Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Subcellular localization of GFP-tagged CP110 and its fragments, CEP97, and CEP97^CP110 chimera. (A) U2OS transiently transfected with the indicated GFP-tagged constructs were fixed and stained with antibodies against CEP192 (magenta), GFP (green), and tyrosinated tubulin (gray). White box highlights region with centrioles, which are enlarged in zoom. (B) U-ExM images of centrioles from U2OS cells overexpressing the indicated constructs and stained for acetylated tubulin (blue), CP110 (magenta), and GFP (green). CP110 full-length and CEP97^CP110 both localize to the distal cap of the mother centriole (white arrowhead) and distal cap of the daughter centriole.

Article Snippet: HEK293T cells (ATCC) and the Flp-In T-REx U2OS host cells ( ) were cultured in DMEM and Ham’s F10 (1:1) supplemented with 10% FCS and 5 U/ml penicillin and 50 μg/ml streptomycin.

Techniques: Transfection, Construct, Staining

Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Characterization of the effects of disrupting CPAP–CP110 interaction on centriole length regulation at interphase. (A) Scheme showing the generation of the inducible transgenic cell lines expressing either GFP-tagged WT full-length CPAP (CPAP-FL WT ) or full-length CPAP with L149A/K150A mutation (CPAP-FL MUT ). U2OS cells (Control) were used to integrate with the Tet repressor, a single FRT site, and the lacZ-Zeocin fusion gene by lentivirus to generate the Flp-In T-REx U2OS host cell line (Host). pcDNA5/FRT/TO vectors for doxycycline-inducible expression of GFP-CPAP-FL WT or GFP-CPAP-FL MUT were co-transfected together with Flp recombinase-encoding pOG44 vector into the Flp-In T-REx U2OS host cell line to induce their integration into the FRT site of the host cell genome in a Flp recombinase-dependent manner. The expression of GFP-CPAP-FL WT or GFP-CPAP-FL MUT was controlled by the inducible hybrid human cytomegalovirus (CMV)/Tet operator 2 (TetO2) promoter. The endogenous CPAP gene was knocked out using a CRISPR/Cas9–based approach. (B) Mean ± SD of the normalized CPAP levels based on western blots shown in ( n = 3 trials). Cell lines used for quantification are shown in magenta, where cell line pairs 1 and 2 (p1 and p2, respectively) are highlighted. Nonsignificant (ns), P > 0.05 calculated using an unpaired two-tailed Mann–Whitney U test. (C and E) Immunofluorescence images acquired using Airyscan 2 confocal microscope of centrioles at G1/S (C) and G2/M (E) and stained for acetylated tubulin (blue), CP110 (green), and GFP-CPAP (magenta). (D) Median ± IQR of mother centriole length at G1/S measured from proximal end of centriole (determined by acetylated tubulin) to distal end (determined by the geometric center of CP110 signal) (scheme in panel F). n , number of analyzed centrioles: control cell line, n = 113; host, n = 105; CPAP-FL WT#3 , n = 132; CPAP-FL WT#4 , n = 131; CPAP-FL MUT#1, n = 84; CPAP-FL MUT#5, n = 170; CPAP-FL MUT#4, n = 81; nonsignificant (ns); and ****P < 0.001 calculated using Kruskal–Wallis ANOVA test. (F) Median ± IQR of centriole length at G2/M measured as in D. n , number of analyzed mother centrioles (MC) and daughter centrioles (DC): control cells, n = 80 MC, 75 DC; host, n = 72 MC, 59 DC; CPAP-FL WT#3 , n = 67 MC, 69 DC; CPAP-FL WT#4 , n = 64 MC, 57 DC; CPAP-FL MUT#1 , n = 71 MC, 80 DC; CPAP-FL MUT#5 , n = 78 MC, 79 DC; CPAP-FL MUT#4 , n = 79 MC, 77 DC; nonsignificant (ns); and ****P < 0.001 calculated using Kruskal–Wallis ANOVA test.

Article Snippet: HEK293T cells (ATCC) and the Flp-In T-REx U2OS host cells ( ) were cultured in DMEM and Ham’s F10 (1:1) supplemented with 10% FCS and 5 U/ml penicillin and 50 μg/ml streptomycin.

Techniques: Transgenic Assay, Expressing, Mutagenesis, Control, Transfection, Plasmid Preparation, CRISPR, Western Blot, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Microscopy, Staining

Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Key resources table

Article Snippet: HEK293T cells (ATCC) and the Flp-In T-REx U2OS host cells ( ) were cultured in DMEM and Ham’s F10 (1:1) supplemented with 10% FCS and 5 U/ml penicillin and 50 μg/ml streptomycin.

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Expressing, Software, Imaging

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: Relative USP22 expression in osteosarcoma cells and tissues. (A) Relative mRNA levels of USP22 in 30 pairs of osteosarcoma (OS) and corresponding non‐tumour tissues, assessed by qRT‐PCR analysis. * p < 0.05. (B) Determination and quantification of USP22 protein levels in osteosarcoma (OS) and corresponding non‐tumour tissues by western blotting assay. GAPDH was used as a loading control. (C, D) Representative images (C) and quantification (D) of USP22 staining in 30 paired osteosarcoma (OS) and noncancer tissues. * p < 0.05. (E, F) mRNA and protein levels of USP22 in osteosarcoma (OS) cells (143B, HOS, MG‐63, U2‐OS) and the immortalised normal cells (hfoBI‐19) line.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

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

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: Effects of USP22 on osteosarcoma growth in vitro and in vivo. (A, B) the mRNA (A) and protein (B) levels of USP22 in 143B cells after transfection with shUSP22 or shNC (control). (C, D) CCK‐8 assay showing proliferation of OS cancer cells following overexpression (right) or knockdown (left) of USP22. * p < 0.05. (E, F) Representative images (left) and quantification (right) of EDU assays of osteosarcoma cells transfected with p‐USP22 or shUSP22. Scale bar, 50 μm. * p < 0.05. (G, H) Representative images of colony formation assays of osteosarcoma (OS) cells transfected with shUSP22 (G) or p‐USP22 (H). (I, J) The apoptosis rate of OS cells was detected by flow cytometry and was significantly increased in the shUSP22 cells but decreased in the p‐USP22 cells. (K–M) 143B/shUSP22 cells were subcutaneously injected into nude mice, and tumour volumes were measured on the indicated days; at the experimental endpoint, tumours were dissected, photographed and weighed ( n = 5, * p < 0.05).

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: In Vitro, In Vivo, Transfection, Control, CCK-8 Assay, Over Expression, Knockdown, Flow Cytometry, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: USP22 promotes aerobic glycolysis in osteosarcoma cells. (A, B) ATP levels, Cellular G6P levels, glucose consumption and lactate production in 143B/shUSP22 cells (A) or U2OS/p‐USP22 cells (B). Three independent experiments were performed. * p < 0.05 versus control. (C, E) ECAR data showing the glycolytic rate and capacity in USP22‐silenced (C) or USP22‐overexpressing (E) osteosarcoma cells. Glucose (10 mM), the oxidative phosphorylation inhibitor oligomycin (1.0 μM) and the glycolytic inhibitor 2‐deoxyglucose (2‐DG, 50 mM) were sequentially injected into each well at the indicated time points. All measurements were normalised to the cell number calculated using crystal violet assay at the end of the experiment. * p < 0.05 versus control. (D, F) OCR results showing the basal respiration and maximum respiration in 143B/shUSP22 cells (D) or U2OS/p‐USP22 cells (F). Oligomycin (1.0 μM), the mitochondrial uncoupler carbonyl cyanide p‐trifluoromethoxy phenylhydrazone (FCCP, 1.0 μM) and the mitochondrial complex I inhibitor rotenone plus the mitochondrial complex III inhibitor antimycin A (Rote/AA, 0.5 μM) were sequentially injected. All measurements were normalised to the cell number calculated using crystal violet assay at the end of the experiment. * p < 0.05 versus control.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Control, Phospho-proteomics, Injection, Crystal Violet Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: Stable knockdown of USP22 decreased HK2 expression in osteosarcoma cells. (A, B) Western blotting and qRT‐PCR analyses of HK2 expression levels in 143B cells stably transfected with shNC or shUSP22 plasmid. * p < 0.05. (C, D) Western blotting and qRT‐PCR analyses of HK2 expression levels in U2OS cells stably transfected with control vector or p‐USP22 plasmid. * p < 0.05. (E, F) Determination of HK2 protein levels in osteosarcoma tissues ( n = 30) and paired non‐tumour tissues ( n = 30) by western blotting. GAPDH was used as a loading control. (G) Representative images of HK2 staining in 30 paired osteosarcoma (OS) and noncancer tissues. (H) Determination of HK2 mRNA levels in osteosarcoma tissues ( n = 30) and paired non‐tumour tissues ( n = 30) by qRT‐PCR. (I) Scatter plots of USP22 and HK2 mRNA expression in osteosarcoma. (J) Quantification of HK2 protein levels in osteosarcoma (OS) and corresponding non‐tumour tissues by western blotting assay. (K) Scatter plots of USP22 and HK2 protein expression in osteosarcoma.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Expressing, Western Blot, Quantitative RT-PCR, Stable Transfection, Transfection, Plasmid Preparation, Control, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: Tumour‐suppressive effects of USP22 silencing in osteosarcoma cells partially reversed by HK2 overexpression. (A) Western blotting of USP22 or HK2 in 143B cells stably transfected with shUSP22 in the presence or absence of p‐HK2. (B) CCK‐8 assays showing proliferation capacity of 143B cells stably transfected with shUSP22 in the presence or absence of p‐HK2. * p < 0.05. (C) Representative images of EdU assays of 143B cells stably transfected with shUSP22 in the presence or absence of p‐HK2. (D) ATP levels, Cellular G6P levels, glucose consumption and lactate production in 143B cells stably transfected with shUSP22 in the presence or absence of p‐HK2. * p < 0.05. (E) ECAR of USP22‐silenced 143B cells with and without HK2 overexpression. * p < 0.05. (F) OCR values of USP22 silenced 143B cells with and without HK2 overexpression. * p < 0.05.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Over Expression, Western Blot, Stable Transfection, Transfection, CCK-8 Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: USP22 regulates HK2 expression through β‐catenin in osteosarcoma. (A) Co‐immunoprecipitation (Co‐IP) showing that endogenous USP22 and HK2 were not directly bound. (B) Protein and mRNA levels of β‐catenin assessed by western blotting and qRT‐PCR in osteosarcoma cells transfected with shUSP22 or shNC. (C) Protein and mRNA levels of β‐catenin assessed by western blotting and qRT‐PCR in osteosarcoma cells transfected with p‐USP22 or control vector. (D, E) The total and nuclear protein levels of β‐catenin were assessed by western blotting in USP22‐silencing 143B cells (D) or USP22‐overexpression U2OS cells (E). GAPDH and Histone 3 were used as a loading control, respectively. (F) The protein levels of β‐catenin and HK2 were assessed by western blotting in USP22‐overexpression U2OS cells following treatment with shβ‐catenin. (G) Quantification for CCK‐8 assays of USP22‐overexpression U2OS cells transfected with shβ‐catenin. * p < 0.05. (H, I) Quantification (H) and representative images (I) for EDU assays of USP22‐overexpression U2OS cells transfected with shβ‐catenin. * p < 0.05. (J) ECAR of USP22‐overexpression U2OS cells transfected with shβ‐catenin. (K) OCR values of USP22‐overexpression U2OS cells transfected with shβ‐catenin.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Over Expression, CCK-8 Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: USP22 Promotes Osteosarcoma Progression by Stabilising β‐Catenin and Upregulating HK2 and Glycolysis

doi: 10.1111/jcmm.70239

Figure Lengend Snippet: USP22 stabilises β‐catenin by regulating the ubiquitination of β‐catenin in osteosarcoma cells. (A) Co‐immunoprecipitation (Co‐IP) showing direct binding of endogenous USP22 and β‐catenin in osteosarcoma cells. (B, C) Osteosarcoma cells were treated with MG132 (15 μmol/L) for the indicated times, and levels of β‐catenin were determined. (D, E) Representative results of β‐catenin protein level in USP22‐silencing cells. The cells were treated with cycloheximide (CHX, 100 mg/mL) for indicated time points were subjected to western blot analysis. (F, G) OS cells transduced with shUSP22 (F) or p‐USP22 (G) were treated with 10 μM MG132. Cells were collected at 6 h and immunoblotted with the antibodies indicated. (H, I) Lysates from OS cells transduced with shUSP22 (H) or p‐USP22 (I) were immunoprecipitated with the anti‐Ub and immunoblotted with the anti‐β‐catenin. Cells were treated with MG132 for 6 h before collection. (J) Proposed model by which ubiquitin‐specific protease USP22 promotes osteosarcoma growth and aerobic glycolysis by upregulating HK2 via stabilisation of β‐catenin.

Article Snippet: Human osteosarcoma cell lines (including MG‐63, 143 B, U2‐OS and HOS) and normal human osteoblast (hfoBI‐19; control) were obtained from the American Type Culture Collection (ATCC).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Transduction