Journal: The Journal of Experimental Medicine

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

doi: 10.1084/jem.20171960

Figure Lengend Snippet: Identification of USP28 as negative regulator of ERK signaling. (A) Third round selection of DUB screen. Immunoblot analysis of 293T cells expressing shRNA vectors targeting the indicated DUBs. ( B) Immunoblot analysis of WM164 melanoma cells treated with indicated concentrations of PLX4032 and probed with the indicated antibodies. (C) Immunoblot analysis of 293T cells expressing shRNA vectors (A–D) targeting USP28 and probed with the indicated antibodies. (D) Immunoblot analysis of 293T cells expressing USP28 shRNA vectors (C and D). Whole cell extracts were probed with the indicated antibodies. (E) Immunoblot analysis in 293T cells expressing Flag-USP28 or Flag-USP28DD. Whole cell extracts were probed with the indicated antibodies. (F) Immunoblot analysis showing 293T cells expressing Myc-FBW7 and BRAF. Whole cell extracts were probed with the indicated antibodies. (G–I) Immunoprecipitation of endogenous USP28 in 293T cells and an immunoblot analysis of indicated proteins BRAF (G), ARAF (H), and CRAF (I). (J) Immunoblot analysis of 293T cells expressing BRAF (V600E), BRAF (V600E) CPD, and wild-type Myc-FBW7 and immunoprecipitated with an anti-BRAF antibody. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.

Article Snippet: The following antibodies were used for immunoblotting: HA 1:1,000 (Y11, Santa-Cruz Biotech); Myc 1:1,000 (9E10, A14, Santa-Cruz Biotech); Flag 1:3,000 (Sigma); phospho-ERK 1:1,000 (T202/Tyr204, 9101, Cell Signaling); ERK1/2 1:1000 (9102, Cell Signaling); USP28 1:1,000 (HPA006778, Sigma); 1:1,000 (ab56900, Abcam); β-actin 1:10,000 (Sigma); BRAF 1:1,000 (F3, Santa-Cruz Biotech); A-RAF 1:1000 (4432, Cell Signaling); Ubiquitin 1:1,000 (P4D1, Santa-Cruz Biotech); C-RAF (9422, Cell Signaling); Bim (2819, Cell Signaling); Cleaved Caspase 3 (9661, Cell Signaling); and Cleaved PARP (9681, Cell Signaling).

Techniques: Selection, Western Blot, Expressing, shRNA, Immunoprecipitation

Journal: The Journal of Experimental Medicine

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

doi: 10.1084/jem.20171960

Figure Lengend Snippet: USP28/FBW7 complex regulates BRAF stability. (A) Representative images of immunoblot analysis of 293T cells expressing BRAF, Flag-USP28, or Flag-USP28 DD. Whole cell extracts were probed with the indicated antibodies. (B) Immunoblot analysis of 293T cells overexpressing Flag-USP28 or Flag-USP28 DD. Whole cell extracts were probed with the indicated antibodies. (C) Immunoblot analysis of 293T cells expressing mutant BRAF (V600E) and Flag-USP28. (D) Immunoblot analysis of 293T cells overexpressing BRAF, Myc-FBW7, or Myc-FBW7(R505L). Whole cell extracts were probed with the indicated antibodies. (E) Immunoblot analysis of 293T cells expressing shRNA vectors against USP28 (C and D). Whole cell extracts were probed with the indicated antibodies. (F) Immunoblot analysis in 293T cells expressing shRNA vectors targeting FBW7. (G) Immunoprecipitation with anti-BRAF resin in 293T cells overexpressing BRAF, Myc-FBW7, and Myc-FBW7 (R505L). Immunoblot analysis of indicated proteins is shown. (H) Immunoprecipitation with anti-BRAF resin in 293T cells expressing BRAF and an shRNA targeting FBW7 treated with proteasome inhibitor, MG132. Immunoblot analysis of indicated proteins is shown. (I) Immunoprecipitation with anti-BRAF in 293T cells expressing BRAF, shRNA vector targeting USP28, and HA-Ub, treated with proteasome inhibitor, MG132. Immunoblot analysis of indicated proteins is shown. (J) Immunoblot analysis of WM164 melanoma cells treated with PLX4032 at indicated concentrations for 18 h. Immunoblot analysis of indicated proteins is shown. (K) Immunoblot analysis of WM164 melanoma cells stably expressing shRNA vectors against USP28 or FBW7 and treated with PLX4032 (5 µM) for 18 h. Whole cell extracts were probed with the indicated antibodies. (L) Graph representing the percentage of BRAF degradation from three independent experiments after vemurafenib treatment as in K. Data shown are representative of three independent and reproducible experiments. Figure G, H, and I were performed in duplicate. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.

Article Snippet: The following antibodies were used for immunoblotting: HA 1:1,000 (Y11, Santa-Cruz Biotech); Myc 1:1,000 (9E10, A14, Santa-Cruz Biotech); Flag 1:3,000 (Sigma); phospho-ERK 1:1,000 (T202/Tyr204, 9101, Cell Signaling); ERK1/2 1:1000 (9102, Cell Signaling); USP28 1:1,000 (HPA006778, Sigma); 1:1,000 (ab56900, Abcam); β-actin 1:10,000 (Sigma); BRAF 1:1,000 (F3, Santa-Cruz Biotech); A-RAF 1:1000 (4432, Cell Signaling); Ubiquitin 1:1,000 (P4D1, Santa-Cruz Biotech); C-RAF (9422, Cell Signaling); Bim (2819, Cell Signaling); Cleaved Caspase 3 (9661, Cell Signaling); and Cleaved PARP (9681, Cell Signaling).

Techniques: Western Blot, Expressing, Mutagenesis, shRNA, Immunoprecipitation, Plasmid Preparation, Stable Transfection

Journal: The Journal of Experimental Medicine

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

doi: 10.1084/jem.20171960

Figure Lengend Snippet: USP28 is down-regulated in melanoma and confers poor prognosis. (A) Oncomine box plot of USP28 in melanoma. (B) Matrix heat map generated using cBioportal showing genetic alterations of BRAF, NRAS, NF1, USP28, and FBW7 in melanoma patients ( n = 287; TCGA). (C) Beeswarm plot demonstrating relative copy number variation of USP28 in melanoma patients, along with their respective mutational status of BRAF (blue), NRAS (red), NF1 (green), and USP28 (X) genes, respectively ( n = 118). (D) Kaplan-Meier curves showing probability of overall survival of melanoma patients with lower copy number of USP28 is significantly less than those with higher level of USP28 (P = 0.05; HR = 8.15). (E) Kaplan-Meier curves showing probability of tumor free survival of melanoma patients with low levels of USP28 is significantly less than those with high levels of USP28 (P = 0.0065; HR = 14.45). (F) Kaplan-Meier survival analysis of melanoma patients harboring BRAF V600E mutation in respect to expression of USP28. Lower expression of USP28 confers poorer overall survival to melanoma patients carrying BRAF 600E mutation (P = 0.046; HR = 3.8).

Article Snippet: The following antibodies were used for immunoblotting: HA 1:1,000 (Y11, Santa-Cruz Biotech); Myc 1:1,000 (9E10, A14, Santa-Cruz Biotech); Flag 1:3,000 (Sigma); phospho-ERK 1:1,000 (T202/Tyr204, 9101, Cell Signaling); ERK1/2 1:1000 (9102, Cell Signaling); USP28 1:1,000 (HPA006778, Sigma); 1:1,000 (ab56900, Abcam); β-actin 1:10,000 (Sigma); BRAF 1:1,000 (F3, Santa-Cruz Biotech); A-RAF 1:1000 (4432, Cell Signaling); Ubiquitin 1:1,000 (P4D1, Santa-Cruz Biotech); C-RAF (9422, Cell Signaling); Bim (2819, Cell Signaling); Cleaved Caspase 3 (9661, Cell Signaling); and Cleaved PARP (9681, Cell Signaling).

Techniques: Generated, Mutagenesis, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

doi: 10.1084/jem.20171960

Figure Lengend Snippet: Down-regulation of USP28 leads to BRAF inhibitor resistance. (A–C) Representative images of immunoblot analysis of BRAF (V600E) mutant melanoma cell lines A373C.6 (A), WM164 (B), and SK-MEL-28 (C) infected with scrambled or USP28 shRNA lentivirus. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (D) Immunoblot analysis of WM164 or WM164 USP28CRSP cells. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (E) Correlation between USP28 and BRAF protein levels in melanoma patients ( n = 98). Statistical significance was determined by an χ 2 test (P = 0.023). R is the correlation coefficient (R=−0.18; top). Immunohistochemical staining of BRAF and USP28 on sequential sections of ME2082B (Biomax) melanoma tissue microarray. Red staining indicates positive immunoreactivity. Bars, 50 µm. Dashed boxes indicate zoomed area. (F) WM164 or WM164 USP28CRSP cells treated with escalating doses of vemurafenib (PLX4032) for 72 h. Viability was assessed using CellTiter Glo as described by the manufacturer. Data represent the mean of six replicates. (G) A373C.6 cells or A373C.6 USP28 knockdown cells treated with escalating doses of vemurafenib (PLX4032) for 72 h. Viability was assessed using CellTiter Glo as described by the manufacturer. Data represent the mean of six replicates. (H) Immunoblot analysis of WM164 or WM164 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 1 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. (I) Immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 1 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. For respective immunoblots proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.

Article Snippet: The following antibodies were used for immunoblotting: HA 1:1,000 (Y11, Santa-Cruz Biotech); Myc 1:1,000 (9E10, A14, Santa-Cruz Biotech); Flag 1:3,000 (Sigma); phospho-ERK 1:1,000 (T202/Tyr204, 9101, Cell Signaling); ERK1/2 1:1000 (9102, Cell Signaling); USP28 1:1,000 (HPA006778, Sigma); 1:1,000 (ab56900, Abcam); β-actin 1:10,000 (Sigma); BRAF 1:1,000 (F3, Santa-Cruz Biotech); A-RAF 1:1000 (4432, Cell Signaling); Ubiquitin 1:1,000 (P4D1, Santa-Cruz Biotech); C-RAF (9422, Cell Signaling); Bim (2819, Cell Signaling); Cleaved Caspase 3 (9661, Cell Signaling); and Cleaved PARP (9681, Cell Signaling).

Techniques: Western Blot, Mutagenesis, Infection, shRNA, Immunohistochemical staining, Staining, Microarray

Journal: The Journal of Experimental Medicine

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

doi: 10.1084/jem.20171960

Figure Lengend Snippet: Down-regulation of USP28 impairs apoptosis induced by vemurafenib. (A) Representative images of immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with 2 µM vemurafenib (PLX4032) for indicated time points. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of two independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls. (B) Representative images of immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with different concentrations of vemurafenib (PLX4032) for 72 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of two independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls. (C) Representative images of cell-cycle analysis of A373C.6 or A373C.6 USP28 knockdown cells after 72 h of treatment with vemurafenib (2 µM). Data shown are representative of three independent and reproducible experiments. (D) Quantification of sub-G 1 population after treatment with vemurafenib as indicated, mean ± SEM of three independent experiments. A two-tailed Student’s t test compares the treated populations; **, P < 0.01.

Article Snippet: The following antibodies were used for immunoblotting: HA 1:1,000 (Y11, Santa-Cruz Biotech); Myc 1:1,000 (9E10, A14, Santa-Cruz Biotech); Flag 1:3,000 (Sigma); phospho-ERK 1:1,000 (T202/Tyr204, 9101, Cell Signaling); ERK1/2 1:1000 (9102, Cell Signaling); USP28 1:1,000 (HPA006778, Sigma); 1:1,000 (ab56900, Abcam); β-actin 1:10,000 (Sigma); BRAF 1:1,000 (F3, Santa-Cruz Biotech); A-RAF 1:1000 (4432, Cell Signaling); Ubiquitin 1:1,000 (P4D1, Santa-Cruz Biotech); C-RAF (9422, Cell Signaling); Bim (2819, Cell Signaling); Cleaved Caspase 3 (9661, Cell Signaling); and Cleaved PARP (9681, Cell Signaling).

Techniques: Western Blot, Cell Cycle Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

doi: 10.1084/jem.20171960

Figure Lengend Snippet: Down-regulation of USP28 impairs the effects of vemurafenib in vivo. (A) Waterfall plot showing the percentage change in tumor volume for the individual tumors at day 15 for untreated controls, day 30 for mice treated twice daily with 35 mg/kg and day 37 for mice treated with 75 mg/kg ( n = 12). (B) Quantification of nude mice bearing xenograft tumors of A373 C.6 or A373 C.6 USP28 knockdown cells ( n = 12). Mice were treated twice daily with vemurafenib (PLX4032; 35 mg/kg, light blue) for 30 d or (75 mg/kg, dark blue) for 37 d (end of experiment). Points indicate mean tumor volume; bars, SE. A two-tailed Student’s t test compares the two treated grouped populations of control cells versus USP28-depleted cells. ****, P < 0.0001. (C) Immunoblot analysis of mouse xenograft A373 C.6 melanoma tumors stably infected with USP28 knockdown vector or relevant controls. Tumor lysates were analyzed with indicated antibodies. Data shown are representative of two independent and reproducible experiments. 1 and 2 indicate two individual mice. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls. (D) Schematic of vemurafenib resistance in USP28 deleted melanoma cells.

Article Snippet: The following antibodies were used for immunoblotting: HA 1:1,000 (Y11, Santa-Cruz Biotech); Myc 1:1,000 (9E10, A14, Santa-Cruz Biotech); Flag 1:3,000 (Sigma); phospho-ERK 1:1,000 (T202/Tyr204, 9101, Cell Signaling); ERK1/2 1:1000 (9102, Cell Signaling); USP28 1:1,000 (HPA006778, Sigma); 1:1,000 (ab56900, Abcam); β-actin 1:10,000 (Sigma); BRAF 1:1,000 (F3, Santa-Cruz Biotech); A-RAF 1:1000 (4432, Cell Signaling); Ubiquitin 1:1,000 (P4D1, Santa-Cruz Biotech); C-RAF (9422, Cell Signaling); Bim (2819, Cell Signaling); Cleaved Caspase 3 (9661, Cell Signaling); and Cleaved PARP (9681, Cell Signaling).

Techniques: In Vivo, Two Tailed Test, Western Blot, Stable Transfection, Infection, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

doi: 10.1084/jem.20171960

Figure Lengend Snippet: Selective synthetic lethality with Rigosertib to USP28 loss. (A) Mean rank-order plot demonstrating fold change from FDA-approved chemical compound screen (316 compounds) in A373C.6 cells versus A373C.6 USP28 knockdown cells. Data represent the mean of three replicates. (B) A373C.6 cells or A373C.6 USP28 knockdown cells treated with escalating doses of rigosertib for 72 h. Viability was assessed using Cell-Titer Glo as described by the manufacturer. Data represent the mean of six replicates. A373 GFP versus A373 USP kd1 ; *, P < 0.0001 (nonlinear regression, extra sum-of-squares test). A373 GFP versus A373 USP kd2 ; *, P < 0.001 (nonlinear regression, extra sum-of-squares test). (C) Representative images of cell-cycle analysis of A373C.6 or A373C.6 USP28 knockdown cells after 48 h of treatment with rigosertib (300 nM). Data shown are representative of three independent and reproducible experiments. (D) Quantification of sub-G 1 population after treatment with rigosertib as indicated, mean ± SEM of three independent experiments. A two-tailed Student’s t test compares the treated populations; **, P < 0.01. (E) Immunoblot analysis of A373C.6 or A373C.6 USP28 knockdown cells treated with rigosertib (300 µM) for 48 h. Whole cell extracts were probed with the indicated antibodies. Data shown are representative of three independent and reproducible experiments. Respective proteins levels were quantified by ImageJ comparing indicated proteins to relevant controls.

Article Snippet: The following antibodies were used for immunoblotting: HA 1:1,000 (Y11, Santa-Cruz Biotech); Myc 1:1,000 (9E10, A14, Santa-Cruz Biotech); Flag 1:3,000 (Sigma); phospho-ERK 1:1,000 (T202/Tyr204, 9101, Cell Signaling); ERK1/2 1:1000 (9102, Cell Signaling); USP28 1:1,000 (HPA006778, Sigma); 1:1,000 (ab56900, Abcam); β-actin 1:10,000 (Sigma); BRAF 1:1,000 (F3, Santa-Cruz Biotech); A-RAF 1:1000 (4432, Cell Signaling); Ubiquitin 1:1,000 (P4D1, Santa-Cruz Biotech); C-RAF (9422, Cell Signaling); Bim (2819, Cell Signaling); Cleaved Caspase 3 (9661, Cell Signaling); and Cleaved PARP (9681, Cell Signaling).

Techniques: Cell Cycle Assay, Two Tailed Test, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Loss of USP28-mediated BRAF degradation drives resistance to RAF cancer therapies

doi: 10.1084/jem.20171960

Figure Lengend Snippet: List of antibodies used in IHC analysis of paraffin sections with related protocols

Article Snippet: The following antibodies were used for immunoblotting: HA 1:1,000 (Y11, Santa-Cruz Biotech); Myc 1:1,000 (9E10, A14, Santa-Cruz Biotech); Flag 1:3,000 (Sigma); phospho-ERK 1:1,000 (T202/Tyr204, 9101, Cell Signaling); ERK1/2 1:1000 (9102, Cell Signaling); USP28 1:1,000 (HPA006778, Sigma); 1:1,000 (ab56900, Abcam); β-actin 1:10,000 (Sigma); BRAF 1:1,000 (F3, Santa-Cruz Biotech); A-RAF 1:1000 (4432, Cell Signaling); Ubiquitin 1:1,000 (P4D1, Santa-Cruz Biotech); C-RAF (9422, Cell Signaling); Bim (2819, Cell Signaling); Cleaved Caspase 3 (9661, Cell Signaling); and Cleaved PARP (9681, Cell Signaling).

Techniques: Blocking Assay, Incubation

Journal: Archives of biochemistry and biophysics

Article Title: Mitochondria-targeted catalase induced cell malignant transformation by the downregulation of p53 protein stability via USP28/miR-200b/PP2A-Cα axis.

doi: 10.1016/j.abb.2024.110047

Figure Lengend Snippet: Fig. 6. mCAT overexpression upregulated USP28 expression, which in turn directly bound to and stabilized c-Jun protein. (A) The relative c-Jun mRNA levels were evaluated by real-time qPCR in Cl41(Vector) and Cl41(mCAT) cells; Gapdh was used as an internal loading control. (B) The expression levels of indicated proteins were analyzed by Western blot in Cl41(GFP-c-Jun/Vector) vs Cl41(GFP-c-Jun/mCAT) cells; β-Actin was presented as the control. (C) c-Jun protein degradation was determined in Cl41(Vector) and Cl41(mCAT) cells in the presence of CHX (50 μg/mL) by Western blot after pretreated with proteasome inhibitor MG-132 (10 μM) for 4 h; GAPDH was used as the protein loading control. (D) The expression levels of indicated proteins were analyzed by Western blot in Cl41(Vector) and Cl41(mCAT) cells; β-Actin was presented as the loading control. (E) Flag-USP28 expression plasmid and its scramble control were transfected into wild-type Cl41 cells. The expression levels of indicated proteins were analyzed by Western blot; β-Actin was used as the protein loading control. (F) shRNA-targeted USP28 and nonsense control vector plasmids were transfected into Cl41(mCAT) cells. The expression levels of indicated proteins were analyzed by Western blot; β-Actin was presented as the control. (G) Co-immunoprecipitation was performed with anti-GFP mAb Agarose in the indicated cells. Immunoprecipitates were then subjected for the detection of USP28. (H) c-Jun protein degradation was determined in Cl41(mCAT/shNon) and Cl41(mCAT/shUSP28) cells in the presence of CHX (50 μg/mL) by Western blot after pretreated with proteasome inhibitor MG-132 (10 μM) for 4 h; GAPDH was used as the protein loading control.

Article Snippet: The antibodies used in this study which were specific against p53 (2524S), p-MDM2 (Ser166) (3521), PP2A-A (2041), PP2A-B (2290), PP2A-C (2259), AKT1 (2938S), AKT2 (3063S), p-AKT (Thr308) (2965S), p-AKT (Thr473) (4060S), JNK1/2 (9152S), p38 (8090S), p-p38 (9211S), ERK1/2 (46953), p-ERK1/2 (4370S), p-IKKα/β (2697P), c-Jun (9165S), p-c-Jun (Ser63) (2361S), p-cJun (Ser73) (3270S), USP4 (2651), Elk1 (9182S), FOXO3a (2497S), cFos (4384), STAT5 (94205) and p-STAT5 (9359) were purchased from Cell Signaling Technology (Boston, USA); antibodies targeting catalase (21260-1-AP), USP8 (27791-1-AP), USP28 (17707-1-AP), ITCH (20920- 1-AP), GAPDH (60004-1-1g) and ACTB (99009-1-Ig) were bought from Proteintech (Wuhan, China); antibody targeting COX-2 (160116) was purchased from Cayman (Ann Arbor, USA), antibody targeting p-PP2AC (Tyr307) (#1155-1) was bought from Epitomics (Burlingame, USA), antibody targeting p-JNK1/2 (44-682G) was purchased from Invitrogen (Carlsbad, USA), and antibody targeting FBW7 (ARP47419_P050) was bought from Aviva Systems Biology (San Diego, USA).

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Western Blot, Transfection, shRNA, Immunoprecipitation

Journal: Cellular and Molecular Life Sciences

Article Title: USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells

doi: 10.1007/s00018-024-05187-2

Figure Lengend Snippet: USP28 regulates MAST1 protein levels. A Transfection of an entire set of sgRNAs targeting individual USP subfamily genes along with Cas9 nuclease into cisplatin-resistant HeLa cells (HeLa-cisR). Transfected cells were treated with a sub-lethal dose (5 µg/mL) of cisplatin. HeLa-cisR cells treated with saline served as the negative control (vehicle) and cisplatin-treated HeLa-cisR cells co-transfected with scrambled sgRNA and Cas9 served as the mock control. Cisplatin-induced cell death was estimated using a cell viability assay and represented as a graph. Data are presented as the mean and standard deviation of three independent experiments (n = 3). B The USP28-depleted cells were treated with an increasing concentrations of cisplatin (5 µg/mL, 10 µg/mL, 15 µg/mL, 20 µg/mL and 25 µg/mL) for 48 h, and cell viability was measured. The IC 50 values of cisplatin in HeLa-CisR mock and USP28-sgRNA transfected cells were 6.32 µg/mL and 3.97 µg/mL, respectively. C Schematic representation of the sgRNAs targeting exon 2 of USP28 gene. The red arrowheads indicate the positions of sgRNAs target site on the sense DNA strand. PAM sequences are indicated in bold blue font; USP28 sgRNA sequences are indicated in red font. D The validation of efficiency of sgRNAs targeting USP28 by transient co-transfection with Cas9 in HEK293 cells and immunoblotting with USP28 antibody. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control (USP28/GAPDH) and presented below the blot. The effect of depleting USP28 on endogenous MAST1 protein was estimated in HEK293 cells. E Validation of sgRNA efficiency targeting USP28 gene by transient co-transfection with Cas9 and sgRNA1 or sgRNA2 into HEK293 cells followed by a T7E1 assay to determine the cleavage efficiency. The cleaved band intensity (indel %) obtained by T7E1 assay was measured using ImageJ software and indicated. Scrambled sgRNA transfected HEK293 cells were used as a control cells. The black arrowhead indicates the cleaved PCR amplicons. A549 cells were transfected with increasing concentrations of F Flag-USP28 and G Flag-USP28CA to validate its effect on endogenous MAST1 protein levels. H The effect of reconstitution of Flag-USP28 on endogenous MAST1 protein in USP28-depleted A549 cells was validated. The protein band intensities for F – H were estimated using ImageJ software with reference to the GAPDH control band (MAST1/GAPDH) and presented below the blot. HEK293 cells were transfected with constant amount of Myc-MAST1 and increasing concentrations of I Flag-USP28 and J Flag-USP28CA to validate its effect on exogenous Myc-MAST1 protein levels. K The effect of reconstitution of Flag-USP28 on Myc-MAST1 protein in USP28-depleted HEK293 cells was validated. The protein band intensities for I – K were estimated using ImageJ software with reference to the GAPDH control band (Myc-MAST1/GAPDH) and presented below the blot

Article Snippet: Flag-tagged USP28 (Addgene #15665) and HA-tagged Ubiquitin (Addgene #18712) plasmids were purchased from Addgene.

Techniques: Transfection, Saline, Negative Control, Viability Assay, Standard Deviation, Cotransfection, Western Blot, Software

Journal: Cellular and Molecular Life Sciences

Article Title: USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells

doi: 10.1007/s00018-024-05187-2

Figure Lengend Snippet: USP28 interacts with MAST1 and extends its half-life. A The interaction between endogenous USP28 and MAST1 protein was analyzed in A549 by immunoprecipitation and immunoblotting using the specific antibodies. B Interaction between ectopically expressed USP28 and MAST1 was analyzed in HEK293 cells. Cells lysates were immunoprecipitated using Myc or Flag antibodies and analyzed by western blot. GAPDH was used as a loading control. C A549 cells were subjected to the Duolink PLA assay to analyze the interaction between USP28 and MAST1 using specific antibodies. The in situ USP28-MAST1 interaction (red PLA dots) was observed when USP28 and MAST1 were immunostained together but not when they were stained with individual antibodies. Scale bar: 10 µm. D–E The effect of USP28 and USP28CA on the half-life of Myc-MAST1 in HEK293 ( D ) and endogenous MAST1 ( E ) protein in A549 cells. CHX (150 μg/mL) was administered for the indicated time, and the cells were then harvested for western blotting with the indicated antibodies. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control. Data are presented as the mean and standard deviation of three independent experiments (n = 3). A two-way ANOVA followed by Tukey's post hoc test was used, and P values are indicated

Article Snippet: Flag-tagged USP28 (Addgene #15665) and HA-tagged Ubiquitin (Addgene #18712) plasmids were purchased from Addgene.

Techniques: Immunoprecipitation, Western Blot, In Situ, Staining, Software, Standard Deviation

Journal: Cellular and Molecular Life Sciences

Article Title: USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells

doi: 10.1007/s00018-024-05187-2

Figure Lengend Snippet: USP28 deubiquitinates MAST1 protein. The ubiquitination and deubiquitination of ectopically expressed Myc-MAST1 were analyzed in HEK293 cells. A The HEK293 cells were transfected with Myc-MAST1 and HA-Ub in a constant amount. Flag-USP28 was transfected in an increasing concentration, followed by immunoprecipitation with Myc antibody and immunoblotting with anti-HA antibody. B The ubiquitination and deubiquitination of ectopically expressed Myc-MAST1 were analyzed by transfecting HEK293 cells with Flag-USP28 and Flag-USP28CA or treatment with DUB-inhibitor PR-619 for 48 h prior to harvest in the HEK293 cells. The cells were harvested, followed by IP with a Myc antibody and immunoblotting with an anti-HA antibody. C The ubiquitination and deubiquitination of ectopically expressed Myc-MAST1 were analyzed by transfecting HEK293 cells with sgRNAs targeting USP28. The cells were harvested, followed by IP with a Myc antibody and immunoblotting with an anti-HA antibody. A – C The relative protein expression of MAST1-(Ub)n with respect to input MAST1 was quantified using ImageJ software and represented as (MAST1-(Ub)n/MAST1) below the blot. D Sanger sequencing data showing the disruption in USP28 gene sequence in A549 (upper panel) and H1299 cells (lower panel). The effect of USP28-KO on the mRNA expression of E USP28 and F MAST1 was evaluated by qRT-PCR with specific primers. The relative mRNA expression levels are shown after normalization to GAPDH mRNA expression. Data are presented as the mean and standard deviation of three independent experiments (n = 3). A two tailed t-test was used, and P values are indicated. G Flow cytometry assay showing the expression of USP28 in mock control vs. USP28-KO in A549 cells (left panel) and H1299 cells (right panel). H Western blot analysis of the endogenous expression of USP28 and MAST1 protein in A549 and H1299 USP28-KO was evaluated. GAPDH was used as the internal loading control. I The TUBEs assay was performed to assess the ubiquitination status of the MAST1 protein in mock control and USP28-KO clones from A549 and H1299 cells. Cell lysates were immunoprecipitated with TUBEs beads, followed by immunoblotting with the indicated antibodies. J–K The effect of USP28-KO on the half-life of MAST1 in A549 cells. The mock control, USP28-KO and USP28-KO cells reconstituted with ( J ) Flag-USP28 and K Flag-USP28CA was treated with CHX (150 μg/mL) for the indicated time, and the cells were then harvested for western blotting with the indicated antibodies. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control

Article Snippet: Flag-tagged USP28 (Addgene #15665) and HA-tagged Ubiquitin (Addgene #18712) plasmids were purchased from Addgene.

Techniques: Transfection, Concentration Assay, Immunoprecipitation, Western Blot, Expressing, Software, Sequencing, Disruption, Quantitative RT-PCR, Standard Deviation, Two Tailed Test, Flow Cytometry, Clone Assay

Journal: Cellular and Molecular Life Sciences

Article Title: USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells

doi: 10.1007/s00018-024-05187-2

Figure Lengend Snippet: Correlation between USP28 and MAST1 expression in various cancers tissues. A Box plot showing difference between USP28 expression in tumor and normal tissues in LUSC, CESC, LIHC and ESCA cancer types. B Box plot showing difference between MAST1 expression in tumor and normal tissues in LUSC, CESC, LIHC and ESCA cancer types. The box plots (A-B) were generated using online bioinformatics tool GEPIA 2 ( http://gepia2.cancer-pku.cn/#index ). C A heat map showing mRNA expression levels of USP28 and MAST1 in different cancer cell lines derived from the CCLE database. Representative samples are arranged from high to low mRNA levels of USP28, and corresponding MAST1 values are sorted. D A scatterplot showing the expression correlation between USP28 and MAST1 mRNA levels in different cancer cell lines derived from the CCLE database. Pearson correlations (r) quantifying the relationship between USP28 and MAST1 are given. E Endogenous protein expression patterns of USP28 and MAST1 in different cancer and non-cancer cell lines were assessed by Western blotting. GAPDH was used as the loading control. F–H Representative immunohistochemical (IHC) staining images of endogenous USP28 and MAST1 in F human lung cancer (n = 27), G breast cancer (n = 18) and H colon cancer (n = 24) tissues. All IHC images were quantified with an H-score and difference in expression of MAST1 and USP28 in normal and tumor samples was represented graphically. Scale bar = 30 µm

Article Snippet: Flag-tagged USP28 (Addgene #15665) and HA-tagged Ubiquitin (Addgene #18712) plasmids were purchased from Addgene.

Techniques: Expressing, Generated, Derivative Assay, Western Blot, Immunohistochemical staining, Immunohistochemistry

Journal: Cellular and Molecular Life Sciences

Article Title: USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells

doi: 10.1007/s00018-024-05187-2

Figure Lengend Snippet: Loss of USP28 suppresses cell viability and promotes DNA damage and apoptosis. Mock control, USP28-KO, and USP28-KO cells reconstituted with either USP28 or MAST1 were used to perform the following experiments. A Western blot analysis to validate the expression of USP28 and MAST1 using USP28- and MAST1-specific antibodies in A549 cells. The cells from A were subjected to the following experiments. B A549 cells were treated with an increasing concentration of cisplatin (5 µg/mL, 10 µg/mL, 15 µg/mL, 20 µg/mL and 25 µg/mL) for 48 h, and cell viability was assayed using CCK-8 reagent. Data are presented as the mean and standard deviation of three independent experiments (n = 3). The IC 50 values of cisplatin in A549 mock, USP28-KO, and USP28-KO reconstituted with USP28 and USP28-KO reconstituted with MAST1 were 3.56 µg/mL, 2.13 µg/mL, 3.36 µg/mL, and 3.44 µg/mL, respectively. C A549 cells were treated with cisplatin (2 µg/mL) for 48 h and subjected to flow cytometry to measure the DNA content using PI staining and Data are presented as the mean and standard deviation of three independent experiments (n = 3). D A549 cells were treated with either vehicle or Cisplatin (2 µg/mL) for 48 h and subjected to immunofluorescence analysis to estimate γH2AX foci formation. Green, γH2AX; blue, nucleus stained by DAPI. Scale bar = 100 µm. The right panel depicts the percentage of γH2AX-positive cells. Data are presented as the mean and standard deviation of three independent experiments (n = 3). E A549 cells treated with cisplatin (2 µg/mL) for 48 h were subjected to immunoblotting analysis with the indicated antibodies. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control (γ-H2AX/GAPDH) and presented below the blot. F A549 cells were treated with the indicated concentrations of USP28 inhibitor (AZ1) with either vehicle or cisplatin (2 µg/mL) for 48 h and subjected to immunofluorescence analysis to estimate γH2AX foci formation. Green, γH2AX; blue, nucleus stained by DAPI. Scale bar = 100 µm. The right panel depicts the percentage of γH2AX-positive cells. Data are presented as the mean and standard deviation of three independent experiments (n = 3). G A549 cells were treated with cisplatin (2 µg/mL) for 48 h. Flow cytometry analysis was performed to analyze annexin-V and PI positive cells and graphically represented. Data are presented as the means and standard deviations of 3 independent experiments. H The effect of USP28 depletion or USP28 inhibitor (AZ1) on MEK pathway in A549 cells treated with cisplatin (2 µg/mL) by western blotting with indicated antibodies. GAPDH was used as the internal loading control

Article Snippet: Flag-tagged USP28 (Addgene #15665) and HA-tagged Ubiquitin (Addgene #18712) plasmids were purchased from Addgene.

Techniques: Western Blot, Expressing, Concentration Assay, CCK-8 Assay, Standard Deviation, Flow Cytometry, Staining, Immunofluorescence, Software

Journal: Cellular and Molecular Life Sciences

Article Title: USP28 promotes tumorigenesis and cisplatin resistance by deubiquitinating MAST1 protein in cancer cells

doi: 10.1007/s00018-024-05187-2

Figure Lengend Snippet: Loss of US28 inhibits tumorigenesis in vitro and in vivo. Mock control, USP28-KO, and USP28-KO cells reconstituted with either USP28 or MAST1 were used to perform the following experiments. The cells from (5A) were treated with either vehicle or cisplatin and were subjected to the following experiments . A Colony formation was measured after 14 days in A549 cells . The colony numbers were quantified and are presented graphically. Scale bar, 500 µm. B The transwell cell invasion assay was performed with the groups mentioned in A549 cells. The number of invaded cells were quantified using ImageJ software and represented graphically. Data are presented as the means and standard deviations of 3 independent experiments. Scale bar, 100 µm. C The migration potential of above mentioned groups was assessed by an in vitro scratch assay in A549 cells. The migration potential was quantified by ImageJ software and are presented graphically. Scale bar, 200 µm. Data are presented as the means and standard deviations of 3 independent experiments. D Xenografts were generated by subcutaneously injecting the mentioned cell groups into the right flanks of NSG mice (n = 4/group). Mice were i.p. injected with either saline (vehicle) or cisplatin (2 mg/kg) twice a week beginning 7 days after xenograft implantation, and tumor size was monitored. Tumor volumes were recorded, and tissues were stored for IHC experiments. The right panel shows the tumors excised from the mice after the experiment. E Tumor volume and tumor weight were measured and are presented graphically. Data are presented as the mean and standard deviation (n = 4 mice per group). Statistical power is 80% and was calculated post experiment using G*Power software. Two-way ANOVA followed by Tukey's post-hoc test was used, and the exact P values are indicated on the figures ( P < 0.05, P < 0.01, P < 0.001, P < 0.0001 were considered as significant, and P > 0.05 considered as non-significant). F Xenograft tumors were embedded in paraffin and sectioned. IHC analyses were performed with the indicated antibodies. Scale bar = 30 µm

Article Snippet: Flag-tagged USP28 (Addgene #15665) and HA-tagged Ubiquitin (Addgene #18712) plasmids were purchased from Addgene.

Techniques: In Vitro, In Vivo, Invasion Assay, Software, Migration, Wound Healing Assay, Generated, Injection, Saline, Standard Deviation