Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A and B ) After 48 h of transfection, the scratch wound-healing assay was performed to evaluate the effect of USP22 on cell migration. The representative images of cell migration were shown in (A), and the width of wounds at the indicated times was shown in (B). ( C ) Invasion of CAL-62 and 8505C cells analyzed by transwell assay. The invaded cells were stained with DAPI. Scale bar: 5 μm. ( D ) Number of DAPI-positive cells per field counted under a fluorescent microscopy. ( E ) Immunofluorescence staining was performed to analyze the effect of USP22 on the expression of E-cadherin and vimentin in 8505C cells. Scale bar: 5 μm. ( F and G ) Expression of invasion-related proteins containing BMI-1, E-cadherin, vimentin, and snail in ATC cells was measured by qPCR (F) and western blot (G). GAPDH and β-actin were used as endogenous controls, respectively. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with the group at 0 h after transfection (B). ## P < 0.01, as compared with mock or pSilencer group at 24 h after transfection (B). * P < 0.05, as compared with mock or pSilencer group (D and F).

Article Snippet: Blotting was performed with primary antibodies targeting USP22, Akt, phosphorylated (p)-Akt (Ser473), Rb, and p-Rb (Ser811) (all from Cell Signaling Technology, Danvers, MA, USA), cyclin D2, BMI-1, and E-cadherin (all from Abcam, Cambridge, UK), vimentin, snail, Bid, Bax, and Bcl-2 (all from Abnova, Taiwan, China), cl-caspase-3, caspase-3, and β-actin (all from Sigma), followed by horseradish peroxidase-conjugated secondary antibody (Sigma).

Techniques: Transfection, Plasmid Preparation, Wound Healing Assay, Migration, Transwell Assay, Staining, Microscopy, Immunofluorescence, Expressing, Western Blot

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: Female six-week-old SCID mice were inoculated subcutaneously into right hind flanks or injected via tail vein with stably expressed pLKO.1 or pLKO.1-shUSP22 8505C-luc cells. Mock-treatment was used as control. ( A ) Tumor growth progression was measured by in vivo luciferase imaging of the xenografts at days 1, 5, 9, and 12 d after inoculation. ( B ) Representative gross photos of tumors 22 d after subcutaneous xenografting ( n = 6). ( C ) Tumor volumes of subcutaneous implantation models of ATC were monitored and calculated after 0, 7, 11, 13, 17, 20, and 22 d of inoculation. D–F. After 28 d of injection through tail vein, the lungs were removed and photographed ( D ), the number of metastatic nodules in lungs was counted ( E ), and the weight of lungs was measured ( F ). ( G ) Fluorescence TUNEL assay was carried out to determine cell apoptosis in the same tumor tissues as indicated above. The rate of TUNEL-positive cells was calculated. Scale bar: 10 μm. ( H ) qPCR assay was performed to detect the mRNA expression of USP22 in tumor tissues from (B). ( I ) Protein expression of USP22 in tumor tissues was analyzed by western blot. ( J ) Representative results of western blot analyses of cyclin D2, Akt, p-Akt, E-cadherin, vimentin, Bax, Bcl-2, cl-caspase-3, and caspase-3 in tumor tissues. (I and J) β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pLKO.1 group (C and E–H).

Article Snippet: Blotting was performed with primary antibodies targeting USP22, Akt, phosphorylated (p)-Akt (Ser473), Rb, and p-Rb (Ser811) (all from Cell Signaling Technology, Danvers, MA, USA), cyclin D2, BMI-1, and E-cadherin (all from Abcam, Cambridge, UK), vimentin, snail, Bid, Bax, and Bcl-2 (all from Abnova, Taiwan, China), cl-caspase-3, caspase-3, and β-actin (all from Sigma), followed by horseradish peroxidase-conjugated secondary antibody (Sigma).

Techniques: Injection, Stable Transfection, Control, In Vivo, Luciferase, Imaging, Fluorescence, TUNEL Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: CAL-62 and 8505C cells were untransfected (Mock) or transfected with pSilencer or pSi-shUSP22-1 plasmid. ( A ) Apoptosis of 8505C cells analyzed by flow cytometry. ( B ) Apoptosis rate in (A) was calculated. ( C ) Fluorescent TUNEL assay conducted to determine the apoptosis of 8505C cells. Scale: 10 μm. ( D ) Percentage of TUNEL-positive cells in (C). ( E ) CAL-62 and 8505C cell apoptosis evaluated by nucleosomal fragmentation assay. ( F ) Quantification of caspase-3 activity in CAL-62 and 8505C cells. ( G ) Western blot analyses of apoptosis-related protein (Bid, Bax, cl-caspase-3, caspase-3, and Bcl-2) expressions in CAL-62 and 8505C cells. β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pSilencer group (B and D–F).

Article Snippet: Blotting was performed with primary antibodies targeting USP22, Akt, phosphorylated (p)-Akt (Ser473), Rb, and p-Rb (Ser811) (all from Cell Signaling Technology, Danvers, MA, USA), cyclin D2, BMI-1, and E-cadherin (all from Abcam, Cambridge, UK), vimentin, snail, Bid, Bax, and Bcl-2 (all from Abnova, Taiwan, China), cl-caspase-3, caspase-3, and β-actin (all from Sigma), followed by horseradish peroxidase-conjugated secondary antibody (Sigma).

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, TUNEL Assay, Activity Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Targeting ubiquitin-specific protease 22 suppresses growth and metastasis of anaplastic thyroid carcinoma

doi: 10.18632/oncotarget.9098

Figure Lengend Snippet: Female six-week-old SCID mice were inoculated subcutaneously into right hind flanks or injected via tail vein with stably expressed pLKO.1 or pLKO.1-shUSP22 8505C-luc cells. Mock-treatment was used as control. ( A ) Tumor growth progression was measured by in vivo luciferase imaging of the xenografts at days 1, 5, 9, and 12 d after inoculation. ( B ) Representative gross photos of tumors 22 d after subcutaneous xenografting ( n = 6). ( C ) Tumor volumes of subcutaneous implantation models of ATC were monitored and calculated after 0, 7, 11, 13, 17, 20, and 22 d of inoculation. D–F. After 28 d of injection through tail vein, the lungs were removed and photographed ( D ), the number of metastatic nodules in lungs was counted ( E ), and the weight of lungs was measured ( F ). ( G ) Fluorescence TUNEL assay was carried out to determine cell apoptosis in the same tumor tissues as indicated above. The rate of TUNEL-positive cells was calculated. Scale bar: 10 μm. ( H ) qPCR assay was performed to detect the mRNA expression of USP22 in tumor tissues from (B). ( I ) Protein expression of USP22 in tumor tissues was analyzed by western blot. ( J ) Representative results of western blot analyses of cyclin D2, Akt, p-Akt, E-cadherin, vimentin, Bax, Bcl-2, cl-caspase-3, and caspase-3 in tumor tissues. (I and J) β-actin was used as endogenous control. The data were from three independent experiments. Bar graph represented mean ± SD. Statistical significance: * P < 0.05, ** P < 0.01, as compared with mock or pLKO.1 group (C and E–H).

Article Snippet: Blotting was performed with primary antibodies targeting USP22, Akt, phosphorylated (p)-Akt (Ser473), Rb, and p-Rb (Ser811) (all from Cell Signaling Technology, Danvers, MA, USA), cyclin D2, BMI-1, and E-cadherin (all from Abcam, Cambridge, UK), vimentin, snail, Bid, Bax, and Bcl-2 (all from Abnova, Taiwan, China), cl-caspase-3, caspase-3, and β-actin (all from Sigma), followed by horseradish peroxidase-conjugated secondary antibody (Sigma).

Techniques: Injection, Stable Transfection, Control, In Vivo, Luciferase, Imaging, Fluorescence, TUNEL Assay, Expressing, Western Blot

Journal: American Journal of Cancer Research

Article Title: MiR-101 targets USP22 to inhibit the tumorigenesis of papillary thyroid carcinoma

doi:

Figure Lengend Snippet: USP22 overexpression reduced the inhibitory effects of miR-101 on PTC cell migration and invasion. K1 cells were transfected with miR-NC, miR-101 mimics, or miR-101 mimics + USP22-expressing plasmid. Transwell assays were performed to detect cell migration (A) and invasion (B), and the number of migrated and invaded cells was calculated. (C) Expression of USP22 and invasion-related proteins containing E-cadherin, vimentin, and snail was measured by western blot analysis. β-actin was used as endogenous control. All data are shown as means ± SD of three separate experiments. *P < 0.05, as compared with miR-NC group; #P < 0.05, as compared with miR-101 group.

Article Snippet: Western blot was performed with primary antibodies targeting USP22, Rb (from Cell Signaling Technology, Danvers, MA, USA), cyclin D2, E-cadherin (from Abcam, Cambridge, UK), vimentin, snail, Bax, Bcl-2 (all from Abnova, Taiwan, China), cleaved (cl)-caspase-3, caspase-3, and β-actin (all from Sigma), followed by horseradish peroxidase-conjugated secondary antibody (Sigma).

Techniques: Over Expression, Migration, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: American Journal of Cancer Research

Article Title: MiR-101 targets USP22 to inhibit the tumorigenesis of papillary thyroid carcinoma

doi:

Figure Lengend Snippet: MiR-101 overexpression or USP22 knockdown suppressed tumor growth and metastasis and promoted apoptosis of PTC cells in vivo. SCID mice were injected subcutaneously or via their tail veins with K1-luc cells infected with a control lentivirus (Lenti-pGCsi or Lenti-pLKO.1) or a recombinant lentivirus expressing a miR-101 precursor (Lenti-pGCsi-miR-101) or shUSP22 (Lenti-shUSP22). A. In vivo luciferase imaging of the xenografts at 5 weeks after implanted with K1-luc cells. B. Representative gross photos of tumors after 12 weeks of implantation. C. Tumor volume was measured and calculated every two weeks. D. The numbers of metastatic foci in the lungs of mice from various groups at 8 weeks after tail vein injection. E. TUNEL assay was performed to detect the percentage of apoptotic cells. F. Representative results of western blot analyses of USP22, cyclin D2, Rb, E-cadherin, snail, cl-caspase-3, and caspase-3 in tumor tissues. β-actin was used as endogenous control. All data are shown as means ± SD of three separate experiments. *P < 0.05.

Article Snippet: Western blot was performed with primary antibodies targeting USP22, Rb (from Cell Signaling Technology, Danvers, MA, USA), cyclin D2, E-cadherin (from Abcam, Cambridge, UK), vimentin, snail, Bax, Bcl-2 (all from Abnova, Taiwan, China), cleaved (cl)-caspase-3, caspase-3, and β-actin (all from Sigma), followed by horseradish peroxidase-conjugated secondary antibody (Sigma).

Techniques: Over Expression, In Vivo, Injection, Infection, Recombinant, Expressing, Luciferase, Imaging, TUNEL Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: USP22 promotes the proliferation and Sorafenib resistance of hepatocellular carcinoma cells via its deubiquitinase activity

doi: 10.1002/ctm2.70324

Figure Lengend Snippet: Ubiquitin‐specific protease 22 (USP22) catalyses the deubiquitination of cyclin‐dependent kinase 11B (CDK11B). (A) Western blotting was used to detect the protein level of CDK11B in control and USP22 knockout cells (HepG2/Hep3B/Huh7). (B) The mRNA level of CDK11B in control and USP22 knockdown HepG2 cells was detected by quantitative real‐time RT‐PCR assays. Data are mean ± SD for n = 3; NS: p > .05 (Student's t ‐test). (C) Empty vectors, 0.5, 1 or 2 µg Flag‐USP22 expression plasmids were transfected into HepG2 cells, and the protein levels of USP22 and CDK11B were detected by Western blotting. (D) Half‐lives of CDK11B protein were examined in control and USP22 knockout cells using cycloheximide (CHX) pulse‐chase assays. (E) Half‐lives of CDK11B protein were examined in control and USP22 overexpressing cells using CHX pulse‐chase assays. (F) Protein level of CDK11B was detected by Western blotting in control and USP22 knockout HepG2 cells treated with dimethyl sulphoxide (DMSO), MG132 (10 µM) or chloroquine (CQ, 20 µM) for 4 h. (G) Schematic diagram of mutation sites in USP22 enzymatic mutant. (H) HEK‐293FT cells were transfected with HA‐ubiquitin expression plasmids together with Flag‐USP22, Flag‐USP22 enzymatic mutant or control plasmids. Forty‐eight hours post‐transfection, the cells were treated with MG132 (10 µM) for 4 h and then the ubiquitination of CDK11B was detected by Western blotting using anti‐Ub after immunoprecipitation by anti‐CDK11B. (I) HA‐ubiquitin expression plasmids were transfected into USP22 knockout HepG2 cells and control cells. Forty‐eight hours post‐transfection, the cells were treated with MG132 (10 µM) for 4 h and the ubiquitination of CDK11B was detected by Western blotting using anti‐Ub after immunoprecipitation by anti‐CDK11B.

Article Snippet: Antibodies and reagents were purchased from the following sources: anti‐USP22 (ab195289), anti‐histone H2A (ab177308) and anti‐histone H2B (ab52484) from Abcam Ltd.; anti‐CDK11B (A12830), anti‐TFRC (A5865) and anti‐β‐actin (AC038) from ABclonal Technology Co., Ltd.; anti‐H2BK120ub (5546), anti‐His (12698), anti‐Ki67 (9027) and anti‐GAPDH (2118) from Cell Signalling Technology Inc.; anti‐H2AK119ub (PTM‐1121) from PTM Biolabs Inc.; anti‐ubiquitin (U7258) from Sigma‒Aldrich Corp.; anti‐Flag (M2, F3165) from Merck KGaA Co.; horseradish peroxidase (HRP)‐conjugated secondary antibodies (5220‐0341 and 5220‐0336) from SeraCare Life Sciences Inc.; fluorescein‐conjugated (111‐095‐003) or rhodamine‐conjugated (111‐025‐003 and 115‐025‐003) secondary antibodies from Jackson ImmunoResearch Laboratories Inc.; Sorafenib (HY‐10201, final concentration 10 μM), Erastin (HY‐15763, final concentration 10 μM), RSL3 (HY‐100218A, final concentration 1 μM) and DFO (HY‐D0903, final concentration 3 μM) from MedChemExpress; Fer‐1 (T6500, final concentration 5 μM) from Target Molecule Corp.; CHX (S7418, final concentration 50 μM) and MG132 (S2619, final concentration 10 μM) from Selleck Chemicals.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Knock-Out, Knockdown, Quantitative RT-PCR, Expressing, Transfection, Pulse Chase, Mutagenesis, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: USP22 promotes the proliferation and Sorafenib resistance of hepatocellular carcinoma cells via its deubiquitinase activity

doi: 10.1002/ctm2.70324

Figure Lengend Snippet: Ubiquitin‐specific protease 22 (USP22) catalyses the deubiquitination of cyclin‐dependent kinase 11B (CDK11B). (A) Western blotting was used to detect the protein level of CDK11B in control and USP22 knockout cells (HepG2/Hep3B/Huh7). (B) The mRNA level of CDK11B in control and USP22 knockdown HepG2 cells was detected by quantitative real‐time RT‐PCR assays. Data are mean ± SD for n = 3; NS: p > .05 (Student's t ‐test). (C) Empty vectors, 0.5, 1 or 2 µg Flag‐USP22 expression plasmids were transfected into HepG2 cells, and the protein levels of USP22 and CDK11B were detected by Western blotting. (D) Half‐lives of CDK11B protein were examined in control and USP22 knockout cells using cycloheximide (CHX) pulse‐chase assays. (E) Half‐lives of CDK11B protein were examined in control and USP22 overexpressing cells using CHX pulse‐chase assays. (F) Protein level of CDK11B was detected by Western blotting in control and USP22 knockout HepG2 cells treated with dimethyl sulphoxide (DMSO), MG132 (10 µM) or chloroquine (CQ, 20 µM) for 4 h. (G) Schematic diagram of mutation sites in USP22 enzymatic mutant. (H) HEK‐293FT cells were transfected with HA‐ubiquitin expression plasmids together with Flag‐USP22, Flag‐USP22 enzymatic mutant or control plasmids. Forty‐eight hours post‐transfection, the cells were treated with MG132 (10 µM) for 4 h and then the ubiquitination of CDK11B was detected by Western blotting using anti‐Ub after immunoprecipitation by anti‐CDK11B. (I) HA‐ubiquitin expression plasmids were transfected into USP22 knockout HepG2 cells and control cells. Forty‐eight hours post‐transfection, the cells were treated with MG132 (10 µM) for 4 h and the ubiquitination of CDK11B was detected by Western blotting using anti‐Ub after immunoprecipitation by anti‐CDK11B.

Article Snippet: Antibodies and reagents were purchased from the following sources: anti‐USP22 (ab195289), anti‐histone H2A (ab177308) and anti‐histone H2B (ab52484) from Abcam Ltd.; anti‐CDK11B (A12830), anti‐TFRC (A5865) and anti‐β‐actin (AC038) from ABclonal Technology Co., Ltd.; anti‐H2BK120ub (5546), anti‐His (12698), anti‐Ki67 (9027) and anti‐GAPDH (2118) from Cell Signalling Technology Inc.; anti‐H2AK119ub (PTM‐1121) from PTM Biolabs Inc.; anti‐ubiquitin (U7258) from Sigma‒Aldrich Corp.; anti‐Flag (M2, F3165) from Merck KGaA Co.; horseradish peroxidase (HRP)‐conjugated secondary antibodies (5220‐0341 and 5220‐0336) from SeraCare Life Sciences Inc.; fluorescein‐conjugated (111‐095‐003) or rhodamine‐conjugated (111‐025‐003 and 115‐025‐003) secondary antibodies from Jackson ImmunoResearch Laboratories Inc.; Sorafenib (HY‐10201, final concentration 10 μM), Erastin (HY‐15763, final concentration 10 μM), RSL3 (HY‐100218A, final concentration 1 μM) and DFO (HY‐D0903, final concentration 3 μM) from MedChemExpress; Fer‐1 (T6500, final concentration 5 μM) from Target Molecule Corp.; CHX (S7418, final concentration 50 μM) and MG132 (S2619, final concentration 10 μM) from Selleck Chemicals.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Knock-Out, Knockdown, Quantitative RT-PCR, Expressing, Transfection, Pulse Chase, Mutagenesis, Immunoprecipitation