Journal: Theranostics

Article Title: A novel USP9X substrate TTK contributes to tumorigenesis in non-small-cell lung cancer

doi: 10.7150/thno.22901

Figure Lengend Snippet: TMT-based quantitative proteomics identifies TTK as a candidate substrate of USP9X. (A) Flow diagram of the TMT-based quantitative proteomics platform applied to identify the substrates of USP9X. A549 cells were stably transfected with 3 different shRNAs targeting USP9X (KD1, KD2, KD3) or control shRNA (Con1, Con2, Con3) and the whole cellular proteins were extracted and quantified. Following trypsin digestion of equal amount of proteins, the resolved peptides were labeled with 6-plex TMT reagents, fractionated by HPLC and analyzed by mass spectrometry. (B) Summary of the TMT labeling assay results. 7471 proteins identified by TMT assay are plotted in the volcano plot, in which the logarithmic ratio of protein intensities in the shUSP9X/control shRNA samples are plotted against negative logarithmic P values of the t-test performed from three replicates. 22 proteins were significantly down-regulated (green), 53 proteins were up-regulated (red) (fold change > 1.5, students' t test P value < 0.05). (C) Decreased USP9X expression correlates with decreased TTK protein level. (D) Validation of protein expression of USP9X and TTK in stably expressing A549 cells by immunoblotting.

Article Snippet: Incubation with antibodies against USP9X (1:100 dilution, Cell Signaling, #14898) or TTK (1:1000 dilution, Merck Millipore, MABE571) was carried out at 4 ºC overnight.

Techniques: Stable Transfection, Transfection, shRNA, Labeling, Mass Spectrometry, Expressing, Western Blot

Journal: Theranostics

Article Title: A novel USP9X substrate TTK contributes to tumorigenesis in non-small-cell lung cancer

doi: 10.7150/thno.22901

Figure Lengend Snippet: USP9X is functionally linked to the stability of TTK. (A) GFP, empty vector control (EV), and flag-TTK were transfected into 293T cells. The cells were extracted for immunoprecipitation with the anti-flag agarose and the proteins were analyzed by immunoblotting. (B) Immunoprecipitated proteins with anti-USP9X antibodies or control IgG from 293T extracts were analyzed by western blotting. Endogenous TTK interacting with USP9X was detected using an anti-TTK antibody. (C) A549 cells were transfected with either two different siRNAs targeting USP9X or a control siRNA. After 72 h, cells were harvested and proteins were analyzed by immunoblotting. (D) The effect of USP9X depletion on TTK in HeLa cells as in (C). (E) Flag-TTK, HA-Ub, siRNA or siUSP9X were co-transfected into 293T cells. The cells were treated with or without MG132 as indicated. Then, cells were extracted for immunoprecipitation with anti-flag agarose and analyzed by western blotting. (F) Flag-TTK, ubiquitin K48-only plasmids, siRNA or siUSP9X were co-transfected into 293T cells. The indicated cells were treated with MG132 overnight. Then, cells were extracted for immunoprecipitation with anti-flag agarose and analyzed by western blotting. (G) A549 cells were transfected with either control or USP9X siRNAs. After 48 h, 200 μg/mL CHX was added and cells were harvested at the indicated times. Protein samples were analyzed by immunoblotting. Quantification of TTK levels relative to β-actin are presented. (H) Half-life analysis of TTK in constructed stable A549 cells. (I-J) HeLa (I) or 293FT (J) cells were transfected with plasmids expressing functional V5-USP9X (WT), catalytically dead C1566S (CS) USP9X or empty vector (EV). After 36 h, 200 μg/mL CHX was added and cells were harvested for immunoblotting.

Article Snippet: Incubation with antibodies against USP9X (1:100 dilution, Cell Signaling, #14898) or TTK (1:1000 dilution, Merck Millipore, MABE571) was carried out at 4 ºC overnight.

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Construct, Expressing, Functional Assay

Journal: Theranostics

Article Title: A novel USP9X substrate TTK contributes to tumorigenesis in non-small-cell lung cancer

doi: 10.7150/thno.22901

Figure Lengend Snippet: Knock down of USP9X or TTK inhibits cell migration and invasion. (A) A549 cells were transfected with either two different siRNAs targeting USP9X or a control siRNA. After 48 h, cell migration was measured by a wound-healing assay. The wound edges are indicated by black lines. Representative images are shown. The quantitative results are shown on the right. The y-axis represents the percentage of wound closure. Data are represented as mean ± s.d. (n = 3, *** P < 0.001, t-test). Scale bars, 300 µM. (B) The migration ability of A549 cells with depletion of TTK examined via the wound-healing assay as in (A). Data are represented as mean ± s.d. (n = 3, *** P < 0.001, t-test). Scale bars, 300 µM. (C) Stable knockdown of either USP9X or TTK decreased A549 cells' invasion ability, which was measured by a transwell assay. The blue dye indicates the transwell cells. The quantitative results by counting cell numbers are shown on the right. Data are represented as mean ± s.d. (n = 3, *** P < 0.001, t-test). Scale bars, 100 µM. (D) Stable knockdown of either USP9X or TTK decreased A549 cells' colony forming ability, which was measured by a colony formation assay. The quantitative results by counting cell numbers are shown on the right. Data are represented as mean ± s.d. (n = 3, *** P < 0.001, t-test). (E) WP1130, an inhibitor of DUBs, could decrease A549 cells' colony forming ability. A549 cells were treated with the indicated drug concentration for the colony formation assay. After two weeks, cells were subjected to crystal violet staining. Data are represented as mean ± s.d. (n = 3, ** P < 0.01, t-test). (F) HeLa cells were induced into mitosis by treating with nocdazole, then the cells were treated with or without WP1130 as the indicated times and the cells were harvested for immunoblotting. Quantification of TTK levels relative to β-actin are shown. (G) Depletion of USP9X or TTK increased A549 cells' sensitivity to WP1130. A549 cells stably expressing shGFP, shUSP9X or shTTK were seeded into 96-well plates and then treated with different concentrations of WP1130 as indicated. The optical density (OD) at 490 nm for each well was detected. Data are represented as mean ± s.d. (n = 5, *** P < 0.001, t-test).

Article Snippet: Incubation with antibodies against USP9X (1:100 dilution, Cell Signaling, #14898) or TTK (1:1000 dilution, Merck Millipore, MABE571) was carried out at 4 ºC overnight.

Techniques: Migration, Transfection, Wound Healing Assay, Transwell Assay, Colony Assay, Concentration Assay, Staining, Western Blot, Stable Transfection, Expressing

Journal: Theranostics

Article Title: A novel USP9X substrate TTK contributes to tumorigenesis in non-small-cell lung cancer

doi: 10.7150/thno.22901

Figure Lengend Snippet: USP9X and TTK knockdown partially suppres cell proliferation and NSCLC . (A) A549 cells stably expressing shGFP, shUSP9X or shTTK were subjected to a cell proliferation assay. Each point represents the mean ± s.d. for biological quintuplicate experiments (n = 5, *** P < 0.001, two-way ANOVA). (B) Tumor images of nude mice (BALB/c; Slac Laboratory Animal, Shanghai, China) with subcutaneous injection of 5×10 6 A549 cells stably transfected with shGFP, shUSP9X or shTTK at day 28 after implantation. (C) Weights of tumors in (B) were measured. Each bar represents the mean ± s.d. for different mice groups (n = 6, *** P < 0.001, one-way ANOVA). (D) Tumor volumes were measured weekly. Each bar represents the mean ± s.d. for different mice groups (n = 6, *** P < 0.001, two-way ANOVA).

Article Snippet: Incubation with antibodies against USP9X (1:100 dilution, Cell Signaling, #14898) or TTK (1:1000 dilution, Merck Millipore, MABE571) was carried out at 4 ºC overnight.

Techniques: Stable Transfection, Expressing, Proliferation Assay, Injection, Transfection

Journal: Theranostics

Article Title: A novel USP9X substrate TTK contributes to tumorigenesis in non-small-cell lung cancer

doi: 10.7150/thno.22901

Figure Lengend Snippet: The expression levels of USP9X and TTK are increased and correlated in NSCLC . (A) Representative immunohistochemical staining for USP9X in NSCLC tissues (n = 55) and matched adjacent normal tissues (n = 55). Scale bar, 50 μm. (B) USP9X expression levels in tumors and normal tissues. (C) TTK expression levels in tumors and normal tissues. Statistical significance in (B-C) was calculated by the chi-square test. R is the Pearson correlation coefficient. (D) Positive correlation between USP9X and TTK expression levels in NSCLC (** P < 0.01, chi-square test). (E) Correlation between USP9X protein expression level and the clinical stages of NSCLC. (F) Correlation between TTK protein expression level and the clinical stages of NSCLC (*** P < 0.001, one-way ANOVA).

Article Snippet: Incubation with antibodies against USP9X (1:100 dilution, Cell Signaling, #14898) or TTK (1:1000 dilution, Merck Millipore, MABE571) was carried out at 4 ºC overnight.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel USP9X substrate TTK contributes to tumorigenesis in non-small-cell lung cancer

doi: 10.7150/thno.22901

Figure Lengend Snippet: USP9X-mediated stabilization of TTK promotes tumorigenesis in NSCLC. TTK is targeted for proteasome-dependent degradation by E3 ubiquitin ligase APC-c. USP9X decreases ubiquitination of TTK, and thereby enhances its half-life. Overexpression of USP9X would increase TTK levels and subsequently promote cell proliferation, migration and invasion. Immunohistochemistry experiments on clinical samples of NSCLC showed that elevated USP9X in NSCLC patients dramatically increases TTK levels and thus promotes tumor growth.

Article Snippet: Incubation with antibodies against USP9X (1:100 dilution, Cell Signaling, #14898) or TTK (1:1000 dilution, Merck Millipore, MABE571) was carried out at 4 ºC overnight.

Techniques: Over Expression, Migration, Immunohistochemistry

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A) Acetylation-mimetic MCL1 K40Q enhances the interaction between MCL1 and USP9X. Shown is IB analysis of WCLs and anti-FLAG immunoprecipitates derived from 293T cells transfected with the indicated constructs. (B) USP9X depletion reverses the ubiquitination levels of acetylation-mimetic MCL1 K40Q. HeLa cells stably expressing the indicated lentiviral shRNA were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated overnight with MG132 (10 μM) before harvesting. (C) Treatment with the USP9X inhibitor reverses ubiquitination of acetylation-mimetic MCL1 K40Q. HeLa cells were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, the cells were treated with MG132 (10 μM) in the presence or absence of WP1130 (5 μM) for 4 h before harvesting. Left: His-Ub-conjugated proteins were captured with Ni-NTA agarose beads. Right: quantification of the Myc-poly-ubiquitination band intensities of IB replicates. Data are presented as mean ± SD, n = 3 independent experiments, *p < 0.05, **p < 0.01. (D) USP9X depletion abolishes stabilization of MCL1 K40Q. HeLa cells stably expressing the lentiviral shRNA specific for GFP or USP9X were transfected with the indicated Myc-MCL1 constructs. 36 h after transfection, cells were treated with 100 μg/mL CHX for the indicated periods before harvesting. (E) Quantification of the Myc band intensities of IB replicates in (D). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01. (F and G) Treatment with the USP9X inhibitor WP1130 efficiently downregulates acetylation-mimetic MCL1 K40Q. Shown is IB analysis of WCLs derived from the MCL1-reintroduced CRISPR-Cas9-mediated MCL1 knockout (KO) HeLa (F) and HCT116 (G) cells presented in . These cells were treated with WP1130 (10 μM) for the indicated periods before harvesting. (H and I) Treatment with the USP9X inhibitor WP1130 abrogates the anti-apoptotic effect of acetylation-mimetic MCL1 K40Q. These cells were treated with the indicated concentrations of WP1130 for 24 h and then subjected to cell viability assays. Data are presented as mean ± SD; n = 3 biological replicates. Data in (A), (B), (F), and (G) are representative of at least two independent experiments. See also .

Article Snippet: Anti-MCL1 (94296), anti-pS64-MCL1 (13297), anti-pS159/T163-MCL1 (4579), anti-pT163-MCL1 (14765), anti-Bcl-2 (4223), anti-Bcl-xL (2764), anti-Acetylated-Lysine (9441), anti-p300 (86377), anti-CBP (7389), anti-PCAF (3378), anti-GCN5L2 (3305), anti-USP9X (14898), anti-SIRT1 (8469), anti-SIRT2 (12650), anti-SIRT3 (5490), anti-Cleaved PARP (5625), anti-Cleaved Caspase-3 (9661), anti-COX IV (4850), anti-Lamin A/C (4777), anti-GFP (2955), polyclonal anti-Myc tag (2278), monoclonal anti-Myc-tag (2276) antibodies, anti-Myc-tag Sepharose beads (3400) were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Transfection, Construct, Stable Transfection, Expressing, shRNA, CRISPR, Knock-Out

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A) SIRT1, SIRT2, and SIRT3 efficiently deacetylate MCL1 in vitro . Shown is IB analysis of MCL1 acetylation after the in vitro deacetylation reaction and WCLs derived from HeLa cells transfected with the indicated Myc-MCL1 and FLAG-SIRT constructs for MCL1 and SIRT protein purification by FLAG and Myc immunoprecipitates . (B) MCL1 interacts with SIRT3 at the endogenous level. Shown is IB analysis of WCLs (input) and anti-MCL1 immunoprecipitates derived from 293T cells. (C) SIRT3 depletion results in the accumulation of MCL1 protein abundance. Shown is IB analysis of WCLs derived from HeLa cells stably expressing the lentiviral shRNA specific for GFP, SIRT1, SIRT2 , or SIRT3 . (D) SIRT3 depletion extends MCL1 protein half-life. HeLa cells stably expressing the lentiviral shRNA specific for GFP or SIRT3 presented in (C) were treated with 100 μg/mL CHX for the indicated period before harvesting. (E) SIRT3 depletion results in impairment of MCL1 poly-ubiquitination. HeLa cells stably expressing the lentiviral shRNA specific for GFP, SIRT1, SIRT2 , or SIRT3 were transfected with Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated with MG132 (10 μM) overnight before harvesting. (F) SIRT3 depletion accumulates endogenous Ac-K40-MCL1. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells stably expressing the indicated lentiviral shRNA specific for GFP or SIRT3 . (G) Ectopic SIRT3 expression increases ubiquitination of WT MCL1 but not K40R. 293T cells were transfected with Myc-MCL1, FLAG-SIRT3, and His-Ub constructs as indicated. 36 h after transfection, the cells were treated with MG132 (20 μM) for 5 h before harvesting. Left: His-Ub-conjugated proteins were captured with Ni-NTA agarose beads. Right: a schematic model of SIRT3-mediated MCL1 ubiquitination through K40 deacetylation followed by USP9X dissociation. (H and I) SIRT3 depletion confers resistance to doxorubicin-induced apoptosis through MCL1 stabilization. CRISPR-Cas9-mediated MCL1 KO and its parental HeLa cells were infected with the lentiviral shRNA specific for GFP or SIRT3 . These cells were treated with the indicated concentrations of doxorubicin for 24 h and then subjected to IB analysis (H) and a cell viability assay (I). Data are presented as mean ± SD; n = 3 biological replicates; **p < 0.01, ***p < 0.001. Data in (A)–(H) are representative of at least two independent experiments. See also .

Article Snippet: Anti-MCL1 (94296), anti-pS64-MCL1 (13297), anti-pS159/T163-MCL1 (4579), anti-pT163-MCL1 (14765), anti-Bcl-2 (4223), anti-Bcl-xL (2764), anti-Acetylated-Lysine (9441), anti-p300 (86377), anti-CBP (7389), anti-PCAF (3378), anti-GCN5L2 (3305), anti-USP9X (14898), anti-SIRT1 (8469), anti-SIRT2 (12650), anti-SIRT3 (5490), anti-Cleaved PARP (5625), anti-Cleaved Caspase-3 (9661), anti-COX IV (4850), anti-Lamin A/C (4777), anti-GFP (2955), polyclonal anti-Myc tag (2278), monoclonal anti-Myc-tag (2276) antibodies, anti-Myc-tag Sepharose beads (3400) were purchased from Cell Signaling Technology.

Techniques: In Vitro, Derivative Assay, Transfection, Construct, Protein Purification, Stable Transfection, Expressing, shRNA, CRISPR, Infection, Viability Assay

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A) MCL1 acetylation levels correlate with p300 expression in breast and prostate cancer cell lines. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from a panel of breast and prostate cancer cell lines. (B) p300 depletion impairs MCL1 protein abundance in breast cancer cells with high p300 and acetylated MCL1 levels. Shown is IB analysis of WCLs derived from breast cancer cell lines stably expressing the lentiviral shRNA specific for GFP or p300 . (C) Treatment with the p300/CBP inhibitor A-485 results in decreased MCL1 protein levels in breast and prostate cancer cells with high p300 and acetylated MCL1 levels. Shown is IB analysis of WCLs derived from breast and prostate cancer cell lines treated with A-485 (3 μM) for 48 h before harvesting. (D) Representative images of MCL1 and p300 expression in breast tumor cells as assessed by immunohistochemistry (IHC). MCL1 and p300 levels were classified as low, moderate, or high, based on the intensities of the IHC staining, and a Spearman correlation test was conducted. Scale bar, 50 μm. See also .) (E and F) Treatment with the p300/CBP inhibitor A-485 reduces MCL1 acetylation and promotes dissociation of USP9X from MCL1. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from SKBR3 (E) and BT-20 (F) treated overnight with the indicated concentrations of A-485 before harvesting. (G and H) The USP9X inhibitor WP1130 effectively induces activation of the apoptotic pathway in cells with higher levels of acetylated MCL1. Shown is IB analysis of WCLs derived from the indicated breast cancer (G) and prostate cancer (H) cell lines. These cells were treated with WP1130 (5 μM) for the indicated periods before harvesting. (I and J) High acetylated MCL1 levels correlate with increased sensitivity to WP1130 in breast and prostate cancer cell lines. Breast cancer (I) and prostate cancer (J) cell lines were treated with the indicated concentrations of WP1130 for 24 h and subjected to cell viability assays. Data are presented as mean ± SD; n = 3 biological replicates; ***p < 0.001. Data in (A)–(C) and (E)–(H) are representative of at least two independent experiments. See also .

Article Snippet: Anti-MCL1 (94296), anti-pS64-MCL1 (13297), anti-pS159/T163-MCL1 (4579), anti-pT163-MCL1 (14765), anti-Bcl-2 (4223), anti-Bcl-xL (2764), anti-Acetylated-Lysine (9441), anti-p300 (86377), anti-CBP (7389), anti-PCAF (3378), anti-GCN5L2 (3305), anti-USP9X (14898), anti-SIRT1 (8469), anti-SIRT2 (12650), anti-SIRT3 (5490), anti-Cleaved PARP (5625), anti-Cleaved Caspase-3 (9661), anti-COX IV (4850), anti-Lamin A/C (4777), anti-GFP (2955), polyclonal anti-Myc tag (2278), monoclonal anti-Myc-tag (2276) antibodies, anti-Myc-tag Sepharose beads (3400) were purchased from Cell Signaling Technology.

Techniques: Expressing, Derivative Assay, Stable Transfection, shRNA, Immunohistochemistry, Activation Assay

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: MCL1 is an unstable protein that is targeted for ubiquitination. p300-directed MCL1 acetylation at K40 leads to enhanced interaction with USP9X, facilitating MCL1 deubiquitination and stabilization. SIRT3 is the potential deacetylase counteracting the acetylation-dependent MCL1 stabilization. Phosphorylation also plays important roles in regulating the interaction with USP9X and certain E3s. Elevated MCL1 acetylation status may correlate with sensitivity to USP9X and p300 inhibitors.

Article Snippet: Anti-MCL1 (94296), anti-pS64-MCL1 (13297), anti-pS159/T163-MCL1 (4579), anti-pT163-MCL1 (14765), anti-Bcl-2 (4223), anti-Bcl-xL (2764), anti-Acetylated-Lysine (9441), anti-p300 (86377), anti-CBP (7389), anti-PCAF (3378), anti-GCN5L2 (3305), anti-USP9X (14898), anti-SIRT1 (8469), anti-SIRT2 (12650), anti-SIRT3 (5490), anti-Cleaved PARP (5625), anti-Cleaved Caspase-3 (9661), anti-COX IV (4850), anti-Lamin A/C (4777), anti-GFP (2955), polyclonal anti-Myc tag (2278), monoclonal anti-Myc-tag (2276) antibodies, anti-Myc-tag Sepharose beads (3400) were purchased from Cell Signaling Technology.

Techniques: Histone Deacetylase Assay

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-MCL1 (94296), anti-pS64-MCL1 (13297), anti-pS159/T163-MCL1 (4579), anti-pT163-MCL1 (14765), anti-Bcl-2 (4223), anti-Bcl-xL (2764), anti-Acetylated-Lysine (9441), anti-p300 (86377), anti-CBP (7389), anti-PCAF (3378), anti-GCN5L2 (3305), anti-USP9X (14898), anti-SIRT1 (8469), anti-SIRT2 (12650), anti-SIRT3 (5490), anti-Cleaved PARP (5625), anti-Cleaved Caspase-3 (9661), anti-COX IV (4850), anti-Lamin A/C (4777), anti-GFP (2955), polyclonal anti-Myc tag (2278), monoclonal anti-Myc-tag (2276) antibodies, anti-Myc-tag Sepharose beads (3400) were purchased from Cell Signaling Technology.

Techniques: Purification, Recombinant, Mutagenesis, Cell Fractionation, Mass Spectrometry, Western Blot, CRISPR, Software