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SPRYD3 is a new interactor of the E3 ligase MYCBP2. A , Flag-SPRYD3 and MYC-MYCBP2 or Flag-MYCBP2 and MYC-SPRYD3 were co-transfected into HEK293T WT cells as indicated and overexpressed for 24 h ( left ) or 48 h ( right ). Cells were harvested for Flag-immunoprecipitation (Flag-IP). Western blot analyses demonstrate reciprocal interaction of ectopically expressed MYCBP2 and SPRYD3. ∗ indicates MYCBP2. B , endogenous SPRYD3 was immunoprecipitated by incubating HEK293T WT cell lysates with anti-IgG ( control ) or anti-SPRYD3 antibodies and Protein A sepharose. Western blot confirms co-immunoprecipitation of endogenous MYCBP2. ∗ indicates MYCBP2 or SPRYD3 respectively. C , recombinant MBP or MBP-SPRYD3 was combined with GST-MYCBP2 2550 to 2825 to perform an MBP pull-down. Western blot analysis confirms direct interaction between recombinant SPRYD3 and MYCBP2 2550 to 2825 ( D ) <t>U2OS</t> or RPE1 cells were transfected twice with 20 nM siRNA targeting GL2 ( control ), SPRYD3 and/or MYCBP2 24 h and 48 h after seeding. 72 h after the first transfection, cells were treated with nocodazole or paclitaxel. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA with Dunette post hoc test, n = 3.

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1) Product Images from "The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly"

Article Title: The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly

Journal: The Journal of Biological Chemistry

doi: 10.1016/j.jbc.2025.110785

Figure Legend Snippet: SPRYD3 is a new interactor of the E3 ligase MYCBP2. A , Flag-SPRYD3 and MYC-MYCBP2 or Flag-MYCBP2 and MYC-SPRYD3 were co-transfected into HEK293T WT cells as indicated and overexpressed for 24 h ( left ) or 48 h ( right ). Cells were harvested for Flag-immunoprecipitation (Flag-IP). Western blot analyses demonstrate reciprocal interaction of ectopically expressed MYCBP2 and SPRYD3. ∗ indicates MYCBP2. B , endogenous SPRYD3 was immunoprecipitated by incubating HEK293T WT cell lysates with anti-IgG ( control ) or anti-SPRYD3 antibodies and Protein A sepharose. Western blot confirms co-immunoprecipitation of endogenous MYCBP2. ∗ indicates MYCBP2 or SPRYD3 respectively. C , recombinant MBP or MBP-SPRYD3 was combined with GST-MYCBP2 2550 to 2825 to perform an MBP pull-down. Western blot analysis confirms direct interaction between recombinant SPRYD3 and MYCBP2 2550 to 2825 ( D ) U2OS or RPE1 cells were transfected twice with 20 nM siRNA targeting GL2 ( control ), SPRYD3 and/or MYCBP2 24 h and 48 h after seeding. 72 h after the first transfection, cells were treated with nocodazole or paclitaxel. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA with Dunette post hoc test, n = 3.

Techniques Used: Transfection, Immunoprecipitation, Western Blot, Control, Recombinant, Live Cell Imaging

USP11 promotes mitotic slippage in presence of MT-poisons. U2OS or RPE1 cells were transfected twice with 20 nM siRNA targeting GL2 ( control ) or USP11 24 h and 48 h after seeding. 72 h after the first transfection, cells were treated with nocodazole or paclitaxel. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA with Dunette post hoc test, n = 3.

Figure Legend Snippet: USP11 promotes mitotic slippage in presence of MT-poisons. U2OS or RPE1 cells were transfected twice with 20 nM siRNA targeting GL2 ( control ) or USP11 24 h and 48 h after seeding. 72 h after the first transfection, cells were treated with nocodazole or paclitaxel. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA with Dunette post hoc test, n = 3.

Techniques Used: Transfection, Control, Live Cell Imaging

SPRYD3-MYCBP2 functions in the same pathway as USP11 to promote bipolar spindle formation. A , U2OS or RPE1 cells were transfected twice with 50 nM siRNA targeting GL2 ( control ), SPRYD3, MYCBP2 or USP11 using two different siRNAs for each protein of interest 24 h and 48 h after seeding. 48 h after the first transfection, cells were incubated with nocodazole for 24 h. After nocodazole wash-out, cells were treated with MG132 for additional 90 min before fixation and staining . For each experiment, 55 (U2OS) or 65 (RPE1) mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ∗ indicates SPRYD3 or MYCBP2 respectively; + indicates unspecific band. B , U2OS cells were co-transfected with Flag-USP11 and MYC-MYCBP2 C4520S or MYC-SPRYD3ΔSPRY2 as indicated. 24 h after transfection, cells were treated with nocodazole and MG132 as described in ( A ). After fixation and staining, 65 mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ∗ indicates MYC-MYCBP2 C4520S. C , U2OS cells were transfected once with a total of 50 nM siRNA targeting GL2 ( control ) or SPRYD3 and MYCBP2 in combination. 24 h after siRNA transfection, cells were transfected with Flag-EV or Flag-USP11 as indicated. 48 h after siRNA transfection, cells were incubated with nocodazole for further 24 h and treated with MG132 as described in ( A ). For each experiment, 65 mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗ p < 0.05, ∗∗p < 0.01. ∗ indicates MYCBP2.

Figure Legend Snippet: SPRYD3-MYCBP2 functions in the same pathway as USP11 to promote bipolar spindle formation. A , U2OS or RPE1 cells were transfected twice with 50 nM siRNA targeting GL2 ( control ), SPRYD3, MYCBP2 or USP11 using two different siRNAs for each protein of interest 24 h and 48 h after seeding. 48 h after the first transfection, cells were incubated with nocodazole for 24 h. After nocodazole wash-out, cells were treated with MG132 for additional 90 min before fixation and staining . For each experiment, 55 (U2OS) or 65 (RPE1) mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ∗ indicates SPRYD3 or MYCBP2 respectively; + indicates unspecific band. B , U2OS cells were co-transfected with Flag-USP11 and MYC-MYCBP2 C4520S or MYC-SPRYD3ΔSPRY2 as indicated. 24 h after transfection, cells were treated with nocodazole and MG132 as described in ( A ). After fixation and staining, 65 mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ∗ indicates MYC-MYCBP2 C4520S. C , U2OS cells were transfected once with a total of 50 nM siRNA targeting GL2 ( control ) or SPRYD3 and MYCBP2 in combination. 24 h after siRNA transfection, cells were transfected with Flag-EV or Flag-USP11 as indicated. 48 h after siRNA transfection, cells were incubated with nocodazole for further 24 h and treated with MG132 as described in ( A ). For each experiment, 65 mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗ p < 0.05, ∗∗p < 0.01. ∗ indicates MYCBP2.

Techniques Used: Transfection, Control, Incubation, Staining

SPRYD3-MYCBP2-mediated ubiquitylation of USP11 C318 promotes bipolar spindle formation and mitotic slippage. A , HEK293T WT cells were co-transfected with Flag-USP11, MYC-MYCBP2 or MYC-SPRYD3 and HA-Ubiquitin as indicated and incubated for 24 h. Cells were harvested for Flag-IP. IP-samples were denatured in 2x Laemmli buffer ± DTT. Western blot analysis demonstrates USP11 ubiquitination status under ± DTT conditions. ∗ indicates MYCBP2. B , Flag-USP11 WT or Flag-USP11 C318S , MYC-MYCBP2 or MYC-SPRYD3 und HA-Ubiquitin were ectopically expressed as indicated in HEK293T WT cells for 24 h. Cells were harvested for Flag-IP. IP-samples were denatured in 2x Laemmli buffer without DTT. Western blot analysis demonstrates USP11 ubiquitination status under -DTT conditions. USP11 ubiquitination status was quantified using Western blot signals after normalization with the respective Flag-IP signal. Flag-USP11 WT was used as 100% reference. Relative values were log-transformed to obtain parametric distribution and applied to one sample t and Wilcoxon test, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ∗ indicates MYCBP2. C , HEK293T WT cells were transfected with Flag-USP11 WT or Flag-USP11 C318S and incubated for 24 h. Cells were harvested for Western blot analysis. Flag-USP11 protein levels were quantified using Western blot signals after α-tubulin-normalization. Flag-USP11 WT was used as reference. Relative values were log-transformed to obtain parametric distribution and applied to one sample t and Wilcoxon test, n = 3. ∗ p < 0.05. D , U2OS cells were transfected with Flag-USP11 WT or Flag-USP11 C318S as indicated. Cells were treated with nocodazole for 24 h and MG132 for additional 90 min prior to harvest . Subsequent to fixation and staining, 65 mitotic cells per condition were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001. E , U2OS cells were transfected twice with 50 nM siRNA targeting GL2 ( control ) or USP11 24 h and 30 h after seeding. 24 h after the first siRNA transfection, U2OS cells were transfected with Flag-USP11 WT siRes or Flag-USP11 C318S siRes as indicated. After 24 h of incubation, cells were treated with nocodazole and MG132 as described in ( D ). Subsequent to fixation and staining, 65 mitotic cells per condition were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ns > 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. F , U2OS cells were transfected with Flag-USP11 WT or Flag-USP11 C318S as indicated and treated with nocodazole or paclitaxel 48 h after transfection. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA with Dunette post hoc test, n = 3. l.e., longer exposure; siRes, siRNA-resistant.

Figure Legend Snippet: SPRYD3-MYCBP2-mediated ubiquitylation of USP11 C318 promotes bipolar spindle formation and mitotic slippage. A , HEK293T WT cells were co-transfected with Flag-USP11, MYC-MYCBP2 or MYC-SPRYD3 and HA-Ubiquitin as indicated and incubated for 24 h. Cells were harvested for Flag-IP. IP-samples were denatured in 2x Laemmli buffer ± DTT. Western blot analysis demonstrates USP11 ubiquitination status under ± DTT conditions. ∗ indicates MYCBP2. B , Flag-USP11 WT or Flag-USP11 C318S , MYC-MYCBP2 or MYC-SPRYD3 und HA-Ubiquitin were ectopically expressed as indicated in HEK293T WT cells for 24 h. Cells were harvested for Flag-IP. IP-samples were denatured in 2x Laemmli buffer without DTT. Western blot analysis demonstrates USP11 ubiquitination status under -DTT conditions. USP11 ubiquitination status was quantified using Western blot signals after normalization with the respective Flag-IP signal. Flag-USP11 WT was used as 100% reference. Relative values were log-transformed to obtain parametric distribution and applied to one sample t and Wilcoxon test, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ∗ indicates MYCBP2. C , HEK293T WT cells were transfected with Flag-USP11 WT or Flag-USP11 C318S and incubated for 24 h. Cells were harvested for Western blot analysis. Flag-USP11 protein levels were quantified using Western blot signals after α-tubulin-normalization. Flag-USP11 WT was used as reference. Relative values were log-transformed to obtain parametric distribution and applied to one sample t and Wilcoxon test, n = 3. ∗ p < 0.05. D , U2OS cells were transfected with Flag-USP11 WT or Flag-USP11 C318S as indicated. Cells were treated with nocodazole for 24 h and MG132 for additional 90 min prior to harvest . Subsequent to fixation and staining, 65 mitotic cells per condition were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001. E , U2OS cells were transfected twice with 50 nM siRNA targeting GL2 ( control ) or USP11 24 h and 30 h after seeding. 24 h after the first siRNA transfection, U2OS cells were transfected with Flag-USP11 WT siRes or Flag-USP11 C318S siRes as indicated. After 24 h of incubation, cells were treated with nocodazole and MG132 as described in ( D ). Subsequent to fixation and staining, 65 mitotic cells per condition were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ns > 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. F , U2OS cells were transfected with Flag-USP11 WT or Flag-USP11 C318S as indicated and treated with nocodazole or paclitaxel 48 h after transfection. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA with Dunette post hoc test, n = 3. l.e., longer exposure; siRes, siRNA-resistant.

Techniques Used: Transfection, Ubiquitin Proteomics, Incubation, Western Blot, Transformation Assay, Staining, Control, Live Cell Imaging

Stable Transfection:

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α
Article Snippet: Blots were then imaged using the LI-COR Odyssey imaging system in 700 and 800 nm channels and processed for brightness and contrast in Adobe Photoshop CC (2019). .. U2OS (HTB-96; ATCC), HCT116 (CCL-247; ATCC), DLD-1 (CCL-221; ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647; ATCC), mouse fibroblast L cells (CRL-2648; ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit [K650001; Thermo Fisher Scientific] and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007; Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in DMEM (Gibco) containing 10% FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza), and 2 mM L-glutamine (Lonza). ..

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α
Article Snippet: Blots were then imaged using the LI-COR Odyssey imaging system in 700 and 800 nm channels and processed for brightness and contrast in Adobe Photoshop CC (2019). .. U2OS (HTB-96, ATCC), HCT116 (CCL-247, ATCC), DLD-1 (CCL-221, ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647, ATCC), mouse fibroblast L cells (CRL-2648, ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit (K650001, Thermo Fisher Scientific) and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007, Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) containing 10 % FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza) and 2 mM L-glutamine (Lonza). ..

Modification:

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α
Article Snippet: Blots were then imaged using the LI-COR Odyssey imaging system in 700 and 800 nm channels and processed for brightness and contrast in Adobe Photoshop CC (2019). .. U2OS (HTB-96, ATCC), HCT116 (CCL-247, ATCC), DLD-1 (CCL-221, ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647, ATCC), mouse fibroblast L cells (CRL-2648, ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit (K650001, Thermo Fisher Scientific) and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007, Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) containing 10 % FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza) and 2 mM L-glutamine (Lonza). ..

Image Search Results

Journal: The Journal of Biological Chemistry

Article Title: The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly

doi: 10.1016/j.jbc.2025.110785

Figure Lengend Snippet: SPRYD3 is a new interactor of the E3 ligase MYCBP2. A , Flag-SPRYD3 and MYC-MYCBP2 or Flag-MYCBP2 and MYC-SPRYD3 were co-transfected into HEK293T WT cells as indicated and overexpressed for 24 h ( left ) or 48 h ( right ). Cells were harvested for Flag-immunoprecipitation (Flag-IP). Western blot analyses demonstrate reciprocal interaction of ectopically expressed MYCBP2 and SPRYD3. ∗ indicates MYCBP2. B , endogenous SPRYD3 was immunoprecipitated by incubating HEK293T WT cell lysates with anti-IgG ( control ) or anti-SPRYD3 antibodies and Protein A sepharose. Western blot confirms co-immunoprecipitation of endogenous MYCBP2. ∗ indicates MYCBP2 or SPRYD3 respectively. C , recombinant MBP or MBP-SPRYD3 was combined with GST-MYCBP2 2550 to 2825 to perform an MBP pull-down. Western blot analysis confirms direct interaction between recombinant SPRYD3 and MYCBP2 2550 to 2825 ( D ) U2OS or RPE1 cells were transfected twice with 20 nM siRNA targeting GL2 ( control ), SPRYD3 and/or MYCBP2 24 h and 48 h after seeding. 72 h after the first transfection, cells were treated with nocodazole or paclitaxel. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ns > 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, one-way ANOVA with Dunette post hoc test, n = 3.

Article Snippet: HEK293T WT (Cat#: ACC 635; DSMZ, Braunschweig), HEK293T ΔFBXO45 (Hoffmann lab, DKFZ Heidelberg), U2OS (HTB-96; ATCC) and U2OS Flp-In-T-REX MYC-SPRYD3 cells were cultured in Dulbecco’s modified Eagle’s medium (Cat#: 41965-039; Gibco). hTERT RPE1 (CRL-4000; ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (Cat#: D8900; Sigma-Aldrich).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Recombinant, Live Cell Imaging

Journal: The Journal of Biological Chemistry

Article Title: The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly

doi: 10.1016/j.jbc.2025.110785

Figure Lengend Snippet: USP11 promotes mitotic slippage in presence of MT-poisons. U2OS or RPE1 cells were transfected twice with 20 nM siRNA targeting GL2 ( control ) or USP11 24 h and 48 h after seeding. 72 h after the first transfection, cells were treated with nocodazole or paclitaxel. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA with Dunette post hoc test, n = 3.

Techniques: Transfection, Control, Live Cell Imaging

Journal: The Journal of Biological Chemistry

Article Title: The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly

doi: 10.1016/j.jbc.2025.110785

Figure Lengend Snippet: SPRYD3-MYCBP2 functions in the same pathway as USP11 to promote bipolar spindle formation. A , U2OS or RPE1 cells were transfected twice with 50 nM siRNA targeting GL2 ( control ), SPRYD3, MYCBP2 or USP11 using two different siRNAs for each protein of interest 24 h and 48 h after seeding. 48 h after the first transfection, cells were incubated with nocodazole for 24 h. After nocodazole wash-out, cells were treated with MG132 for additional 90 min before fixation and staining . For each experiment, 55 (U2OS) or 65 (RPE1) mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ∗ indicates SPRYD3 or MYCBP2 respectively; + indicates unspecific band. B , U2OS cells were co-transfected with Flag-USP11 and MYC-MYCBP2 C4520S or MYC-SPRYD3ΔSPRY2 as indicated. 24 h after transfection, cells were treated with nocodazole and MG132 as described in ( A ). After fixation and staining, 65 mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ∗ indicates MYC-MYCBP2 C4520S. C , U2OS cells were transfected once with a total of 50 nM siRNA targeting GL2 ( control ) or SPRYD3 and MYCBP2 in combination. 24 h after siRNA transfection, cells were transfected with Flag-EV or Flag-USP11 as indicated. 48 h after siRNA transfection, cells were incubated with nocodazole for further 24 h and treated with MG132 as described in ( A ). For each experiment, 65 mitotic cells were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗ p < 0.05, ∗∗p < 0.01. ∗ indicates MYCBP2.

Techniques: Transfection, Control, Incubation, Staining

Journal: The Journal of Biological Chemistry

Article Title: The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly

doi: 10.1016/j.jbc.2025.110785

Figure Lengend Snippet: SPRYD3-MYCBP2-mediated ubiquitylation of USP11 C318 promotes bipolar spindle formation and mitotic slippage. A , HEK293T WT cells were co-transfected with Flag-USP11, MYC-MYCBP2 or MYC-SPRYD3 and HA-Ubiquitin as indicated and incubated for 24 h. Cells were harvested for Flag-IP. IP-samples were denatured in 2x Laemmli buffer ± DTT. Western blot analysis demonstrates USP11 ubiquitination status under ± DTT conditions. ∗ indicates MYCBP2. B , Flag-USP11 WT or Flag-USP11 C318S , MYC-MYCBP2 or MYC-SPRYD3 und HA-Ubiquitin were ectopically expressed as indicated in HEK293T WT cells for 24 h. Cells were harvested for Flag-IP. IP-samples were denatured in 2x Laemmli buffer without DTT. Western blot analysis demonstrates USP11 ubiquitination status under -DTT conditions. USP11 ubiquitination status was quantified using Western blot signals after normalization with the respective Flag-IP signal. Flag-USP11 WT was used as 100% reference. Relative values were log-transformed to obtain parametric distribution and applied to one sample t and Wilcoxon test, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ∗ indicates MYCBP2. C , HEK293T WT cells were transfected with Flag-USP11 WT or Flag-USP11 C318S and incubated for 24 h. Cells were harvested for Western blot analysis. Flag-USP11 protein levels were quantified using Western blot signals after α-tubulin-normalization. Flag-USP11 WT was used as reference. Relative values were log-transformed to obtain parametric distribution and applied to one sample t and Wilcoxon test, n = 3. ∗ p < 0.05. D , U2OS cells were transfected with Flag-USP11 WT or Flag-USP11 C318S as indicated. Cells were treated with nocodazole for 24 h and MG132 for additional 90 min prior to harvest . Subsequent to fixation and staining, 65 mitotic cells per condition were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ∗∗p < 0.01, ∗∗∗p < 0.001. E , U2OS cells were transfected twice with 50 nM siRNA targeting GL2 ( control ) or USP11 24 h and 30 h after seeding. 24 h after the first siRNA transfection, U2OS cells were transfected with Flag-USP11 WT siRes or Flag-USP11 C318S siRes as indicated. After 24 h of incubation, cells were treated with nocodazole and MG132 as described in ( D ). Subsequent to fixation and staining, 65 mitotic cells per condition were analyzed for mitotic spindle pole number. Results were subjected to one-way ANOVA with Dunette post hoc test, n = 3. ns > 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. F , U2OS cells were transfected with Flag-USP11 WT or Flag-USP11 C318S as indicated and treated with nocodazole or paclitaxel 48 h after transfection. Live cell imaging was performed to determine mitotic cell fate of 60 cells. ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA with Dunette post hoc test, n = 3. l.e., longer exposure; siRes, siRNA-resistant.

Techniques: Transfection, Ubiquitin Proteomics, Incubation, Western Blot, Transformation Assay, Staining, Control, Live Cell Imaging

A subset of microtubules has an expanded lattice in cells. (A) Tomogram slice (thickness: 10 nm) showing two selected microtubule (MT) backbones in an untreated U2OS cell (red and pink). (B and C) Power spectra of the masked and transformed MT segments from the red MT (B) and pink MT (C) shown in A. (D) Overlay of the layer line plots of the power spectra of the MT segments from the compacted (red) and expanded (pink) MTs in A. Arrows indicate the location of the layer line peaks and their related lattice spacing. (E) Violin plot showing the distribution of lattice spacings in untreated U2OS cells ( N = 31, 12 tomograms, 7 cells), from microtubules assembled in vitro from GTP-bound soluble tubulin yielding dynamic microtubules ( N = 40, 6 tomograms), in the presence of Taxol ( N = 32, 3 tomograms), or from GMPCPP-bound soluble tubulin ( N = 33, 14 tomograms). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. (F) Simplified cartoon showing the long-range effect of a compacted or an expanded MT lattice. Scale bar: 100 nm (A).

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: A subset of microtubules has an expanded lattice in cells. (A) Tomogram slice (thickness: 10 nm) showing two selected microtubule (MT) backbones in an untreated U2OS cell (red and pink). (B and C) Power spectra of the masked and transformed MT segments from the red MT (B) and pink MT (C) shown in A. (D) Overlay of the layer line plots of the power spectra of the MT segments from the compacted (red) and expanded (pink) MTs in A. Arrows indicate the location of the layer line peaks and their related lattice spacing. (E) Violin plot showing the distribution of lattice spacings in untreated U2OS cells ( N = 31, 12 tomograms, 7 cells), from microtubules assembled in vitro from GTP-bound soluble tubulin yielding dynamic microtubules ( N = 40, 6 tomograms), in the presence of Taxol ( N = 32, 3 tomograms), or from GMPCPP-bound soluble tubulin ( N = 33, 14 tomograms). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. (F) Simplified cartoon showing the long-range effect of a compacted or an expanded MT lattice. Scale bar: 100 nm (A).

Article Snippet: The U2OS Flp-In-T-Rex cell line that upon doxycycline-induction expresses hKif5b(1–560)G234A-mNeongreen-mNeongreen, a kinesin-1 rigor construct, here referred to as StableMARK , was derived from the U2OS Flp-In-T-Rex cell line by transfection with the pCDN5/FRT/TO vector (Invitrogen) and pOG44 vector (Invitrogen).

Techniques: Transformation Assay, In Vitro

Taxol treatment induces a hyperexpanded lattice within cells. (A) Tomogram slice showing a representative image of Taxol-treated microtubules in WT U2OS cells. Scale bar: 100 nm. (B) Violin plot showing the lattice spacing distribution in Taxol treated cells ( N = 30, 6 tomograms, 5 cells) and in untreated cells ( N = 31, 12 tomograms, 7 cells, same data as , included for comparison). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. Taxol distribution is significantly different from the untreated distribution (****P value <0.0001, unpaired t test based permutation test). (C) Microtubule average shows that Taxol-treated microtubules consist of 13 PFs. Central volume slices (28 nm thick) from top (left) and side (right) views. Scale bar: 5 nm.

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Taxol treatment induces a hyperexpanded lattice within cells. (A) Tomogram slice showing a representative image of Taxol-treated microtubules in WT U2OS cells. Scale bar: 100 nm. (B) Violin plot showing the lattice spacing distribution in Taxol treated cells ( N = 30, 6 tomograms, 5 cells) and in untreated cells ( N = 31, 12 tomograms, 7 cells, same data as , included for comparison). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. Taxol distribution is significantly different from the untreated distribution (****P value <0.0001, unpaired t test based permutation test). (C) Microtubule average shows that Taxol-treated microtubules consist of 13 PFs. Central volume slices (28 nm thick) from top (left) and side (right) views. Scale bar: 5 nm.

Techniques: Comparison

Correlation of FM to SEM data using an integrated cryo-FM. (A) Cartoon describing the FM-SEM correlation with FM data obtained after milling. Correlation is confirmed using extracellular beads. (B) FIB image of an intact U2OS cell (9° tilted side view). (C) Untilted SEM image of the same grid square as shown in B. The beads used to confirm FM-SEM correlation are indicated with white and black arrows in C and D, respectively. (D) Untilted SEM image of the polished lamella of the cell shown in C. (E) Scatterplot of correlation errors from leave-one-out calculations; each dataset has a unique color, grey circles mark the 1xSD and 2xSD boundaries (10 datasets, 51 beads), dx = difference in x, dy = difference in y. (F) Boxplot showing the distribution of scaling factors (mean = 0.584, standard deviation = 0.005, N = 10). (G) Scaled FM image of the extracellular beads used to guide FM-SEM overlay, beads used to confirm FM-SEM correlation are indicated with black arrows, similar to C and D. (H) Scaled FM image of fBSA-Au 5 beads used for subsequent FM-TEM correlation (see ). (I) Scaled FM image of the StableMARK signal. Scalebars: 10 µm (B–D and G–I).

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Correlation of FM to SEM data using an integrated cryo-FM. (A) Cartoon describing the FM-SEM correlation with FM data obtained after milling. Correlation is confirmed using extracellular beads. (B) FIB image of an intact U2OS cell (9° tilted side view). (C) Untilted SEM image of the same grid square as shown in B. The beads used to confirm FM-SEM correlation are indicated with white and black arrows in C and D, respectively. (D) Untilted SEM image of the polished lamella of the cell shown in C. (E) Scatterplot of correlation errors from leave-one-out calculations; each dataset has a unique color, grey circles mark the 1xSD and 2xSD boundaries (10 datasets, 51 beads), dx = difference in x, dy = difference in y. (F) Boxplot showing the distribution of scaling factors (mean = 0.584, standard deviation = 0.005, N = 10). (G) Scaled FM image of the extracellular beads used to guide FM-SEM overlay, beads used to confirm FM-SEM correlation are indicated with black arrows, similar to C and D. (H) Scaled FM image of fBSA-Au 5 beads used for subsequent FM-TEM correlation (see ). (I) Scaled FM image of the StableMARK signal. Scalebars: 10 µm (B–D and G–I).

Techniques: Standard Deviation

siRNA-mediated TIM knockdown results in increased DNA damage and ssDNA gap accumulation. ( A ) Schematic of the DNA fiber assay with S1 endonuclease. ( B ) Western blotting (WB) confirming the knockdown of TIM in U2OS cells using two independent siRNA oligonucleotides (versus negative control, Ctrl). ( C ) Dot plot of the DNA fiber IdU track lengths from U2OS cells depleted of TIM by siRNA. Where indicated, 20 U/ml S1 nuclease was treated for 30 min. 10 μM olaparib was treated for 2 h as a positive control to induce DNA gaps. Red bars indicate the median value of at least 150 tracks. n = 3, **** P < 0.0001, ** P < 0.01, Mann–Whitney. ( D ) Dot plot of the DNA fiber IdU track lengths from Flp-In cells reconstituted with Flag-tagged TIM WT or EQ/EQ/TD via siRNA-mediated TIM knockdown and doxycycline (dox)-dependent expression of siRNA-resistant cDNA. Where indicated, cells were treated with 20 U/ml S1 nuclease for 30 min. Red bar: median, n = 3, **** P < 0.0001, ** P < 0.001, ns: not significant, Mann–Whitney. ( E ) Representative images of poly(ADP-ribose) or pADPr signals in U2OS cells knocked down of TIM using two independent siRNA. Where indicated, cells were treated with 10 μM PARGi for 30 min before fixation. Scale bar: 10 μm. ( F ) Quantification of pADPr immunofluorescence signals in either EdU negative or EdU positive cells. At least 250 cells were analyzed in each condition. Red bar: median, n = 3, **** P < 0.0001, ns: not significant, Mann–Whitney. ( G ) WB analysis of cellular pADPr levels in U2OS cells transfected with siRNA TIM (versus Ctrl) in the presence or absence of 10 μM PARG inhibitor (PARGi). ( H ) WB analysis of DNA damage in U2OS cells transfected with siRNA TIM (versus Ctrl) for 72 h.

Journal: Nucleic Acids Research

Article Title: The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

doi: 10.1093/nar/gkae445

Figure Lengend Snippet: siRNA-mediated TIM knockdown results in increased DNA damage and ssDNA gap accumulation. ( A ) Schematic of the DNA fiber assay with S1 endonuclease. ( B ) Western blotting (WB) confirming the knockdown of TIM in U2OS cells using two independent siRNA oligonucleotides (versus negative control, Ctrl). ( C ) Dot plot of the DNA fiber IdU track lengths from U2OS cells depleted of TIM by siRNA. Where indicated, 20 U/ml S1 nuclease was treated for 30 min. 10 μM olaparib was treated for 2 h as a positive control to induce DNA gaps. Red bars indicate the median value of at least 150 tracks. n = 3, **** P < 0.0001, ** P < 0.01, Mann–Whitney. ( D ) Dot plot of the DNA fiber IdU track lengths from Flp-In cells reconstituted with Flag-tagged TIM WT or EQ/EQ/TD via siRNA-mediated TIM knockdown and doxycycline (dox)-dependent expression of siRNA-resistant cDNA. Where indicated, cells were treated with 20 U/ml S1 nuclease for 30 min. Red bar: median, n = 3, **** P < 0.0001, ** P < 0.001, ns: not significant, Mann–Whitney. ( E ) Representative images of poly(ADP-ribose) or pADPr signals in U2OS cells knocked down of TIM using two independent siRNA. Where indicated, cells were treated with 10 μM PARGi for 30 min before fixation. Scale bar: 10 μm. ( F ) Quantification of pADPr immunofluorescence signals in either EdU negative or EdU positive cells. At least 250 cells were analyzed in each condition. Red bar: median, n = 3, **** P < 0.0001, ns: not significant, Mann–Whitney. ( G ) WB analysis of cellular pADPr levels in U2OS cells transfected with siRNA TIM (versus Ctrl) in the presence or absence of 10 μM PARG inhibitor (PARGi). ( H ) WB analysis of DNA damage in U2OS cells transfected with siRNA TIM (versus Ctrl) for 72 h.

Article Snippet: U2OS Flp-In T-REx TIM WT and EQ/EQ/TD cell lines were generated by co-transfecting pcDNA5/FRT/TO Flag-TIM and the pOG44 plasmid encoding the Flp recombinase (Invitrogen) into the host U2OS cell line stably expressing the Tet-repressor (T-REx) and carrying a single FRT locus, followed by hygromycin selection and recovery of stably transfected cells after 4 weeks.

Techniques: Knockdown, Western Blot, Negative Control, Positive Control, MANN-WHITNEY, Expressing, Immunofluorescence, Transfection

Physical disruption of the TIM–PARP1 interaction impairs PARP1-dependent OF processing. ( A ) Schematic depicting a strategy to disrupt the TIM–PARP1 interaction in an inducible manner. The C-terminal PARP1-binding region (PAB) of TIM fused to a destabilization domain (DD) is short-lived via rapid proteasomal degradation but stabilized by a synthetic ligand Shield-1 (Shld1), thus competing with endogenous TIM to disrupt its interaction with PARP1. ( B ) WB to show the induction of myc-tagged DD-PAB wildtype (WT) or EQ/EQ/TD mutant in cells treated with 1 μM Shld1 for the indicated times. ( C ) Representative images of the TIM–PARP1 PLA foci from U2OS DD-PAB WT or EQ/EQ/TD mutant cells following treatment of 1 μM Shld1 for the indicated times. Scale bar: 10 μm. ( D ) Quantification of cells positive for the TIM–PARP1 PLA foci. n = 3, mean ± SD, *** P < 0.001, ns: not significant, Student's t -test. ( E ) Dot plot of the DNA fiber IdU track lengths of DD-TIM-PAB WT or EQ/EQ/TD mutant cells following treatment of 1 μM Shld1 for the indicated times. Red bar: median, n = 2, **** P < 0.0001, ns: not significant, Mann–Whitney. ( F ) Dot plot representing the quantification of S phase-specific pADPr intensity from EdU-positive DD-TIM-PAB WT or EQ/EQ/TD mutant cells treated with 1 μM Shld1 for the indicated times. Red bar: median, n = 2, **** P < 0.0001, * P < 0.05, ns: not significant, Mann–Whitney. ( G ) WB to visualize cellular pADPr levels in DD-TIM-PAB WT or EQ/EQ/TD mutant cells treated with 10 μM FEN1i for 4 h and/or 1 μM Shld1 for 8 h. 10 μM PARGi was treated for 20 min before harvest. ( H ) Representative images of pADPr signals in DD-TIM-PAB WT or EQ/EQ/TD mutant cells transfected with siRNA LIG1 for 66 h and/or treated with 1 μM Shld1 for 8 h. 10 μM PARGi was treated for 20 min before fixation. (I) Dot plot representing S phase-specific XRCC1 foci intensity in EdU-positive DD-TIM-PAB WT or EQ/EQ/TD mutant cells transfected with siRNA LIG1 for 66 h and/or treated with 1 μM Shld1 for 8 h. Red bar: median, n = 2, **** P < 0.0001, * P < 0.05, ns: not significant, Mann–Whitney.

Journal: Nucleic Acids Research

Article Title: The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

doi: 10.1093/nar/gkae445

Figure Lengend Snippet: Physical disruption of the TIM–PARP1 interaction impairs PARP1-dependent OF processing. ( A ) Schematic depicting a strategy to disrupt the TIM–PARP1 interaction in an inducible manner. The C-terminal PARP1-binding region (PAB) of TIM fused to a destabilization domain (DD) is short-lived via rapid proteasomal degradation but stabilized by a synthetic ligand Shield-1 (Shld1), thus competing with endogenous TIM to disrupt its interaction with PARP1. ( B ) WB to show the induction of myc-tagged DD-PAB wildtype (WT) or EQ/EQ/TD mutant in cells treated with 1 μM Shld1 for the indicated times. ( C ) Representative images of the TIM–PARP1 PLA foci from U2OS DD-PAB WT or EQ/EQ/TD mutant cells following treatment of 1 μM Shld1 for the indicated times. Scale bar: 10 μm. ( D ) Quantification of cells positive for the TIM–PARP1 PLA foci. n = 3, mean ± SD, *** P < 0.001, ns: not significant, Student's t -test. ( E ) Dot plot of the DNA fiber IdU track lengths of DD-TIM-PAB WT or EQ/EQ/TD mutant cells following treatment of 1 μM Shld1 for the indicated times. Red bar: median, n = 2, **** P < 0.0001, ns: not significant, Mann–Whitney. ( F ) Dot plot representing the quantification of S phase-specific pADPr intensity from EdU-positive DD-TIM-PAB WT or EQ/EQ/TD mutant cells treated with 1 μM Shld1 for the indicated times. Red bar: median, n = 2, **** P < 0.0001, * P < 0.05, ns: not significant, Mann–Whitney. ( G ) WB to visualize cellular pADPr levels in DD-TIM-PAB WT or EQ/EQ/TD mutant cells treated with 10 μM FEN1i for 4 h and/or 1 μM Shld1 for 8 h. 10 μM PARGi was treated for 20 min before harvest. ( H ) Representative images of pADPr signals in DD-TIM-PAB WT or EQ/EQ/TD mutant cells transfected with siRNA LIG1 for 66 h and/or treated with 1 μM Shld1 for 8 h. 10 μM PARGi was treated for 20 min before fixation. (I) Dot plot representing S phase-specific XRCC1 foci intensity in EdU-positive DD-TIM-PAB WT or EQ/EQ/TD mutant cells transfected with siRNA LIG1 for 66 h and/or treated with 1 μM Shld1 for 8 h. Red bar: median, n = 2, **** P < 0.0001, * P < 0.05, ns: not significant, Mann–Whitney.

Techniques: Disruption, Binding Assay, Mutagenesis, MANN-WHITNEY, Transfection

The TIM–PARP1 interaction is necessary for engaging PARP1 to ssDNA gaps behind replication forks. ( A ) HCT116-mAID cells were treated with 10 μM FEN1i for 6 h and/or 1 μM 5-Ph-IAA for 6 h. Following 125 μM EdU pulse and click reaction to conjugate EdU-labeled forks with biotin, replication fork-associated proteins were isolated by iPOND and analyzed by WB. ( B ) Quantification of the numbers of PARP1-EdU PLA foci from cells containing more than two PLA foci. U2OS DD-PAB WT cells were treated with 1 μM Shld1 for 24 h and 10 μM FEN1i for 6 h followed by a brief pulse with 125 μM EdU for 12 min and chase with 1 mM thymidine for another 10 min to label DNA behind replication forks. 10 μM PARGi was treated during the last 20 min of FEN1i incubation and during the EdU pulse. Dashed lines indicate Q1, median, and Q3. n = 2, **** P < 0.0001, Mann–Whitney. ( C ) Representative images of the PARP1-EdU PLA foci. Scale bar: 10 μm. ( D ) Quantification of the numbers of XRCC1-EdU PLA foci. Cells were treated as (B). Dashed lines indicate Q1, median, and Q3. n = 2, **** P < 0.0001, Mann–Whitney. ( E ) Representative images of the XRCC1-EdU PLA foci. Scale bar: 10 μm.

Journal: Nucleic Acids Research

Article Title: The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

doi: 10.1093/nar/gkae445

Figure Lengend Snippet: The TIM–PARP1 interaction is necessary for engaging PARP1 to ssDNA gaps behind replication forks. ( A ) HCT116-mAID cells were treated with 10 μM FEN1i for 6 h and/or 1 μM 5-Ph-IAA for 6 h. Following 125 μM EdU pulse and click reaction to conjugate EdU-labeled forks with biotin, replication fork-associated proteins were isolated by iPOND and analyzed by WB. ( B ) Quantification of the numbers of PARP1-EdU PLA foci from cells containing more than two PLA foci. U2OS DD-PAB WT cells were treated with 1 μM Shld1 for 24 h and 10 μM FEN1i for 6 h followed by a brief pulse with 125 μM EdU for 12 min and chase with 1 mM thymidine for another 10 min to label DNA behind replication forks. 10 μM PARGi was treated during the last 20 min of FEN1i incubation and during the EdU pulse. Dashed lines indicate Q1, median, and Q3. n = 2, **** P < 0.0001, Mann–Whitney. ( C ) Representative images of the PARP1-EdU PLA foci. Scale bar: 10 μm. ( D ) Quantification of the numbers of XRCC1-EdU PLA foci. Cells were treated as (B). Dashed lines indicate Q1, median, and Q3. n = 2, **** P < 0.0001, Mann–Whitney. ( E ) Representative images of the XRCC1-EdU PLA foci. Scale bar: 10 μm.

Techniques: Labeling, Isolation, Incubation, MANN-WHITNEY

Disruption of the TIM–PARP1 interaction triggers synergistic replication fork instability in cells deficient in the canonical OF processing pathway. ( A ) Quantification of cellular viability measured by ATP-dependent luminescence. TIM-mAID cells were treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM 5-Ph-IAA (versus control) for 3 more days. n = 3, mean ± SD, **** P < 0.0001, *** P < 0.001, Student's t -test. ( B ) Quantification of clonogenic survival of TIM-mAID cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM 5-Ph-IAA (versus control) for 11 more days. n = 3, mean ± SD, **** P < 0.0001, Student's t -test. ( C ) Representative images of colony formation in (B). ( D ) Quantification of cellular viability measured by ATP-dependent luminescence. U2OS DD-PAB WT cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM Shld1 (versus control) for 3 more days. n = 3, mean ± SD, **** P < 0.0001, Student's t -test. (E) Quantification of clonogenic survival of U2OS DD-PAB WT cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM Shld1 (versus control) for 11 more days. n = 3, mean ± SD, **** P < 0.0001, * P < 0.05, Student's t -test. ( F ) Representative images of colony formation in (E). ( G ) TIM-mAID cells were treated with 1 μM 5-Ph-IAA for 48 h and/or 10 μM FEN1i for the first 24 h, and DNA damage and cell death were analyzed by WB. ( H ) As (G) except where indicated cells were treated with 50 μM mirin for 12 h before harvest. ( I ) U2OS DD-PAB WT cells were first treated with 10 μM FEN1i for 24 h and then replenished with media containing 1 μM Shld1 for additional 48 h. Cell lysates were analyzed by WB.

Journal: Nucleic Acids Research

Article Title: The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation

doi: 10.1093/nar/gkae445

Figure Lengend Snippet: Disruption of the TIM–PARP1 interaction triggers synergistic replication fork instability in cells deficient in the canonical OF processing pathway. ( A ) Quantification of cellular viability measured by ATP-dependent luminescence. TIM-mAID cells were treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM 5-Ph-IAA (versus control) for 3 more days. n = 3, mean ± SD, **** P < 0.0001, *** P < 0.001, Student's t -test. ( B ) Quantification of clonogenic survival of TIM-mAID cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM 5-Ph-IAA (versus control) for 11 more days. n = 3, mean ± SD, **** P < 0.0001, Student's t -test. ( C ) Representative images of colony formation in (B). ( D ) Quantification of cellular viability measured by ATP-dependent luminescence. U2OS DD-PAB WT cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM Shld1 (versus control) for 3 more days. n = 3, mean ± SD, **** P < 0.0001, Student's t -test. (E) Quantification of clonogenic survival of U2OS DD-PAB WT cells treated with DMSO or 10 μM FEN1i for the first 24 h followed by 1 μM Shld1 (versus control) for 11 more days. n = 3, mean ± SD, **** P < 0.0001, * P < 0.05, Student's t -test. ( F ) Representative images of colony formation in (E). ( G ) TIM-mAID cells were treated with 1 μM 5-Ph-IAA for 48 h and/or 10 μM FEN1i for the first 24 h, and DNA damage and cell death were analyzed by WB. ( H ) As (G) except where indicated cells were treated with 50 μM mirin for 12 h before harvest. ( I ) U2OS DD-PAB WT cells were first treated with 10 μM FEN1i for 24 h and then replenished with media containing 1 μM Shld1 for additional 48 h. Cell lysates were analyzed by WB.

Techniques: Disruption, Control

Review

u2os flp (ATCC)

Structured Review

Images

1) Product Images from "The E3 ubiquitin ligase SPRYD3-MYCBP2(PAM) regulates mitotic cell fate and ubiquitination of USP11 to control spindle assembly"

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