Journal: Nature Communications

Article Title: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression

doi: 10.1038/s41467-025-59407-9

Figure Lengend Snippet: a 293T cells were co-transfected with His-Ub and wild-type (WT) HA-cyclin D1, or various HA-cyclin D1 mutants (T286A and T288A), or a Vector control for 48 h. Then the cells were lysed with ubiquitination assay lysis buffer, followed by pull-down using Ni-NTA beads, and the precipitates were analyzed by western blot. b Schematic of the screening workflow to identify FBXO32 as a protein candidate interacting with dephosphorylated cyclin D1. c Co-immunoprecipitation (Co-IP) assay was utilized to determine the interaction between endogenous FBXO32 and cyclin D1 in Huh7 cells. IgG indicates the control antibody group. d Co-IP analysis of the interaction between Flag-tagged FBXO32 and HA-tagged cyclin D1 in Huh7 cells transfected with the indicated expression constructs. e Huh7 or CFPAC-1 cells were transfected with Flag-FBXO32 plasmid or control Vector for 48 h. Immunofluorescence assay was utilized to measure the colocalization of FBXO32 and cyclin D1. Hoechst was used to stain DNA. Scale bar, 10 μm. f Huh7 cells transfected with HA-cyclin D1 construct were lysed and incubated with GST or GST-FBXO32 conjugated to beads. Pull-down samples and 5% of the input were analyzed by western blot and Ponceau S staining. g SPR assay revealed the kinetic interaction between His-tagged recombinant human FBXO32 and cyclin D1. h 293T cells were co-transfected with WT HA-cyclin D1 and various domain-deleted Flag-FBXO32 plasmids for 48 h. MG132 (10 μM) was added to incubate with the cells for 4 h before being harvested. Then co-IP assay was performed using an anti-HA antibody to pull down HA-tagged cyclin D1, followed by western blot analysis. i WT HA-cyclin D1 and cyclin D1 truncation mutants were co-transfected with Flag-FBXO32 into 293T cells for 48 h. MG132 was added to incubate with the cells for 4 h before being harvested. Then, cells were collected for the co-IP assay using anti-Flag antibody, followed by immunoblotting. Representative immunoblot was shown from 3 biologically independent experiments ( a , c , d , f , h , i ). Source data are provided as a Source Data file. WCE whole cell extraction, OE overexpression.

Article Snippet: Antibodies against Cyclin D1 (sc-450, 1:500 for IF), Cyclin D2 (sc-452, 1:1000 for WB), Cyclin D3 (sc-6283, 1:1000 for WB), Rb (sc-102, 1:1000 for WB), FBXL8 (sc-390682, 1:1000 for WB) were purchased from Santa Cruz.

Techniques: Transfection, Plasmid Preparation, Control, Ubiquitin Proteomics, Lysis, Western Blot, Co-Immunoprecipitation Assay, Expressing, Construct, Immunofluorescence, Staining, Incubation, SPR Assay, Recombinant, Extraction, Over Expression

Journal: Nature Communications

Article Title: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression

doi: 10.1038/s41467-025-59407-9

Figure Lengend Snippet: a Huh7 and CFPAC-1 cells were transfected with the indicated concentrations of plasmids expressing Flag-FBXO32 and negative control for 48 h, followed by western blot analysis to detect the protein levels of cyclin D1. b After transfection with FBXO32-specific shRNA or negative control (sh-NC) for 48 h, Huh7 and CFPAC-1 cells were lysed for western blot analysis to determine cyclin D1 protein levels. c 293T cells were co-transfected with His-Ub and HA-cyclin D1 expression plasmid together with either a Vector control, Flag-FBXO32 or FBXO32ΔF plasmid for 48 h, followed by ubiquitination assay. d , e Huh7 ( d ) and CFPAC-1 ( e ) cells were transfected with Flag-tagged FBXO32 expression plasmid (Flag-FBXO32) or negative control (Vector) for 48 h, followed by treatment with 100 μg/mL cycloheximide (CHX) for the indicated times. The protein level of cyclin D1 was detected by western blot and quantified using Image J software. f , g After co-transfection with plasmids expressing HA-cyclin D1 and Flag-FBXO32 or negative control (Vector) for 48 h, Huh7 ( f ) and CFPAC-1 ( g ) cells were treated as in ( d ). The protein level of HA-cyclin D1 was measured by western blot and quantified using Image J software. h , i Plasmids expressing HA-cyclin D2 or HA-cyclin D3 were co-transfected with Flag-FBXO32 or negative control (Vector) into Huh7 cells for 48 h, followed by treatment as in ( d ). The protein levels of HA-cyclin D2 ( h ) and HA-cyclin D3 ( i ) were measured by western blot and quantified using Image J software. j 293T cells were co-transfected with Flag-FBXO32 and HA-cyclin D1 together with WT His-Ub or various His-Ub mutants (M1, K6, K11, K27, K29, K33, K48, K63) for 48 h, followed by ubiquitination assay. k 293T cells were co-transfected with Flag-FBXO32 and HA-cyclin D1 together with WT His-Ub or His-Ub mutant (K27R), followed by ubiquitination assay. Representative immunoblot was shown from 3 biologically independent experiments ( a – k ). Data are presented as mean ± SEM, and P -values were calculated using two-tailed unpaired Student’s t -test ( d – i ). Source data are provided as a Source Data file. LV lentivirus.

Article Snippet: Antibodies against Cyclin D1 (sc-450, 1:500 for IF), Cyclin D2 (sc-452, 1:1000 for WB), Cyclin D3 (sc-6283, 1:1000 for WB), Rb (sc-102, 1:1000 for WB), FBXL8 (sc-390682, 1:1000 for WB) were purchased from Santa Cruz.

Techniques: Transfection, Expressing, Negative Control, Western Blot, shRNA, Plasmid Preparation, Control, Ubiquitin Proteomics, Software, Cotransfection, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression

doi: 10.1038/s41467-025-59407-9

Figure Lengend Snippet: a Schematic of the workflow for identifying the potential ubiquitination sites of cyclin D1 influenced by FBXO32 with mass spectrometry analysis. Schematic illustration was created in BioRender ( https://BioRender.com/m10t423 ). b Lysine 58 (K58) ubiquitination sites in cyclin D1 identified by mass spectrometry analysis. c 293T cells were co-transfected with plasmids expressing Flag-FBXO32 and His-Ub together with WT HA-cyclin D1 or various mutants HA-cyclin D1 (K58R, K72R, K114R, K123R, K180R) for 48 h. Then the cells were lysed with ubiquitination lysis buffer, followed by pull-down using Ni-NTA beads, and the precipitates were analyzed by western blot. d Flag-FBXO32 or control plasmid was co-transfected with HA-cyclin D1 WT or K58R in Huh7 and CFPAC-1 cells, followed by immunoblotting with indicated antibodies. e , f Huh7 cells ( e ) and CFPAC-1 cells ( f ) were co-transfected with Flag-FBXO32 and WT HA-cyclin D1 or mutant HA-cyclin D1 K58R for 48 h, followed by treatment with 100 μg/mL CHX for the indicated times. The protein levels of HA-cyclin D1 and HA-cyclin D1 K58R were detected by western blot and quantified using Image J software. The immunoblotting experiments were repeated three times with similar results ( c – f ). Data are presented as mean ± SEM, and P -values were calculated using two-tailed unpaired Student’s t -test ( e , f ). Source data are provided as a Source Data file.

Article Snippet: Antibodies against Cyclin D1 (sc-450, 1:500 for IF), Cyclin D2 (sc-452, 1:1000 for WB), Cyclin D3 (sc-6283, 1:1000 for WB), Rb (sc-102, 1:1000 for WB), FBXL8 (sc-390682, 1:1000 for WB) were purchased from Santa Cruz.

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Transfection, Expressing, Lysis, Western Blot, Control, Plasmid Preparation, Mutagenesis, Software, Two Tailed Test

Journal: Nature Communications

Article Title: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression

doi: 10.1038/s41467-025-59407-9

Figure Lengend Snippet: a 293T cells were co-transfected with Flag-FBXO32 and WT HA-cyclin D1, or various HA-cyclin D1 mutants (T286A, T288A) or a Vector control for 48 h, followed by co-IP assay. b 293T cells were co-transfected with Flag-FBXO32 and His-Ub together with WT HA-cyclin D1, various HA-cyclin D1 mutants (T286A, T288A) or a Vector control for 48 h, then the cells were lysed to detect ubiquitinated cyclin D1. c , d Huh7 and CFPAC-1 cells were co-transfected with Flag-FBXO32 and WT HA-cyclin D1 or various HA-cyclin D1 mutants for 48 h, followed by treatment with 100 μg/mL CHX for the indicated times, then cyclin D1 was determined by western blotting and quantified by Image J software. e , f After co-transfection with His-Ub and HA-cyclin D1 T286A (or HA-cyclin D1 T288A ) together with Flag-FBXO32 or a Vector control for 48 h, 293T cells were lysed for ubiquitination assay. g , h Huh7 cells were co-transfected with HA-cyclin D1 T286A ( g ) or HA-cyclin D1 T288A ( h ) and Flag-FBXO32 or a Vector control for 48 h, followed by a CHX pulse-chase assay. i 293T cells were co-transfected with HA-cyclin D1 and His-Ub together with Flag-FBXO32 or a Vector control for 36 h, followed by treatment with GSK-3β inhibitor TWS119 (5 μM), DYRK1B inhibitor AZ191 (1 μM) or MEK1/2 inhibitor trametinib (100 nM) for another 12 h, followed by ubiquitination assay. j After co-transfected with HA-cyclin D1, His-Ub and Flag-FBXO32 together with GSK-3β or negative control siRNA for 48 h, 293T cells were lysed for ubiquitination assay. k 293T cells were co-transfected with HA-cyclin D1, His-Ub and Flag-FBXO32 together with WT GSK-3β or various GSK-3β mutants, followed by ubiquitination assay. l 293T cells were transfected with WT GSK-3β, or various GSK-3β mutants, or negative control vector for 48 h, followed by GSK-3β activity measurement. ( n = 3 independent experiments). Representative immunoblot was shown from 3 biologically independent experiments ( a – k ). Data are presented as mean ± SEM, and P -values were calculated using one-way ANOVA with Dunnett’s ( c , d ) or Tukey’s ( l ) multiple comparisons or two-tailed unpaired Student’s t -test ( g , h ). Source data are provided as a Source Data file.

Article Snippet: Antibodies against Cyclin D1 (sc-450, 1:500 for IF), Cyclin D2 (sc-452, 1:1000 for WB), Cyclin D3 (sc-6283, 1:1000 for WB), Rb (sc-102, 1:1000 for WB), FBXL8 (sc-390682, 1:1000 for WB) were purchased from Santa Cruz.

Techniques: Transfection, Plasmid Preparation, Control, Co-Immunoprecipitation Assay, Western Blot, Software, Cotransfection, Ubiquitin Proteomics, Pulse Chase, Negative Control, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression

doi: 10.1038/s41467-025-59407-9

Figure Lengend Snippet: a – e CCK-8, colony formation and trans-well assays were performed to measure the proliferation ( a – c ), migration ( d ) and invasion ( e ) of Huh7 and CFPAC-1 cells with stably expressing FBXO32 shRNA (sh-FBXO32) or negative control (sh-NC) by lentivirus infection in the presence of palbociclib (10 μM) or not. Scale bar, 100 μm. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons, and data are represented as mean ± SEM ( n = 3 independent experiments). f – i Huh7 and CFPAC-1 cells with stably expressing FBXO32 shRNA (sh-FBXO32) or negative control (sh-NC) were constructed through lentivirus infection, and then were injected subcutaneously to axilla of BALB/c nude mice. After 4 weeks, the masses of tumor formed from Huh7 or CFPAC-1 cells were divided equally into 1 mm 3 pieces and implanted orthotopically into BALB/c nude mice. Both sh-NC and sh-FBXO32 group mice were treated with palbociclib (100 mg/kg) or Vehicle (sodium lactate) daily by oral gavage administration for 14 days. The mice were sacrificed after the last administration. Representative images of primary liver ( f ) and pancreatic ( h ) tumors were presented, and the tumor volumes were monitored. Statistical analysis was performed using one-way ANOVA with Dunnett’s or Tukey’s multiple comparisons, and data are represented as mean ± SEM (For HCC, n = 7 biologically independent animals per group; For PDAC, n = 5 biologically independent animals per group). H&E staining was performed to determine the tumor tissue, and IHC staining was utilized to measure the protein levels of FBXO32 and cyclin D1 in HCC ( g ) and PDAC ( i ) tumor tissues. Scale bar, 50 μm. Source data are provided as a Source Data file.

Article Snippet: Antibodies against Cyclin D1 (sc-450, 1:500 for IF), Cyclin D2 (sc-452, 1:1000 for WB), Cyclin D3 (sc-6283, 1:1000 for WB), Rb (sc-102, 1:1000 for WB), FBXL8 (sc-390682, 1:1000 for WB) were purchased from Santa Cruz.

Techniques: CCK-8 Assay, Migration, Stable Transfection, Expressing, shRNA, Negative Control, Infection, Construct, Injection, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression

doi: 10.1038/s41467-025-59407-9

Figure Lengend Snippet: a , b Representative IHC staining images of FBXO32 in tumor and adjacent non-tumor tissues of HCC ( a ) and PDAC ( b ) ( n = 30 samples). Left scale bar, 200 μm, and right scale bar, 50 μm. c – g Representative IHC images of consecutive sections of HCC tissues stained with antibodies against p-GSK-3β S9 , FBXO32 and cyclin D1, respectively ( c ), followed by scoring according to the staining intensity and positive proportion of target protein ( n = 131 samples). Then the correlations between cyclin D1 and FBXO32 or p-GSK-3β S9 were evaluated ( d , e ). Statistical analysis was performed using two-tailed Spearmanʹs rank correlation test. Kaplan–Meier survival curves were performed to analyze the outcome of 4 subgroups HCC patients with different FBXO32/cyclin D1 (or FBXO32/p-GSK-3β S9 ) protein levels ( f , g ). Statistical analysis was performed using Log-rank statistic test. h – l PDAC samples were utilized to detect the protein levels of p-GSK-3β S9 , FBXO32 and cyclin D1 by IHC staining ( n = 90 samples). Representative images were shown in ( h ). Correlation analysis ( i , j ) between cyclin D1 and FBXO32 or p-GSK-3β S9 and Kaplan–Meier survival analysis ( k , l ) was performed as described in ( c – g ). Scale bar, 50 μm. Statistical analysis was performed using two-tailed Spearman’s rank correlation test and log-rank statistic test, respectively. Source data are provided as a Source Data file.

Article Snippet: Antibodies against Cyclin D1 (sc-450, 1:500 for IF), Cyclin D2 (sc-452, 1:1000 for WB), Cyclin D3 (sc-6283, 1:1000 for WB), Rb (sc-102, 1:1000 for WB), FBXL8 (sc-390682, 1:1000 for WB) were purchased from Santa Cruz.

Techniques: Immunohistochemistry, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: F-box protein FBXO32 ubiquitinates and stabilizes D-type cyclins to drive cancer progression

doi: 10.1038/s41467-025-59407-9

Figure Lengend Snippet: Schematic illustration of the protein stability of cyclin D coordinated by FBXO32 and GSK-3β. In normal cells, cyclin D protein is phosphorylated by GSK-3β kinase at the Thr-288 and Thr-286 site, then recognized and added the K48-linked ubiquitin chains by other E3 ligases, which leads to the degradation of cyclin D in a proteasome-dependent manner. While in cancer cells, GSK-3β is phosphorylated and then inactivated, which contributes to the dephosphorylation of cyclin D. Highly expressed FBXO32 in tumor tissues recognizes dephosphorylated cyclin D and catalyzes the K27-linked polyubiquitination of cyclin D1 at the K58 site, suppressing the degradation of cyclin D1, which leads to uncontrolled cell cycle and cancer progression. Graphical abstract was created in BioRender ( https://BioRender.com/h63r237 ).

Article Snippet: Antibodies against Cyclin D1 (sc-450, 1:500 for IF), Cyclin D2 (sc-452, 1:1000 for WB), Cyclin D3 (sc-6283, 1:1000 for WB), Rb (sc-102, 1:1000 for WB), FBXL8 (sc-390682, 1:1000 for WB) were purchased from Santa Cruz.

Techniques: Ubiquitin Proteomics, De-Phosphorylation Assay