knockdown, One-tailed Test, RNA Sequencing, Control, Cell Cycle Assay, Staining, Flow Cytometry

Journal: Nature Structural & Molecular Biology

Article Title: Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control

doi: 10.1038/s41594-025-01616-3

Figure Lengend Snippet: a , Venn diagram of overlaps between 5´ TOP motif mRNAs ( n = 97) and transcripts with significantly increased A-site pauses in the first 5 codons in ZNF598 RING -expressing hiPSCi ( n = 704), and comparison of 5´ UTR lengths and TPM levels from RNA-Seq in both groups ( p -value from two-sided Wilcoxon test). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range. b , Comparison of ribosome recruitment scores for 5´ UTRs quantified by direct analysis of ribosome targeting (DART) for histone mRNAs with start site pauses ( n = 29), other mRNAs with start site pauses ( n = 351), other well-translated mRNAs ( n = 1340), and other detectable mRNAs ( n = 3791). Box plots: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× the interquartile range. c , Density heatmaps of 50-80 nt ribosome footprints according to length and 5’ end position around CDS start (left) and stop sites (right) for a second biological replicate. d , Density heatmaps of 50-80 nt ribosome footprints according to length and 3’ end position around CDS start (left) and stop sites (right) for a second biological replicate. e , Metagene profiles of 50-80 nt ribosome footprints around CDS start and stop sites in hiPSCi ( n = 2 biological replicates). f , Polysome profiles and immunoblot analysis of uS10 and eS10 in sucrose gradient fractions from control HEK293i or after a short (2.5 min) treatment with 2 µg/ml homoharringtonine (HAR) ( n = 1 biological replicate). g , Metagene profiles of 50-80 nt ribosome footprints around CDS start and stop sites in ZNF598-depleted hiPSCi treated with GCN2 inhibitor (GCN2i, A-92, 1.25 µM ; n = 2 biological replicates). h , Polysome profiles (from Fig. ) and immunoblot analysis of uS3 and uS5 in sucrose gradient fractions from control hiPSCi, after short (2.5 min) or long (2 hours) treatment with 2 µg/ml homoharringtonine, or after treatment with 0.05 mg/l anisomycin (ANS) for 15 minutes ( n = 1 biological replicate). Membranes from Fig. were stripped and re-probed with antibodies against uS3 and uS5. i , Global protein synthesis measurements by OPP labeling in control hiPSCi and/or after treatment with 6.25, 12.5, and 25 µM 4EGI-1 for 1 day. Median fluorescence intensity was quantified by flow cytometry ( > 10,000 cells/ analysis) and normalized to the average value in controls ( n = 2 biological replicates).

Article Snippet: Membranes were destained with PBST (0.1% Tween-20), blocked for 1 h in 5% milk in PBST (0.1% Tween-20) and further incubated with primary antibodies in blocking solution overnight at 4 °C (ZNF598: 1:1,000, Abcam, ab135921; PELO F-4: 1:1,000, Santa Cruz Biotechnology, sc-393418; HBS1L: 1:1,000, Atlas Antibodies, HPA029729; ASCC3: 1:1,000, Bethyl Laboratories, A304-015A; eIF2α: 1:1,000, Cell Signaling, 9722; eIF2α-p S51: 1:1,000, Abcam, ab32157; p38: 1:1,000, Cell Signaling, 9212; p38-p T180/Y182: 1:1,000, Cell Signaling, 9211; eS10: 1:1,000, Abcam, ab151550; uS10: 1:1,000, Abcam, ab133776; uS5: 1:1,000, Bethyl Laboratories, A303-794A; uS3: 1:1,000, Bethyl Laboratories, A303-840A).

Techniques: Expressing, Comparison, RNA Sequencing, Western Blot, Control, Labeling, Fluorescence, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

doi: 10.1093/nar/gky1093

Figure Lengend Snippet: ALS- and SMA-causative proteins in the RNAP II/U1 snRNP machinery

Article Snippet: Polyclonal antibodies against EWSR1 (cat # A300-418A), MATR3 (cat # A300-591A), HA (cat # A190-108A), ASCC1 (cat # A303-871A), ASCC2 (cat # A304-020A), ASCC3 (cat # A304-014A) and TRIP4 (cat # A300-203A) were from Bethyl (Montgomery, TX, USA), and the polyclonal antibody against TAF15 was from Novus (Littleton, CO) (cat # NB100-567).

Techniques:

Journal: Nucleic Acids Research

Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

doi: 10.1093/nar/gky1093

Figure Lengend Snippet: Disease-causing mutations in FUS or TRIP4 disrupt interactions of ASC-1 complex components with the RNAP II/U1 snRNP machinery. ( A ) Purified recombinant GST-FUS or GST-DDX39B was added to FUS KO extract, and RNAP II was IP’d followed by Westerns with antibodies to the ASC-1 components. The asterisk indicates degradation products of GST-FUS. Endogenous FUS, GST-FUS, and GST-DDX39B were detected by the FUS antibody, as this antibody recognizes GST-DDX39B due to the GST tag common to both proteins. ( B ) Three independent replicates of the data shown in (A) were quantitated. The colored bars in the graph show the mean values of fold change for the indicated proteins. Error bars represent standard deviations. * P < 0.05, n.s., not significant (two-tailed Student's t -test). ( C and D ), same as (A and B), except that GST-FUS G156E or FUSR 514G was used for add-backs. ( E ) HA-DDX39B, HA-TRIP4 or HA-TRIP4 1-254 was expressed in HeLa cells followed by IP/westerns with the indicated antibodies. ( F ) Quantitation of three independent replicates of data shown in (E). ** P < 0.01 (two-tailed Student's t -test.)

Article Snippet: Polyclonal antibodies against EWSR1 (cat # A300-418A), MATR3 (cat # A300-591A), HA (cat # A190-108A), ASCC1 (cat # A303-871A), ASCC2 (cat # A304-020A), ASCC3 (cat # A304-014A) and TRIP4 (cat # A300-203A) were from Bethyl (Montgomery, TX, USA), and the polyclonal antibody against TAF15 was from Novus (Littleton, CO) (cat # NB100-567).

Techniques: Purification, Recombinant, Two Tailed Test, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex

doi: 10.1093/nar/gky1093

Figure Lengend Snippet: ALS- and SMA-causative proteins in the RNAP II/U1 snRNP machinery

Article Snippet: Polyclonal antibodies against EWSR1 (cat # A300-418A), MATR3 (cat # A300-591A), HA (cat # A190-108A), ASCC1 (cat # A303-871A), ASCC2 (cat # A304-020A), ASCC3 (cat # A304-014A) and TRIP4 (cat # A300-203A) were from Bethyl (Montgomery, TX, USA), and the polyclonal antibody against TAF15 was from Novus (Littleton, CO) (cat # NB100-567).

Techniques:

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC2 interacts with polyubiquitylated PCNA and is recruited by PCNA ubiquitylated at K164 to stalled forks. ( A ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS cells transfected with siControl or siRNA against SHPRH, HLTF or RFWD3. The PLA experiments were performed twice independently with reproducible data in this, 2D, and 2E panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 805-846 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( B ) Western blot analyses of U2OS WT and SHPRH-KO cells. Immunoblotting was performed with anti-SHPRH, anti-ASCC2, anti-ASCC3, and anti-γ-tubulin antibodies. ( C ) Representative images of ASCC2-EdU PLA foci in HU-treated U2OS WT and SHPRH-KO cells. ( D ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS WT and SHPRH-KO cells from (C). U2OS ASCC2-KO cells were included as a control. A total of 817-822 cells per condition were analyzed. **** P <0.0001. ( E ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS SHPRH-KO cells expressing various Myc-SHPRH alleles as indicated. A total of 827-832 cells per condition were analyzed. **** P <0.001. ( F ) Representative images of ASCC2-EdU PLA foci in no HU- or HU-treated RPE1 parental (WT), A1 (PCNA-K164R), and B1 (PCNA-K164R) cells. ( G ) Quantification of ASCC2-EdU PLA foci formation in RPE1 WT, A1, B1, A1 revertant (A1.R, PCNA-K164K), and B1 revertant (B1.R, K164K) cells that were treated with or without HU. This PLA experiment was done once. A total of 525-540 cells per condition were analyzed. **** P <0.0001. ( H ) Coimmunoprecipitation with anti-HA antibody in UV-irradiated, ATRi-treated and USP1-depleted HEK293T cells expressing the vector alone, HA-tagged ASCC2, or HA-tagged ASCC2 carrying LLL-AAA mutations. Immunoblotting was performed with anti-Ubiquityl-PCNA (K164), anti-PCNA, and anti-HA antibodies.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Transfection, MANN-WHITNEY, Western Blot, Control, Expressing, Irradiation, Plasmid Preparation

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC2 recruits ASCC3 to stalled forks in a manner dependent upon ASCC2’s ubiquitin binding activity. ( A ) Western blot analyses of U2OS WT, ASCC2-KO, and ASCC3-KO cells. Immunoblotting was performed with anti-ASCC2, anti-ASCC3, and anti-γ-tubulin antibodies. The γ-tubulin blot was used as a loading control in this and subsequent figures except for where the α-tubulin blot was used as a loading control. The asterisk (*) indicates a non-specific band. ( B ) Representative images of endogenous ASCC2-EdU PLA foci formation in U2OS WT and ASCC2-KO that were either treated without or with 4 mM HU for 4 h. Nuclei were stained with DAPI in blue in this and subsequent figures. Scale bars in this and subsequent figures: 5 μm. ( C ) Quantification of endogenous ASCC2-EdU PLA foci formation from (B). The PLA experiments were performed independently twice with reproducible data in this, 1F, 1G, and 1H panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 492-506 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( D ) Quantification of PLA foci formation in U2OS treated with 4 mM HU for 4 h. PLA assays were performed in several conditions as indicated. This PLA experiment was performed once. A total of 492-515 cells per condition were analyzed. **** P <0.0001. ( E ) Representative images of endogenous ASCC3-EdU PLA foci formation in no HU- or HU-treated U2OS WT and ASCC3-KO cells. ( F ) Quantification of endogenous ASCC3-EdU PLA foci formation from (E). A total of 1389-1396 cells per condition were analyzed. **** P <0.0001. ( G ) Quantification of Myc-tagged ASCC2-EdU PLA foci formation in no HU- or HU-treated U2OS ASCC2-KO cells expressing various Myc-ASCC2 alleles as indicated. A total of 603-617 cells per condition were analyzed. **** P <0.0001. ( H ) Quantification of endogenous ASCC3-EdU PLA foci formation in no HU- or HU-treated U2OS ASCC2-KO cells stably expressing various Myc-ASCC2 alleles as indicated. A total of 806-816 cells per condition were analyzed. **** P <0.0001. ( I ) Western blot analyses of U2OS ASCC2-KO cells stably expressing various Myc-ASCC2 alleles as indicated. Immunoblotting was performed with anti-ASCC3 and anti-γ-tubulin antibodies.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Ubiquitin Proteomics, Binding Assay, Activity Assay, Western Blot, Control, Staining, MANN-WHITNEY, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC2 interacts with polyubiquitylated PCNA and is recruited by PCNA ubiquitylated at K164 to stalled forks. ( A ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS cells transfected with siControl or siRNA against SHPRH, HLTF or RFWD3. The PLA experiments were performed twice independently with reproducible data in this, 2D, and 2E panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 805-846 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( B ) Western blot analyses of U2OS WT and SHPRH-KO cells. Immunoblotting was performed with anti-SHPRH, anti-ASCC2, anti-ASCC3, and anti-γ-tubulin antibodies. ( C ) Representative images of ASCC2-EdU PLA foci in HU-treated U2OS WT and SHPRH-KO cells. ( D ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS WT and SHPRH-KO cells from (C). U2OS ASCC2-KO cells were included as a control. A total of 817-822 cells per condition were analyzed. **** P <0.0001. ( E ) Quantification of ASCC2-EdU PLA foci formation in HU-treated U2OS SHPRH-KO cells expressing various Myc-SHPRH alleles as indicated. A total of 827-832 cells per condition were analyzed. **** P <0.001. ( F ) Representative images of ASCC2-EdU PLA foci in no HU- or HU-treated RPE1 parental (WT), A1 (PCNA-K164R), and B1 (PCNA-K164R) cells. ( G ) Quantification of ASCC2-EdU PLA foci formation in RPE1 WT, A1, B1, A1 revertant (A1.R, PCNA-K164K), and B1 revertant (B1.R, K164K) cells that were treated with or without HU. This PLA experiment was done once. A total of 525-540 cells per condition were analyzed. **** P <0.0001. ( H ) Coimmunoprecipitation with anti-HA antibody in UV-irradiated, ATRi-treated and USP1-depleted HEK293T cells expressing the vector alone, HA-tagged ASCC2, or HA-tagged ASCC2 carrying LLL-AAA mutations. Immunoblotting was performed with anti-Ubiquityl-PCNA (K164), anti-PCNA, and anti-HA antibodies.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Transfection, MANN-WHITNEY, Western Blot, Control, Expressing, Irradiation, Plasmid Preparation

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 interacts with RPA and this interaction is further induced by replication stress. ( A ) Anti-Myc coIPs in HEK293T cells transfected with eGFP-ASCC3 together with either the vector alone, Myc-RPA32, or Myc-RPA70. Immunoblotting was done with anti-Myc and anti-GFP antibodies. ( B ) Anti-Myc coIPs in HEK293T cells transfected with HA-RPA70 together with either the vector alone or Myc-ASCC3. Immunoblotting was done with anti-Myc and anti-HA antibodies. ( C ) Anti-Myc coIPs in HEK293T cells transfected with HA-RPA32 together with the vector alone or Myc-ASCC3. Immunoblotting was done with anti-Myc and anti-HA antibodies. ( D ) Coimmunoprecipitations (coIPs) with IgG, anti-ASCC2, or anti-ASCC3 antibodies in HCT116 cells. Immunoblotting was done with anti-ASCC2, anti-ASCC3, anti-RPA70, and anti-RPA32 antibodies. ( E ) Anti-RPA32 coIPs in HCT116 cells. IgG was used as a control. Immunoblotting was performed with anti-RPA32 and anti-ASCC3 antibodies. ( F ) Anti-HA coIPs in no HU- or HU-treated HEK293T cells expressing the vector alone or HA-RPA70. Immunoblotting was done with anti-HA, anti-ASCC2, and anti-ASCC3 antibodies. ( G ) Anti-HA coIPs in HEK293T cells expressing the vector alone or HA-RPA70 in the presence or absence of DNase I. Immunoblotting was done with anti-HA and anti-ASCC3 antibodies. ( H ) Representative images of ASCC3-RPA32 PLA foci formation in no HU- or HU-treated U2OS WT and ASCC3-KO cells. ( I ) Quantification of ASCC3-RPA32 PLA foci formation from (H). The PLA experiments were performed twice independently with reproducible data. Data from one representative experiment are shown as scatter plot graphs with the mean indicated. A total of 402-418 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test. **** P <0.0001.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 unwinds DNA to promote ssDNA-RPA accumulation upon replication stress. ( A ) Quantification of the percentage of cells with ≥ 10 BrdU foci in U2OS WT and ASCC3-KO cells that were pulse-labeled with BrdU for 20 min prior to their treatment with or without 4 mM HU for 4 h. A total of 802-825 cells per condition were scored in blind. SDs from three independent experiments are indicated in this and subsequent panels. *** P <0.001. ( B ) Quantification of the percentage of cells with ≥ 30 BrdU foci in U2OS WT and ASCC3-KO cells that were labeled with BrdU for 20 h prior to their treatment with or without 4 mM HU for 4 h. A total of 800-849 cells per condition were scored in blind. *** P <0.001. ( C ) Quantification of the percentage of cells with ≥ 10 BrdU foci in U2OS ASCC3-KO cells expressing various Myc-ASCC3 alleles as indicated. These cells were pulse-labeled with BrdU for 20 min prior to their treatment with HU. A total of 601-641 cells per condition were scored in blind. *** P <0.001. ( D ) Quantification of the percentage of cells with ≥ 30 BrdU foci in U2OS ASCC3-KO cells expressing various Myc-ASCC3 alleles as indicated. These cells were labeled with BrdU for 20 h prior to their treatment with HU. A total of 502-521 cells per condition were scored in blind. * P <0.05; ** P <0.01. ( E ) Quantification of EdU+ cells with ≥ 30 RPA32 foci in U2OS WT and ASCC3-KO cells that were pulse-labeled with EdU for 10 min prior to treatment with or without 4 mM HU for 6 h. A total of 800-873 cells per condition were scored in blind. *** P <0.001. ( F ) Quantification of the percentage of EdU+ cells with ≥ 30 RPA32 foci in U2OS ASCC3-KO cells expressing various Myc-ASCC3 alleles as indicated. A total of 801-856 cells per condition were scored in blind. *** P <0.001. ( G ) Quantification of the percentage of cells with ≥ 10 BrdU foci in U2OS WT and TRIP4-KO cells that were pulse-labeled with BrdU for 20 min prior to their treatment with or without HU. A total of 601-650 cells per condition were scored in blind. ** P <0.01. ( H ) Quantification of the percentage of EdU+ cells with ≥ 30 RPA32 foci in U2OS WT and TRIP4-KO cells that were pulse-labeled with EdU for 10 min prior to their treatment with or without HU. A total of 742-977 cells per condition were scored in blind. ** P <0.01. ( I ) Schematic diagram of in vitro RPA binding assays. ( J ) Western blot analyses of recombinant RPA70 recovered from streptavidin pulldown of biotinylated ssDNA from indicated reactions. Immunoblotting was done with an anti-RPA70 antibody. ( K ) Quantification of recombinant RPA70 from (J). * P <0.05.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Labeling, Expressing, In Vitro, Binding Assay, Western Blot, Recombinant

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 antagonizes RAD51-mediated recombination. ( A ) Quantification of the CldU/IdU ratio in HU-treated U2OS WT and ASCC3-KO cells expressing control siRNA or siRNA against PRIMPOL (siPRIMPOL). DNA fiber experiments were performed twice independently with reproducible data in this, 6B, and 6C panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this, 6B, and 6C panels. A total of 407-421 fibers per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( B ) Quantification of the CldU/IdU ratio in U2OS WT and ASCC3-KO cells that were treated with HU in the presence or absence of REV1 inhibitor JH-RE-06 (REV1i). A total of 413-414 fibers per condition were analyzed. **** P <0.0001. ( C ) Quantification of the CldU/IdU ratio in HU-treated U2OS WT and ASCC3-KO cells expressing control siRNA or two independent siRNA against RAD51 as indicated. A total of 407-415 fibers per condition were analyzed. **** P <0.0001. ( D ) Quantification of EdU+ cells with ≥ 20 RAD51 foci in U2OS WT and ASCC3-KO cells that were pulse-labeled with EdU for 10 min prior to treatment with or without 4 mM HU for 6 h. A total of 801-853 cells per condition were scored in blind. SDs from three independent experiments are indicated in this, and 6E-6G panels. *** P <0.001. ( E ) Quantification of EdU+ cells with ≥ 20 RAD51 foci in U2OS ASCC3-KO cells expressing various Myc-ASCC3 alleles as indicated. A total of 802-827 cells per condition were scored in blind. ** P <0.01; *** P <0.001. ( F ) Quantification of cells with ≥ 10 IR-induced RAD51 foci in U2OS WT and ASCC3-KO cells that were treated with 10 Gy IR prior to fixation. A total of 503-554 cells per condition were scored in blind. ** P <0.01. ( G ) Quantification of the percentage of cells with restoration of GFP expression following HR-mediated repair of I-SceI-induced DSBs. ** P <0.01. ( H ) Schematic diagram of strand exchange assays. ( I ) In vitro strand exchange assays. Top panel: Recombinant RAD51 (270 nM) was premixed with 1µM of 3’-ssDNA tail for 5 min, followed by further incubation with 350 nM of labeled duplex DNA and increasing concentration (150 nM, 300 nM, 600 nM) of ASCC3 HR WT-TRIP4 or ASCC3 HR -DD-AA-TRIP4 for 15 min. * indicates the 32 P-labeled DNA end. Bottom panel: Quantification of strand exchange efficiency (%) relative to RAD51 alone reaction from top panel. SDs from four independent experiments are shown. * P <0.05; **** P <0.0001.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Expressing, Control, MANN-WHITNEY, Labeling, In Vitro, Recombinant, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: The Ski2 helicase ASCC3 unwinds DNA upon fork stalling to control replication stress responses

doi: 10.1101/2025.07.24.666583

Figure Lengend Snippet: ASCC3 promotes fork reversal upon replication stress. ( A ) Quantification of the percentage of U2OS WT and ASCC3-KO cells with ≥ 10 SMARCAL1 foci. Cells were treated with or without 4 mM HU for 2 h. A total of 501-531 cells per condition were scored in blind. SDs from three independent experiments are indicated in this and 6B panels. *** P <0.001. ( B ) Quantification of the percentage of cells with ≥ 10 SMARCAL1 foci in U2OS ASCC3-KO cells expressing the vector alone, Myc-ASCC3, or Myc-ASCC3-G1354D prior to treatment with HU. A total of 501-537 cells per condition were scored in blind. ** P <0.01. ( C ) Representative images of SMARCAL1-EdU PLA foci formation in no HU- or HU-treated U2OS WT and ASCC3-KO cells. ( D ) Quantification of SMARCAL1-EdU PLA foci formation from (C). The PLA experiments were performed twice independently with reproducible data in this and 5E panels. Data from one representative experiment are shown as scatter plot graphs with the mean indicated in this and subsequent panels. A total of 768-793 cells per condition were analyzed. The P -value was determined using a non-parametric Mann-Whitney rank-sum t -test in this and subsequent panels. **** P <0.0001. ( E ) Quantification of SMARCAL1-EdU PLA foci formation in HU-treated U2OS ASCC3-KO cells expressing the vector alone, Myc-ASCC3, or Myc-ASCC3-G1354D. A total of 612-635 cells per condition were analyzed. **** P <0.0001. ( F ) Representative images of DNA fibers from U2OS WT or ASCC3-KO cells treated with or without HU. ( G ) Quantification of the CldU/IdU ratio from (F). DNA fiber experiments were performed three times independently with reproducible data. A total of 388-418 fibers per condition were analyzed. **** P <0.0001. ( H ) Quantification of the CldU/IdU ratio for HU-treated U2OS ASCC3-KO cells expressing the vector alone, Myc-ASCC3, or Myc-ASCC3-G1354D. DNA fiber experiments were performed twice independently with reproducible data. A total of 333-357 fiber per condition were analyzed. **** P <0.0001. ( I ) Western blot analyses of U2OS WT and ASCC3-KO cells transfected with indicated siRNAs. Immunoblotting was performed with anti-BRCA1 and anti-γ-tubulin antibodies. ( J ) Western blot analysis of U2OS WT and ASCC3-KO cells transfected with indicated siRNA. Immunoblotting was performed with anti-BRCA2 and anti-γ-tubulin antibodies. ( K ) Representative images of DNA fibers from U2OS WT or ASCC3-KO cells transfected with siControl, siBRCA1, or siBRCA2. ( L ) Quantification of the CldU/IdU ratio from (K). DNA fiber experiments were performed twice independently with reproducible data. A total of 400-407 fibers per condition were analyzed. **** P <0.0001.

Article Snippet: Antibodies used include: ASCC2 (1:1000; A304-020A, Bethyl Laboratories); ASCC2 (1:5000; 1152-1-AP, Proteintech); ASCC3 (1:400; 17627-1-AP, Proteintech); ASCC3 (1:1000; PA5-56794, Invitrogen); Biotin (1:100000; A150-109A, Bethyl Laboratory); Biotin (1:100000; 200-002-211, Jackson ImmunoResearch); BrdU (1:100; 347580, BD Biosciences); BrdU (1:500; MAB3222, Millipore); BrdU (BU1/75 [ICR1]) (1:800; NB500-169, Novus Biologicals); 53BP1 (1:2000; 612522, BD Biosciences); BRCA1 (1:5000; 07-434, Millipore); BRCA2 (1:2000; 29450-1-AP, Proteintech); CHK1 (1:250; sc-7898, Santa cruz); CHK1-pS345 (1:1000; 2348S, Cell Signaling); CSB (1:200; ab66598, Abcam); FASN (1:20000; 10624-2-AP, Proteintech); GFP (1:1000; 50430-2-AP, Proteintech); HA (1:500; #2367, Cell Signaling); HLTF (1:1000; A300-229A, Bethyl Laboratory); Myc (1:1000; 9E10, Calbiochem); PCNA (1:2000; sc-56, Santa Cruz); PCNA (1:600 for PLA or 1:4000 for western blot; 10205-2-AP, Proteintech); Ubiquityl-PCNA (K164) (1:1000; 13439T, Cell Signaling); PRIMPOL (1:1000; 29824-1-AP, Proteintech); RAD51 (1:2000, ab63801, Abcam); RFWD3 (1:1000; 19893-1-AP, Proteintech); RPA32 (1:10000; NB100-332, Novus Biologicals); RPA32 (1:200; ab2175, Abcam); RPA32-pS33 (1:50000; A300-246A, Bethyl Laboratories); RPA70 (1:2000; 2267S, Cell Signaling); SHPRH (21995-1-AP, Proteintech); SMARCAL1 (1:100, sc-166209, Santa cruz); SMARCAL1 (1:2000; GTX109468, GenTex); TRIP4 (1:1000; 12324-1-AP, Proteintech); α-tubulin (1:20,000; T9026, Sigma); γ-tubulin (1:20,000; GTU88, Sigma).

Techniques: Expressing, Plasmid Preparation, MANN-WHITNEY, Western Blot, Transfection