Journal: eLife

Article Title: GMCL1 controls 53BP1 stability and modulates taxane sensitivity

doi: 10.7554/eLife.106730

Figure Lengend Snippet: ( A ) Dot plots and graphs show the proportions of RPE1 cells in S, G1, and G2/M phases at the indicated time points following mitotic shake-off. Cells were synchronized in mitosis by nocodazole treatment for 16 hr and were subsequently released into fresh medium. Cell cycle distribution was determined by EdU pulse labeling and PI staining. EdU was added 1 hr prior to each indicated time point. Cells had been transfected 48 hr before the experiment with siNT, si GMCL1 , or co-transfected with si GMCL1 and either si USP28 or si TP53BP1 . Error bars represent standard deviation. Differences among four groups were tested by one-way ANOVA followed by Tukey’s multiple comparisons test (* p <0.05, ** p <0.005, **** p <0.0001). ( B ) Representative immunoblot showing the silencing efficiencies for panel ( A ). ( C ) Dot plots and graphs show the proportions of RPE1 cells stably expressing either an empty vector or V5-germ cell-less protein-like 1 (GMCL1), in S, G1, and G2/M phases at the indicated time points following mitotic shake-off. Cells were synchronized in mitosis by nocodazole treatment for 16 hr and were subsequently released into fresh medium. Cell cycle distribution was determined by EdU pulse labeling and PI staining. EdU was added 1 hr prior to each indicated time point. Error bars represent standard deviation. Differences between EV and V5-GMCL1 were tested by a two-tailed Welch’s t test (**** p <0.0001). ( D ) Representative immunoblot showing the overexpression of V5-GMCL1 for panel ( C ). Figure 3—source data 1. This file contains the uncropped, unprocessed original blot images. Figure 3—source data 2. This source data file contains full, uncropped western blot images, with red boxes marking the regions used in the corresponding figures.

Article Snippet: The following antibodies were used: β-actin (1:5000, Sigma-Aldrich A5441), CUL3 (1:1000, Bethyl Laboratories A301-109A), FLAG (1:2000, Sigma-Aldrich F7425), GMCL1 (1:1,000, Proteintech 15575–1-AP), HA (1:2000, Bethyl Laboratories A190-108A), Histone H3 (1:10,000, Abcam, ab1791), p21 (1:1000, Cell Signaling Technology 2947 S), p53 (1:1000, Proteintech 10442–1-AP), pHistone H3 (D2C8) (Ser10, 1:1000, Cell Signaling Technology, #3377), Ubiquitin (p37) (1:1000, Cell Signaling Technology, 58395 S), USP28 (1:1,000, Proteintech 17707–1-AP), 53BP1 (1:2000, Abcam ab36823), α-tubulin (1:5000, Sigma-Aldrich T6074).

Techniques: Labeling, Staining, Transfection, Standard Deviation, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Two Tailed Test, Over Expression

Journal: eLife

Article Title: GMCL1 controls 53BP1 stability and modulates taxane sensitivity

doi: 10.7554/eLife.106730

Figure Lengend Snippet: ( A–G ) MCF7 ( A ), U2OS ( C ), HeLa ( E ), HEC-1-A ( G ) cells were transfected with GMCL1-targeting siRNAs or non-targeting (NT) control for 72 hr. Cells were treated with DMSO or 100 nM paclitaxel for 48 hr, and cell viability was assessed using the CellTiter-Glo Cell Viability Assay from four or six independent measurements. ( B–H ) Apoptosis was measured in the same conditions, i.e . , MCF7 ( B ), U2OS ( D ), HeLa ( F ), HEC-1-A ( H ), using the RealTime-Glo Annexin V Apoptosis and Necrosis Assay from four or six independent measurements. For comparisons between two independent groups ( A–H ), a two-tailed Welch’s test was applied (* p <0.05, ** p <0.005, *** p <0.001). ( I ) hTERT-RPE1 cells were transfected with a non-targeting (NT) control or GMCL1 -targeting siRNAs alone or in combination with either TP53BP1 or USP28 targeting siRNAs for 72 hr, followed by treatment with 100 nM paclitaxel for 48 hr. Cell viability was assessed using the CellTiter-Glo Cell Viability Assay from five independent measurements. ( J ) Apoptosis was measured in the same conditions , i.e . , RPE1 using the RealTime-Glo Annexin V Apoptosis and Necrosis Assay from five independent measurements. Error bars represent standard deviation. For analysis involving four groups ( I and J ), one-way ANOVA followed by Tukey’s multiple-comparisons test was applied (* p <0.05, ** p <0.005, *** p <0.001). ( K ) Schematic model of this study. During prolonged mitosis, GMCL1 promotes degradation of 53BP1, thereby releasing p53 from the 53BP1-p53-USP28 ternary complex and leading to p53 degradation. As a result, daughter cells proceed through the cell cycle. In the absence of GMCL1, excessive accumulation of 53BP1 results in inheritance of the 53BP1-p53-USP28 ternary complex into daughter cells, where p21 expression is induced and cell cycle progression is arrested.

Article Snippet: The following antibodies were used: β-actin (1:5000, Sigma-Aldrich A5441), CUL3 (1:1000, Bethyl Laboratories A301-109A), FLAG (1:2000, Sigma-Aldrich F7425), GMCL1 (1:1,000, Proteintech 15575–1-AP), HA (1:2000, Bethyl Laboratories A190-108A), Histone H3 (1:10,000, Abcam, ab1791), p21 (1:1000, Cell Signaling Technology 2947 S), p53 (1:1000, Proteintech 10442–1-AP), pHistone H3 (D2C8) (Ser10, 1:1000, Cell Signaling Technology, #3377), Ubiquitin (p37) (1:1000, Cell Signaling Technology, 58395 S), USP28 (1:1,000, Proteintech 17707–1-AP), 53BP1 (1:2000, Abcam ab36823), α-tubulin (1:5000, Sigma-Aldrich T6074).

Techniques: Transfection, Control, Viability Assay, Two Tailed Test, Standard Deviation, Expressing

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 1. Usp28 is expressed in intestinal crypts and controls intestinal differentiation and proliferation. (A and B) Expression and localization analysis of USP28 in murine intestine. (A) Usp28fl/fl mice. Boxed regions are shown at higher magnification at right. Bottom right: Usp28 was expressed in crypt base columnar cells (red arrowhead), whereas Paneth cells were devoid of USP28 staining (green arrow). Top right: Usp28 expression gradually decreased within the transit-amplifying cell compartment and was lost in the upper half of the crypt, which contains differentiated cells. (B) Usp28ΔG mice showed no USP28 staining in intestinal tissue. (C) Staining for GFP to label Lgr5+ stem cells in an Lgr5-GFP mouse. (D) Usp28 expression in crypt cells sorted for Lgr5-GFP. (E and F) Representative sections of villi (E) and quantification of goblet cells (AB/PAS+; F), showing increased numbers of goblet cells in Usp28ΔG intestines. n = 10 per group. (G) Representative crypt sections showing mislocalization of Paneth cells (black arrowheads) in Usp28ΔG animals. (H) Quantification of Paneth cells (lysozyme+) per crypt. n = 10 per group. (I and J) Usp28ΔG animals had fewer proliferating cells (BrdU pulse 2.5 hours) within the crypt. n = 10 per group. (K) qRT-PCR of RNA from isolated Usp28ΔG crypt cells showing decreased progenitor and increased differentiation marker gene expression. Expression was normalized to actin and is shown relative to control Usp28fl/fl cells (assigned as 1.0; dashed line). n = 5 per group. Original magnification, ×10 (E); ×20 (C, top); ×40 (A, left; B; C, bottom; G; and I); ×80 (A, right). (D, F, H, J, and K) ***P < 0.0001, *P < 0.001, Student’s t test. Error bars indicate SEM.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Expressing, Staining, Quantitative RT-PCR, Isolation, Marker, Gene Expression, Control

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 2. Usp28ΔG intestinal tissue shows decreased c-JUN and NICD1 as well as c-MYC protein. (A) Western blots of isolated crypt cell lysates from Usp28ΔG intestines show lower USP28, NICD1, phospho– and total c-MYC, and phospho– and total c-JUN proteins compared with Usp28fl/fl controls. (B) Quantification of A. (C) Levels of USP25 and phospho– and total β-catenin were unchanged in crypt cell lysates from Usp28ΔG intestines compared with Usp28fl/fl controls. USP28 and actin are shown as controls. (D) qRT-PCR of RNA from isolated Usp28ΔG crypt cells showing decreased expression of Usp28 (black bars) and c-JUN, NICD1, and c-MYC target genes (white bars) compared with Usp28fl/fl controls, whereas expression of Notch1 and Myc (gray bars) was unaltered. Dll1, which is negatively regulated by Notch signaling, is an example of mRNA increased in Usp28ΔG cells. Expression was normalized to actin and is shown relative to Usp28fl/fl control (assigned as 1.0; dashed line). n = 3 animals per genotype. Error bars indicate SEM.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Western Blot, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 3. c-JUN and NICD1 are deubiquitinated and stabilized by USP28. (A) NICD1, c-MYC, cyclin E1, and c-JUN protein levels were decreased in HCT116 cells transiently transfected with 2 independent shRNA constructs against USP28 (sh-USP28-1 and sh-USP28-2) compared with nontargeting control shRNA (sh-scrambled). Vinculin was used as a loading control. Images are representative of 2 independent experiments. (B) Time course of cycloheximide (CHX) treatment of HCT116 cells transiently transfected with scrambled control shRNA or shRNA targeting USP28. Protein stability of USP28, NICD1, and c-MYC was analyzed at the indicated times by Western blot. CDK2 and vinculin were used as controls. Images are representative of 3 independent experiments. (C) Endogenous USP28 coimmunoprecipitated NICD1, c-MYC, and c-JUN. Images are representative of 3 independent experiments. (D) USP28 promoted deubiquitination of c-JUN and NICD1. HeLa cells transfected with c-JUN or NICD1 together with His-tagged ubiquitin, with or without overexpres- sion of USP28 or the catalytically impaired USP28 C171A, were treated 2 days after transfection with MG132 for 3 hours and lysed, after which protein com- plexes were immunoprecipitated using nickel-NTA beads. Ubiquitinated (Ubi-) complexes were analyzed by Western blot against c-JUN or NICD1. Images are representative of 3 independent experiments.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Transfection, shRNA, Construct, Control, Western Blot, Ubiquitin Proteomics, Immunoprecipitation

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 4. USP28 is commonly overexpressed in human colorectal carcinomas. (A) Heat map of APC, USP28, and c-MYC expression in human intestinal samples from tumors of mucosa and crypt epithelium (adenoma or carcinoma) and the corresponding normal tissue. Data were obtained from Oncomine and Skrzypczak public databases (see Methods). (B) Human colorectal tissue microarray samples from control sections or grade 1 tumor sections stained with antibodies against USP28, c-JUN, and c-MYC. Bottom row shows higher magnifications of the same samples. Original magnification, ×10 (top); ×40 (bottom). (C) Staining intensity was blind-assessed and quantified (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) in tumor sec- tions of grade 1–3 and control (C) tissue sections from the tissue microarray (total 80 samples). *P < 0.05.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Expressing, Microarray, Control, Staining

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 5. USP28 is a c-MYC target gene that is highly expressed in murine as well as human intestinal tumors. (A) H&E- and USP28-stained sections of the small intestine of an Apcmin/+ animal, including adenomas (dashed outlines). Red and green boxed regions denote tumor and control image areas, respectively. USP28 staining was higher in tumor tissue compared with control crypts and villi. Scale bars: 2.5 mm (gut roll); 100 μm (low-power views); 25 μm (high-power views). (B) qRT-PCR of RNA isolated from Apcmin/+ tumors showed increased Usp28 expression compared with WT tissue. n = 5 per group. Error bars indicate SEM. (C) Human USP28 locus, showing the c-MYC binding site (E-box) upstream of the transcription start site. Small arrows indicate primers used for ChIP in D. (D) ChIP from HCT116 cells. c-MYC efficiently bound to the E-box within the USP28 promoter, but not to an unrelated control locus (see Methods). Data are representative of 3 independent experiments. (E) qRT-PCR validation of c-MYC knockdown and effect on USP28 expression. Error bars indicate SEM. Data are representative of 2 independent experiments. (F) Western blot analysis of HCT116 cells 48 or 72 hours after siRNA transfection. Knockdown of c-MYC decreased USP28 protein levels.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Staining, Control, Quantitative RT-PCR, Isolation, Expressing, Binding Assay, Biomarker Discovery, Knockdown, Western Blot, Transfection

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 6. USP28 deficiency ameliorates tumorigenesis in Apcmin/+ animals. (A) Kaplan-Meier diagram showing increased lifespan of Apcmin/+ Usp28ΔG (n = 14) compared with Apcmin/+ Usp28fl/fl (n = 22) mice. (B) Average tumor size in the small intestine at 18 weeks of age was reduced in Apcmin/+ Usp28ΔG mice. n = 5 per group. (C) Number of tumors in the small intestine and colon at 18 weeks of age is reduced in Apcmin/+ Usp28ΔG mice. n = 5 per group. (D) Quantification of BrdU-incorporating cells per mm2 area of tumor section showing decreased proliferation in Apcmin/+ Usp28ΔG tumors. n = 4 per group. (E) Representative sections of tumors (black dashed outline) of the small intestine stained with H&E or AB/PAS to stain goblet cells. Boxed regions are shown at higher magnification. Quantification of goblet cells is also shown. (F) Sections of tumors (white dashed line) within the small intestine of 18 week-old Apcmin/+ Usp28fl/fl and Apcmin/+ Usp28ΔG animals, stained with H&E (left) and USP28 antibody (right, enlarged). Black dashed box indicates enlarged area. (G) Lysates from isolated small intestinal Apcmin/+ tumors show decreases in the indicated proteins in Usp28ΔG tumors when analyzed by western blot- ting. Actin was used as loading control. (H) qRT-PCR of RNA isolated from small intestinal tumors showing expression of genes positively (Hes1, Hes5) and negatively (Dll1) regulated by NICD1. n = 5 per group. Data represent means. Original magnification, ×5 (E, left and middle, and F, left); ×40 (E and F, right). (B–E and H) Where shown, error bars indicate SEM. *P < 0.05, **P < 0.005.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Staining, Isolation, Western Blot, Control, Quantitative RT-PCR, Expressing

Journal: Journal of Clinical Investigation

Article Title: The deubiquitinase USP28 controls intestinal homeostasis and promotes colorectal cancer

doi: 10.1172/jci73733

Figure Lengend Snippet: Figure 7. Inducible Usp28 deletion reduces tumor burden and improves survival in Apcmin/+ animals. (A) Inducible Usp28 deletion. Cre expression was induced by 5 consecutive days of i.p. tamoxifen injection at 110 days of age, when intestinal tumors were already established. R26CreERt, Rosa26 Cre-ERT2. (B) Kaplan-Meier diagram showing survival of tamoxifen-injected Apcmin/+ Usp28fl/fl and Apcmin/+ Usp28fl/fl Rosa26 Cre-ERT2 mice. n = 5 per genotype. (C) Average tumor size in the small intestine at endpoint. n = 5 per group. (D) Quantification of Ki67+ cells per mm2 in tumors at endpoint. (E) AB/PAS+ goblet cells per mm2 in tumors at endpoint. (F) Number of tumors in the small intestines of mice from A postmortem. n = 5 per group. (G) Acute loss of Usp28 reduced cell proliferation in tumors. Representative immunohistological sections of tumors from tamoxifen-treated Apcmin/+ Usp28fl/fl and Apcmin/+ Usp28fl/fl Rosa26 Cre-ERT2 mice, 2 days after acute Usp28 deletion, stained for phospho–histone 3 (pH3). Scale bars: 50 μm. (H) pH3+ cells per mm2 in tumors 2 days after acute Usp28 deletion. (I) Apcmin/+ tumors stained with cleaved caspase 3, showing increased cell death after acute Usp28 deletion. Scale bars: 50 μm. (J) Cleaved caspase 3+ cells per mm2 in tumors 2 days after acute Usp28 deletion. (C–F, H, and J) Error bars indicate SEM. Symbols in D, E, H, and J indicate individual tumors. ***P < 0.001, *P < 0.05.

Article Snippet: Antibodies against USP28 (Sigma-Aldrich, or Ptglab 17707-1-AP), USP25 (Sigma-Aldrich), p53 (NEB), c-JUN (BD Biosciences), phospho–c-JUN(S63) (Cell Signaling Technology), active NICD1 (Abcam), c-MYC (Millipore, OP31), phospho–c-MYC(T58/S62) (Santa Cruz), β-catenin (SigmaAldrich), phospho–β-catenin (S37/T41) (Millipore), and β-actin (SigmaAldrich) were used.

Techniques: Expressing, Injection, Staining