Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: (A) Schematic summary of genome-wide CRISPR/Cas9 screen. (B) Top 5 GO term enrichment analysis of 585 statistically significant ( P -value < 0.05) candidates identified in genome-wide CRISPR/Cas9 screen. Candidates were categorised into biological process (BP), cellular component (CC), and molecular function (MF), and the x-axis represents the gene counts. (C) Data from DNA sequencing showing sgRNA target sites (highlighted in red) and PAM sequence (highlighted in blue). (D) qRT-PCR analysis of FBXO11 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. FBXO11 mRNA levels were normalised to GAPDH, and fold change relative to WT shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of FBXO11 isoform 4 and 1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple Ccomparison test. (F) Brightfield microscopy showing morphology of WT and FBXO11 −/− macrophages following PMA differentiation. Scale bar corresponds to 100 μm. (G) Live cell imaging showing the percentage of Draq7-positive (dead) WT and FBXO11 −/− macrophages treated with PBS, PVL (62.5 ng/ml), LukAB (62.5 ng/ml), and nigericin (10 μM). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for WT versus FBXO11 −/− (E3) at 15 h post toxin treatment; ** = P < 0.01 for WT versus FBXO11 −/− (E2) at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Genome Wide, CRISPR, DNA Sequencing, Sequencing, Quantitative RT-PCR, Comparison, Western Blot, Quantitative Proteomics, Microscopy, Live Cell Imaging

Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: (A) Live cell imaging showing the percentage of Draq7-positive (dead) WT and C5aR1 −/− macrophages to PBS or PVL (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. ** = P < 0.01 for WT versus C5aR1 −/− at 15 h post toxin treatment; by unpaired t test. (B) Flow cytometric analysis and median fluorescence intensity of C5aR1 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, red and blue lines represent FBXO11 −/− (E2 and E3, respectively), and dark grey line represents C5aR1 −/− macrophages. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Confocal microscopy images of WT, FBXO11 −/− , and C5aR1 −/− macrophages. PFA-fixed cells were stained using anti-C5aR1 (green), and nuclei were stained with DAPI (blue). Scale bar corresponds to 100 μM. (D) Immunoblot analysis of C5aR1 in WT, FBXO11 −/− , and C5aR1 −/− macrophages. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (E) Immunoblot analysis of C5aR1 in WT macrophages that were treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophage. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (F) qRT-PCR analysis of C5aR1 mRNA in WT, FBXO11 −/− , and C5aR1 −/− macrophages. C5aR1 levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (G) Flow cytometric analysis and MFI of recombinant LukS-PV subunit binding to WT and FBXO11 −/− macrophages. Grey line represents isotype, dotted black line represents no LukS-PV negative control, solid black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages treated with LukS-PV (E2 and E3, respectively). *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (H) Western blot analysis of WT and FBXO11 −/− macrophages treated with LukS-PV over time. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages at respective time points. Recombinant LukS-PV is included in the left lane. Mean ± SEM of three independent biological replicates shown. *** = P < 0.001, **** = P < 0.0001;by two-way ANOVA with Sidak’s multiple comparisons test. Source data are available for this figure.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Live Cell Imaging, Fluorescence, Expressing, Negative Control, Comparison, Confocal Microscopy, Staining, Western Blot, Quantitative Proteomics, Quantitative RT-PCR, Recombinant, Binding Assay

Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: Flow cytometric analysis and MFI of C5L2, CD45, and CD11b cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages (E2 and E3, respectively). Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Expressing, Negative Control, Comparison

Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: (A) Graphical summary of the generation of doxycycline inducible C5aR1 overexpression THP1 monocytes using the lentiviral vector pTRE3G-C5aR1. (B) Western blot analysis of C5aR1 in pTRE3G-C5aR1 transfected cells—WT-pTRE3G-C5aR1 and FBXO11 −/− (E2) pTRE3G-C5aR1 THP1 monocytes and macrophages. Cells were treated with or without doxycycline (1 μg/ml) for 24 h to induce C5aR1 expression. Abbreviations: macrophages (mΦ); monocytes (mono); doxycycline (Dox). Protein abundance was normalised to β-actin and represented as fold change compared with untreated cells. Mean ± SEM of three independent biological replicates shown. ns = not significant, * = P < 0.05; by two-way ANOVA with Sidak’s multiple comparisons test. (C) Flow cytometric analysis and MFI of C5aR1 cell surface expression on WT and FBXO11 −/− (E2) macrophages treated with (black line) or without (red line) doxycycline (1 μg/ml). The grey line represents isotype negative control. Representative of three independent experiments. ns = not significant, **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Live cell imaging showing the percentage of Draq7-positive (dead) untransfected WT macrophages, and pTRE3G-C5aR1 transfected WT and FBXO11 −/− (E2) macrophages to PVL (125 ng/ml). Cells were treated with or without doxycycline (1 μg/ml) for 24 h before toxin treatment. Mean ± SEM of three independent experiments shown. ** = P < 0.01 for “+PVL” versus “+PVL +Dox” at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Over Expression, Plasmid Preparation, Western Blot, Transfection, Expressing, Quantitative Proteomics, Negative Control, Live Cell Imaging

Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: (A, B) Flow cytometric analysis and MFI of (A) CD11b and (B) CD45 cell surface expression in WT and FBXO11 −/− (E2) macrophages containing pTRE3G-C5aR1 treated with or without doxycycline (1 μg/ml). Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Expressing

Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: (A) Schematic illustration of THP1 PMA–induced differentiation and LPS treatment. (B) Western blot analysis of C5aR1 in WT and FBXO11 −/− macrophages, treated with or without LPS for 24 h. Protein abundance was normalised to β-actin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05; by two-way ANOVA with Sidak’s multiple comparisons test. (C) qRT-PCR analysis of relative C5aR1 mRNA level, comparing the LPS-treated and -untreated macrophages. The mRNA levels were normalised to the control values of GAPDH, and fold change was LPS-treated cells that were compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Flow cytometric analysis and MFI of C5aR1 in LPS-treated or -untreated WT and FBXO11 −/− macrophages. Grey line represents isotype, black line represents untreated, and red line represents LPS-treated macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; ** = P < 0.01; by two-way ANOVA with Sidak’s multiple comparisons test. (E) Live cell imaging showing the percentage of Draq7-positive (dead) LPS-treated or -untreated WT and FBXO11 −/− macrophages treated with PVL (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01, *** = P < 0.001 for + PVL versus + PVL + LPS at 14 h post toxin treatment; by unpaired t test. Source data are available for this figure.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Western Blot, Quantitative Proteomics, Quantitative RT-PCR, Control, Live Cell Imaging

Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: (A) Western blot analysis of C5aR1 and FBXO11 protein expression in WT and FBXO11 −/− (E2) macrophages, exposed to LPS (100 ng/ml), S. aureus (MOI 10), or heat-killed S. aureus (MOI 10) for 3 h. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (B, C) Flow cytometric analysis and MFI of (B) CD45 and (C) CD11b protein expression in untreated (black line) and LPS-treated (red line) WT, and FBXO11 −/− macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; **** = P < 0.0001; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Live cell imaging showing the percentage of Draq7-positive (dead) LPS-treated WT and FBXO11 −/− macrophages treated with LukAB (62.5 ng/ml). Mean ± SEM of three independent biological replicates shown. * = P < 0.05 for +LukAB versus + LukAB +LPS at 15 h post toxin treatment; by unpaired t test. Source data are available for this figure.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Western Blot, Expressing, Quantitative Proteomics, Live Cell Imaging

Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: (A, B) WT, FBXO11 −/− , and (B) C5aR1 −/− macrophages were primed with LPS (100 ng/ml) for 3 h before PVL (62.5 ng/ml), LukAB (15.6 ng/ml), or nigericin (10 μM) treatment for 2 h. Il-1β in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant,; * = P < 0.05, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. (C) Western blot analysis of pro-IL-1β (34 kD) and cleaved IL-1β (17 kD) in cell lysates and supernatants of WT and FBXO11 −/− macrophages treated with LPS and/or toxins. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Abbreviation: S, short exposure; L, long exposure. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (D) Western blot analysis of pro-IL-1β in whole cell lysates of unprimed THP1 cells. Monocyte (Mono) was differentiated with PMA, and macrophages were cultured in PMA-free media for the indicated amount of time. Protein abundance was normalised to β-actin and represented as fold change compared with monocytes. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) Western blot analysis of NLRP3 in whole-cell lysates of WT and FBXO11 −/− macrophage. Protein abundance was normalised to β-actin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (F) Western blot analysis of pro-IL-1β and MCL-1 in WT and FBXO11 −/− macrophages. Cells were primed with LPS (100 ng/ml) for 2 h, with MG132 (20 μM) and Q-VD-OPh (20 μM) added in the last 30 min alongside. Cells were then treated with CHX (20 μg/ml) with or without MG132 for the indicated amount of time before cell lysate collection. Protein abundance was normalised to α-tubulin and represented as fold change compared with WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant; by two-way ANOVA with Sidak’s multiple comparisons test. (G) Western blot analysis of pro-IL-1β in WT and FBXO11 −/− macrophage cell lysates and TUBE-isolated ubiquitinated proteins. WT* indicates agarose beads only as control. (H) qRT-PCR analysis of relative IL-1β mRNA level. The mRNA levels were normalised to the control values of GAPDH. Mean ± SEM of three independent biological replicates shown. * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. Source data are available for this figure.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative Proteomics, Cell Culture, Comparison, Isolation, Control, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins

doi: 10.26508/lsa.202201735

Figure Lengend Snippet: (A) Western blot analysis of BCL-6 and CD40 in WT and FBXO11 −/− macrophage whole-cell lysate. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages. Mean ± SEM of three independent biological replicates shown. ns = not significant, * = P < 0.05, ** = P < 0.01; by one-way ANOVA followed by Dunnett’s multiple comparison test. (B) qRT-PCR analysis of CD40 mRNA in WT and FBXO11 −/− macrophages. CD40 mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. **** = P < 0.0001; by one-way ANOVA followed by Dunnett’s multiple comparison test. (C) Flow cytometric analysis and MFI of CD40 cell surface expression. Grey line represents isotype negative control, black line represents WT macrophages, and red and blue lines represent FBXO11 −/− macrophages (E2 and E3, respectively). Mean ± SEM of three independent biological replicates shown. ns = not significant; by one-way ANOVA followed by Dunnett’s multiple comparison test. (D) Western blot analysis of BCL-6, CD40, C5aR1, and IL-1β in WT and FBXO11 −/− macrophage whole-cell lysate treated with or without BI-3802 (1 or 5 μM) during recovery period. Protein abundance was normalised to α-tubulin and represented as fold change compared with untreated WT macrophages at respective time points. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01, *** = P < 0.001; by two-way ANOVA with Sidak’s multiple comparisons test. (E) qRT-PCR analysis of CD40, C5aR1, and IL-1β mRNA in WT and FBXO11 −/− macrophages treated with or without BI-3802 (5 μM). mRNA levels were normalised to GAPDH, and fold change relative to WT macrophage shown. Mean ± SEM of three independent biological replicates shown. ns = not significant; * = P < 0.05, ** = P < 0.01; by two-way ANOVA with Sidak’s multiple comparisons test. (F) BI-3802 (5 μM) untreated or treated WT, FBXO11 −/− (E2) macrophages were treated with PVL (62.5 ng/ml), LukAB (62.5 ng/ml) or nigericin (10 μM) for 2 h. Il-1β levels in culture supernatants were determined by ELISA. Mean ± SEM of three independent biological replicates shown. ns = not significant; ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001; by two-way ANOVA followed by Sidak’s multiple comparisons test. Abbreviation: Nig, nigericin. Source data are available for this figure.

Article Snippet: The membranes were then probed with primary antibodies — C5aR1 (#SC-271949; Santa-Cruz), LukS-PV, FBXO11 (# NB100-59826; Novus Biologicals), IL-1β (#AF-401-NA; R&D Systems), BCL-6 (#PA-527390; Invitrogen), or CD40 (#ab224639; Abcam) — in 5% skim milk overnight at 4°C.

Techniques: Western Blot, Quantitative Proteomics, Comparison, Quantitative RT-PCR, Expressing, Negative Control, Enzyme-linked Immunosorbent Assay

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Generation of a neuronal FBXO11 deficient cell model using CRISPR-CAS9 (A) Outline of generation process of FBXO11 heterozygous and complete knockout hIPSC lines is shown, including validation of mutated clones and isogenic controls. (B) Representative western blot of all nine FBXO11 hIPSC lines is shown. Blots were stained with anti-FBXO11 and anti-GAPDH antibodies. (C) Quantification of three independent western blot experiments of FBXO11 hIPSC lines confirmed loss of FBXO11 in HET and KO lines. Individual values are shown as dots and mean values are shown as bars with SEM. Mean of all three WT controls was set to 1. p values were calculated using a one-sample t test with a hypothetical control mean set to 1. For all HET and KO lines compared with the control mean, reduction was significant at p < 0.01. (D) Schematic outline of differentiation protocol from hIPSCs to NPCs and neurons with timeline and culture media used. Below, experiments performed here are indicated at various timepoints. (E) Representative images of immunofluorescence of FBXO11 WT, HET, and KO NPCs stained with antibodies against neural progenitor markers Nestin (NES, red) and PAX6 (green) confirm differentiation to NPCs. Images of all nine NPC lines can be found in Figure S2 . Images were taken on an AxioImager Z2 with Apotome 3 with a 40× objective. Scale bar, 20 μm. (F) Quantification of PAX6-positive cells among NPCs shows comparable levels of PAX6-positive cells for WT, KO, and HET cells. Quantification for individual lines can be found in Figure S3 . For quantification, cells were analyzed using CellProfiler identifying DAPI-positive cells and PAX6 positive cells (fraction of PAX6-positive cells = PAX6 stained cells/DAPI-stained cells).

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: CRISPR, Knock-Out, Biomarker Discovery, Clone Assay, Western Blot, Staining, Control, Immunofluorescence

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Gene expression changes due to loss of FBXO11 in human neurons and fly heads (A) Principal-component analysis (PCA) of three FBXO11 WT and three KO neuron samples showed clear separation of WT and KO samples along the first principal component. (B) Enriched gene ontology (GO) terms among differentially expressed genes were grouped based on function and show a broad enrichment of biological processes involved in, e.g., development, signaling, and migration. Detailed results on individual enriched GO terms can be found in Figure S5 . (C) Integration of GO term analysis of FBXO11-deficient human neuron and Drosophila head transcriptome analysis. The top five biological processes enriched in GO term analysis of human neurons are shown in black. The enrichment of these processes in Fbxo11-deficient Drosophila heads are shown in green. (D) Stacked bar chart grouping genes expressed in FBXO11 KO neurons based on their differential gene expression and colored by corresponding expression changes during neuronal differentiation in a publicly available dataset on gene expression during differentiation from hIPSC to neurons. Increasing expression during differentiation is marked in green, and decreasing expression during differentiation is shown in pink. Unchanged expression is shown in gray. Number of genes with increasing expression during differentiation is increased for genes downregulated in FBXO11 KO neurons. down = downregulated, up = upregulated, not sig = expression not significantly changed, exp. = expression.

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: Gene Expression, Migration, Expressing

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Loss of FBXO11 alters neuronal migration, proliferation, and differentiation (A) Representative images of neurosphere assay on FBXO11 WT, HET, and KO NPCs imaged 48 h after plating. Inner circle represents initial neurosphere size, outer circle represents migration after 48 h. Images were taken on a Nikon Ts2-FL microscope. Scale bar, 100 μm. (B) Quantification of migration as ratio between area occupied at 48 h and plating (0 h). Migration is impacted in HET and more severely in KO neurospheres. Quantification of all nine individual lines can be found in Figure S7 A. At least 30 neurospheres per genotype (≥8 neurospheres per line) from three independent experiments were analyzed. Significance was calculated using a Student’s t test. (C) Representative images of immunofluorescence of FBXO11 WT, HET, and KO NPCs stained with antibodies against proliferation markers Ki-67 (red) and mitotic marker pHH3 (green) are shown. Images were taken on an AxioImager Z2 with a 20× objective. Scale bar, 100 μm. (D) Quantification of Ki67-positive cells among NPCs shows increased levels of Ki67-positive HET and KO cells. Quantification for individual lines can be found in Figure S7 D. For quantification, cells from 15 images were analyzed using CellProfiler identifying DAPI-positive and Ki67 positive cells (fraction of Ki67-positive cells = Ki67 stained cells/DAPI-stained cells). Significance was calculated using a Student’s t test. (E) Proliferation of differentiating neurons (D20-D28) was assessed using an XTT assay. Absorbance was normalized to the absorbance of D20 (NPC stage and first day of measurement). Plotted is the mean (circle) together with a trend line (colored and dashed) and the standard error (gray shading). Proliferation differences between WT vs. HET and HET vs. KO were significant for all three tested timepoints (D23, D26, D28, p < 0.01) and between WT vs. HET for two timepoints (D23 and D26, p < 0.01). The experiment was carried out three times with three technical replicates each. Significance was calculated using a Student’s t test. (F) Representative western blot of 3-week-old neurons (D42) stained against neuronal marker MAP2, FBXO11, and H3 as a loading control. (G) Quantification of MAP2 levels from western blot in (F) showed reduced MAP2 levels (normalized to loading control H3) for HET and KO neurons compared with WT. The experiment was performed three times. Mean expression of three WT controls were set to 1. Significance was calculated using a one-sample t test with a theoretical mean of 1. (H) Representative images of immunofluorescence of FBXO11 WT, HET, and KO 1-week old neurons (D28) stained with antibodies against neuronal markers MAP2 (red) and TUBB3 (green) are shown here and images of all nine neuronal lines can be found in Figure S4 . Images were taken on an AxioImager Z2 with a 40× objective. Scale bar, 40 μm. (I) Quantification of TUBB3-positive cells among neurons showed reduced levels of TUBB3-positive KO cells. Quantification for individual lines can be found in Figure S7 F. For quantification cells from at least 15 images were analyzed using CellProfiler identifying DAPI-positive cells (all cells) and TUBB3-positive cells (fraction of TUBB3-positive cells = TUBB3-stained cells/DAPI-stained cells). Significance was calculated using a Student’s t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: Migration, Neurosphere Assay, Microscopy, Immunofluorescence, Staining, Marker, XTT Assay, Western Blot, Control, Expressing

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Deficiency of Fbxo11 leads to impaired behavior and dendritic branching in Drosophila melanogaster (A) Climbing assay upon pan-neuronal knockdown of Fbxo11 showed impaired locomotor ability for two of the three RNAi lines tested. Individual data points are shown as circles and summarized data are shown as boxplots. At least 200 flies in batches of 10 ( n = 20) were analyzed per condition. Significance was calculated using a Wilcoxon signed rank test. (B) Representative image of traced da neurons from control and knockdown larvae upon da neuron-specific knockdown (477-Gal4; UAS-mCD8GFP driver line) is shown. Images were acquired using a Zeiss LSM 710 confocal microscope with a 20× objective. Scale bar, 100 μm. (C) Quantification of total dendrite length of da neurons. (D) Quantification of number of branches from da neurons. Tracings of da neurons were performed in ImageJ using the NeuronJ plugin. At least 10 da neurons from five different larvae from two independent crosses were analyzed for each line. Statistical significance was calculated using a Student’s t test.

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: Climbing Assay, Knockdown, Control, Microscopy

Journal: Human Genetics and Genomics Advances

Article Title: Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency

doi: 10.1016/j.xhgg.2025.100425

Figure Lengend Snippet: Rescue of FBXO11-deficiency-associated phenotypes with proteasome-activating substances (A) Formulas of tested substances PD169316, R-Verapamil, and Verapamil are shown. (B) Scoring scheme for rescue experiments corresponding to the level of completeness of the rescue. For dark-filled boxes, rescue resulted in almost complete normalization to control levels under the DMSO (75%–100%). For boxes filled with light shades of respective color, rescue levels reached 50%–75%. Different tested substances were supplemented to the fly food or the cell culture medium and are color-coded as follows: black – DMSO control, green – PD169316, red – R-Verapamil, purple – Verapamil. For fly experiments, all substances were used at 1 μM, for cell-based experiments, different concentrations were used (PD169316: 20 μM, R-Verapamil: 15 μM, Verapamil: 10 μM). (C) Climbing assay deficit upon pan-neuronal Fbxo11 knockdown with RNAi 1 could partially be rescued with proteasome-activating substances supplemented to the fly food. Improvement of phenotypes was seen when adding substances at time of egg laying (developmental supp.) or after flies hatched (adult supp.). At least 200 flies in batches of 10 ( n = 20) were analyzed per condition. (D) Total dendrite length of da neurons increased upon addition of proteasome-activating substances to the fly food at time of egg laying. At least five different neurons from five different larvae were analyzed per condition. (E and F) NPCs (E) or D28 neurons (F) were treated with proteasome-activating substances for 2 days (NPCs) or 1 week (neurons) before staining with Ki-67 antibody to assess proliferation. For quantification, cells from at least 15 images were analyzed using CellProfiler identifying DAPI-positive and Ki67-positive cells (% of Ki67-positive cells = Ki67-stained cells/DAPI-stained cells). For all plots, individual data points are shown as circles and summarized data are shown as boxplots. Statistical significance was calculated using either a Wilcoxon signed rank test (climbing assay) or a Student’s t test (da neuron assays and cell-based experiments) with correction for multiple testing. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Blots were stained with antibodies against FBXO11 (1:2,500, NB100-59826, Novus Bio), MAP2 (1:1,000), GAPDH (1:5,000, #2118, Cell Signaling), H3 (1:10,000, #4499, Cell Signaling), c-Myc (1:5,000, M4439, Sigma-Aldrich), c-Myc (1:2,500, #2272, Cell Signaling), FLAG (1:5,000, F7425, Sigma-Aldrich), FLAG (1:1,000, 194502, Addgene, gift from Melina Fan; http://n2t.net/addgene:194502 ; RRID: AB_2924869 ), and HA (1:2,000, H3663, Sigma-Aldrich).

Techniques: Control, Cell Culture, Climbing Assay, Knockdown, Staining

Journal: Bone

Article Title: FBXO11 regulates bone development

doi: 10.1016/j.bone.2023.116709

Figure Lengend Snippet: FBXO11 promotes osteogenic differentiation of osteoblasts in vitro. Overexpression of FBXO11 in MC3T3 cells by lentiviral transfection was confirmed by its mRNA expression (1A). FBXO11 transduced osteoblasts and their controls were cultured in osteogenic induction medium in vitro. ALP staining was performed in 0, 7 day group, and AR mineralization staining was performed in 0, 14 day groups. mRNA expression of osteogenic marker genes Runx-2, OSX, Alp, and BSP were compared between FBXO11 overexpression MC3T3 cells and their controls in 0, 3 days osteogenic induction groups. The stronger osteogenic differentiation was exhibited in FBXO11 overexpressing osteoblasts (1A, 1B). On the other hand, FBXO11-shRNA and the scramble RNA were transduced into osteoblasts with Lenti virus transfection in order to knock down the endogenous FBXO11 expression in the osteoblasts, which confirmed by the reduction of its mRNA expression level (1C). FBXO11 knockdown MC3T3 cells and their controls were differentiated in osteogenic induction medium for 3 days. ALP staining was performed in 0, 7 day group, and AR mineralization staining was performed in 0, 14 day groups. mRNA expression of osteogenic marker genes Runx-2, OSX, ALP, and BSP were compared. It exhibited that FBXO11 gene knockdown inhibited osteogenic differentiation of osteoblast. (1C, 1D). In this Fig. 1, all the experiments were repeated three times, the data present as Mean ± SEM, *, P < 0.05; **, P < 0.01.

Article Snippet: We obtained the primary antibodies from the following sources: Rabbit polyclonal antibody to FBXO11 (Novus Biologics, Centennial, CO), rabbit polyclonal antibody to Snail1 (Cell Signaling, Danvers, MA), mouse monoclonal antibody to α-tubulin, and mouse monoclonal antibody to GAPDH (Sigma-Aldrich, St. Louis, MO).

Techniques: In Vitro, Over Expression, Transfection, Expressing, Cell Culture, Staining, Marker, shRNA, Virus, Knockdown

Journal: Bone

Article Title: FBXO11 regulates bone development

doi: 10.1016/j.bone.2023.116709

Figure Lengend Snippet: Generation of osteoblast-specific conditional FBXO11KO mice. (2A). 2.3kbCol1a1-CreERT2 mice and Bglap2-Cre mice were crossbred with FBXO11 mutant-Flox to generate osteoblast-specific mice. The 3-week-old osteoblast-specific FBXO11KO FBXO11KO mice (Col1a1-CreERT2; T (Tamoxifen)/FBXO−/−) showed smaller body size than their WT and hemizygous (Col1a1-CreERT2; T (Tamoxifen)/FBXO11+/−) littermates (2B). The 1-week-old osteoblast-specific FBXO11KO (Col1a1-CreERT2; T (Tamoxifen)/FBXO11−/−) mice exhibited smaller skeletal size than their WT and hemizygous littermates (2C). The newborn osteoblast-specific FBXO11cKO (Bglap2-FBXO11−/−) mice exhibited smaller skeletal size than their WT and hemizygous littermates (2D). Diagram exhibits the structure of FBXO11 Mutants with Flox sites for FBXO11 mutant mice. Vertical bars represent exons. The targeted exon (exon 4) encodes the F-box. Deletion causes reading frame shift for the downstream exons.

Article Snippet: We obtained the primary antibodies from the following sources: Rabbit polyclonal antibody to FBXO11 (Novus Biologics, Centennial, CO), rabbit polyclonal antibody to Snail1 (Cell Signaling, Danvers, MA), mouse monoclonal antibody to α-tubulin, and mouse monoclonal antibody to GAPDH (Sigma-Aldrich, St. Louis, MO).

Techniques: Mutagenesis

Journal: Bone

Article Title: FBXO11 regulates bone development

doi: 10.1016/j.bone.2023.116709

Figure Lengend Snippet: FBXO11cKO mice show significantly slower bone formation and decrease osteoblastic activity. The Declomycin and Calcein IP injection were given to 1-month-old Bglap2-FBXO11cKO and WT mice 7 days and 3 days prior to sacrifice, respectively. Fluorochrome-based indices of cancellous bone formation was measured in 8-μm-thick sections from methyl methacrylate- embedded undecalcified femur samples. (4A) exhibits undecalcified femur with tetrachrome stain. (4B) shows weaker fluorochrome labeling signals in FBXO11cKO femurs under ultraviolet light. Mineralizing surface, an index of active bone formation, was calculated as the percentage of cancellous (MS/BS) bone surfaces with a double-fluorochrome label (4C). In (4D), mineral apposition rate (MAR), an index of osteoblastic activity, was calculated by dividing the interlabel distance by the time interval between fluorochrome labeling. In (4E), bone formation rate (BFR/BS) was calculated by multiplying MS/BS by MAR (n = 6, Mean ± SEM). In (4F), serum OCN is significantly lower in cKO mice. The results in 4D-4F shows significantly decreased osteoblastic activity in FBXO11cKO mice (*, P < 0.05). (4G) and (4H) shows no significantly difference of OB.S/BS and OB.N/BS between cKO mice and WT mice. In (4I), we compared the mRNA expression of the osteogenic marker genes, Runx-2, OSX, BSP, ALP, and OCN in the long bones harvested from the 8-week-old Col1a1-CreERT2; FBXO11−/− mice, Col1a1-CreERT2; FBXO11+/− mice and WT mice after tamoxifen treatment. Knockdown of FBXO11 gene was confirmed by the significant reduction of FBXO11 gene mRNA expression in FBXO11−/− group and FBXO+/− group. The depletion of FBXO11 in osteoblasts decreased osteogenic marker genes’ expression in the long bones. The significant difference was found between cKO and WT mice in Runx-2, BSP and FBXO11. Data are the results of four long bone mRNA samples in each group and are presented as mean ± SEM. *P < 0.05.

Article Snippet: We obtained the primary antibodies from the following sources: Rabbit polyclonal antibody to FBXO11 (Novus Biologics, Centennial, CO), rabbit polyclonal antibody to Snail1 (Cell Signaling, Danvers, MA), mouse monoclonal antibody to α-tubulin, and mouse monoclonal antibody to GAPDH (Sigma-Aldrich, St. Louis, MO).

Techniques: Activity Assay, Injection, Staining, Labeling, Expressing, Marker, Knockdown

Journal: Bone

Article Title: FBXO11 regulates bone development

doi: 10.1016/j.bone.2023.116709

Figure Lengend Snippet: FBXO11cKO in osteoblastic cells caused osteogenic inhibition through Snail accumulation (6A) shows endogenous Snail1 protein level is lower in FBXO11 overexpression MC3T3 cells, while (6B) shows Snail1 protein level is higher in FBXO11-knockdown cells based on the change in relative protein level observed using western blotting. FBXO11-overexpressing MC3T3 cells and controls were pretreated with MG132 or vehicle for 6 h, and proteins were extracted, IP was performed with anti-Snail1 antibody, and immunoblotting with anti-ubiquitin antibody. Snail1 protein was degraded by FBXO11 through ubiquitination (6C). Snail1 overexpression in osteoblasts snail1 inhibits Overexpression their of osteogenic Snail1 in differentiation. MC3T3 was confirmed by WB with anti-Snail1 antibody (6D). Snail1 overexpression MC3T3 and its control were cultured in osteogenic induction medium in vitro. ALP staining was performed in 0, 7 day group, and AR mineralization staining was performed in 0, 14 day groups. mRNA expression of osteogenic marker genes Runx-2, OSX, Alp, and BSP were compared between Snail1 overexpression MC3T3 cells and their controls in 0, 3 days osteogenic induction groups. The weaker osteogenic differentiation was exhibited in in Snail1 overexpressing osteoblasts. (6E, 6F). In mouse femur bone, the osteoblasts from FBXO11cKO mice exhibits stronger Snail1 immunostaining that WT controls (6G). (X40). In Fig. 5F, all the experiments were repeated three times, the data present as Mean + SD, T-Test; *, P < 0.05; **, P < 0.01. V = control, shFBXO = FBXO11-shRNA.

Article Snippet: We obtained the primary antibodies from the following sources: Rabbit polyclonal antibody to FBXO11 (Novus Biologics, Centennial, CO), rabbit polyclonal antibody to Snail1 (Cell Signaling, Danvers, MA), mouse monoclonal antibody to α-tubulin, and mouse monoclonal antibody to GAPDH (Sigma-Aldrich, St. Louis, MO).

Techniques: Inhibition, Over Expression, Knockdown, Western Blot, Control, Cell Culture, In Vitro, Staining, Expressing, Marker, Immunostaining, shRNA

Journal: Clinical and Translational Medicine

Article Title: Cullin‐associated and neddylation‐dissociated 1 regulate reprogramming of lipid metabolism through SKP1‐Cullin‐1‐F‐box FBXO11 ‐mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma

doi: 10.1002/ctm2.1443

Figure Lengend Snippet: Cullin‐associated and neddylation‐dissociated 1 (CAND1) functions by regulating the SCFFBXO11 complex to recruit hnRNPA2B1. (A) A coimmunoprecipitation (co‐IP) experiment detected binding between CAND1 and CUL1. (B) CUL1 knockdown partially reversed the CAND1 overexpression‐induced promotion of cell invasion. (C) CUL1 knockdown reduced colony formation in CAND1 overexpressing HCC‐LM3 cells. (D) CCK8 assay showing that CUL1 knockdown reduces the proliferation of CAND1‐overexpressing cells. (E) BODIPY staining showed that lipid accumulation clearly decreased in the CUL1 knockdown group. (F) CUL1 knockdown partially reversed the CAND1 overexpression‐induced increase in intracellular triglycerides and cholesterol. (G) Relative quantitative mass spectrometry (MS)‐based proteomic analysis. (H) A co‐IP experiment was performed to detect binding between CUL1 and different F‐box proteins. (I) A co‐IP experiment was performed to detect binding between FBXO11 and hnRNPA2B1, and binding between hnRNPA2B1 and other F‐box proteins. (J) GST pull‐down assay. (K) A co‐IP experiment was performed to detect binding between HA‐FBXO11 and Flag‐hnRNPA2B1. (L) FBXO11 immunolabeling shows colocalization with hnRNPA2B1. All cellular experiments were run in triplicate and repeated three times. p < .05(*), p < .01(**) or p < .001(***).

Article Snippet: Antibodies against FBXO11 (27610‐1‐AP), CUL1 (12895‐1‐AP), FBXO5 (EPR15320‐103), FBW7 (28424‐1‐AP), FBX15 (13024‐1‐AP), hnRNPA2B1 (14813‐1‐AP), Flag (80010‐1‐RR), HA (51064‐2‐AP), His (66005‐1‐Ig), FASN (10624‐2‐AP), ACC (21923‐1‐AP), PPARD (60193‐1‐Ig), PPARG (66936‐1‐Ig), NR2F2 (24573‐1‐AP), TNFR1 (21574‐1‐AP), TRAF2 (26846‐1‐AP), P65 (80979‐1‐RR), Caspase8 (66093‐1‐Ig) and β‐ actin (66009‐1‐Ig) were purchased from Proteintech.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Knockdown, Over Expression, CCK-8 Assay, Staining, Mass Spectrometry, Pull Down Assay, Immunolabeling

Journal: Clinical and Translational Medicine

Article Title: Cullin‐associated and neddylation‐dissociated 1 regulate reprogramming of lipid metabolism through SKP1‐Cullin‐1‐F‐box FBXO11 ‐mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma

doi: 10.1002/ctm2.1443

Figure Lengend Snippet: hnRNPA2B1 mediates cullin‐associated and neddylation‐dissociated 1 (CAND1) function that was antagonized by FBXO11. (A) Protein expression was assessed by a western blot of cells with CAND1 expression knocked down. (B) The expression of hnRNPA2B1, FASN, ACC1 and ACLY increased when CAND1 was overexpressed. (C) Protein expression was assessed with cells in FBXO11 expression knocked down. (D) Protein expression in cells with FBXO11 expression knocked down. (E) A Cell Counting Kit‐8 (CCK‐8) assay suggesting that hnRNPA2B1 overexpression reverses the inhibition of cell proliferation mediated by CAND1 knockdown. (F) Colony formation assays demonstrate that overexpression of hnRNPA2B1 partially reversed the suppressed proliferation induced by CAND1 knockdown. (G‐H) CAND1 expression knockdown effects on cell migration and invasion were partially reversed by hnRNPA2B1 overexpression. (I) CAND1 knockdown downregulates hnRNPA2B1 expression, which was reversed by shFBXO11. (J) CAND1 promotes lipid synthesis, which is reversed by FBXO11. (K) CAND1 overexpression upregulates hnRNPA2B1 expression, which is reversed by overexpression of FBXO11. All cellular experiments were run in triplicate and repeated three times. p < .05(*), p < .01(**) or p < .001(***).

Article Snippet: Antibodies against FBXO11 (27610‐1‐AP), CUL1 (12895‐1‐AP), FBXO5 (EPR15320‐103), FBW7 (28424‐1‐AP), FBX15 (13024‐1‐AP), hnRNPA2B1 (14813‐1‐AP), Flag (80010‐1‐RR), HA (51064‐2‐AP), His (66005‐1‐Ig), FASN (10624‐2‐AP), ACC (21923‐1‐AP), PPARD (60193‐1‐Ig), PPARG (66936‐1‐Ig), NR2F2 (24573‐1‐AP), TNFR1 (21574‐1‐AP), TRAF2 (26846‐1‐AP), P65 (80979‐1‐RR), Caspase8 (66093‐1‐Ig) and β‐ actin (66009‐1‐Ig) were purchased from Proteintech.

Techniques: Expressing, Western Blot, Cell Counting, CCK-8 Assay, Over Expression, Inhibition, Knockdown, Migration

Journal: Clinical and Translational Medicine

Article Title: Cullin‐associated and neddylation‐dissociated 1 regulate reprogramming of lipid metabolism through SKP1‐Cullin‐1‐F‐box FBXO11 ‐mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma

doi: 10.1002/ctm2.1443

Figure Lengend Snippet: Cullin‐associated and neddylation‐dissociated 1 (CAND1) suppresses SCFFBXO11 complex‐mediated hnRNPA2B1 ubiquitination and degradation. (A) FBXO11 overexpression accelerates hnRNPA2B1 degradation. (B) FBXO11 expression knockdown decelerates hnRNPA2B1 degradation. (C) Knocking down CAND1 expression accelerates hnRNPA2B1 degradation. (D) MG132 significantly increases hnRNPA2B1 protein levels. (E) FBXO11‐induced degradation of hnRNPA2B1 is reversed by MG132 treatment. (F) The ubiquitination of hnRNPA2B1 is promoted by CAND1 expression knockdown. (G) The ubiquitination of hnRNPA2B1 is inhibited by CAND1 overexpression. (H) shRNA knockdown of FBXO11 expression levels decreased hnRNPA2B1 ubiquitination. (I) Overexpression of FBXO11 increased hnRNPA2B1 ubiquitination. (J) FBXO11 promotes hnRNPA2B1 ubiquitination in 293T cells. (K) hnRNPA2B1 protein levels are regulated by FBXO11 in a dose‐dependent manner. (L) Ubiquitination levels of hnRNPA2B1 are increased by FBXO11 in a dose‐dependent manner. (M) hnRNPA2B1 ubiquitination is regulated by CAND1 in a dose‐dependent manner. The experiments were dependently repeated three times.

Article Snippet: Antibodies against FBXO11 (27610‐1‐AP), CUL1 (12895‐1‐AP), FBXO5 (EPR15320‐103), FBW7 (28424‐1‐AP), FBX15 (13024‐1‐AP), hnRNPA2B1 (14813‐1‐AP), Flag (80010‐1‐RR), HA (51064‐2‐AP), His (66005‐1‐Ig), FASN (10624‐2‐AP), ACC (21923‐1‐AP), PPARD (60193‐1‐Ig), PPARG (66936‐1‐Ig), NR2F2 (24573‐1‐AP), TNFR1 (21574‐1‐AP), TRAF2 (26846‐1‐AP), P65 (80979‐1‐RR), Caspase8 (66093‐1‐Ig) and β‐ actin (66009‐1‐Ig) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Over Expression, Expressing, Knockdown, shRNA

Journal: Clinical and Translational Medicine

Article Title: Cullin‐associated and neddylation‐dissociated 1 regulate reprogramming of lipid metabolism through SKP1‐Cullin‐1‐F‐box FBXO11 ‐mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma

doi: 10.1002/ctm2.1443

Figure Lengend Snippet: FBXO11 directly binds to and promotes K27‐ and K48‐linked ubiquitination of hnRNPA2B1. (A) Domain architectures in FBXO11 proteins. (B) FBXO11 and mutant FBXO11 were overexpressed, and ubiquitination of hnRNPA2B1 was detected. (C, D) A co‐IP experiment was performed to detect the binding of hnRNPA2B1 to FBXO11 and mutant FBXO11. (E) Domains shown in the structure diagram of hnRNPA2B1. (F) Diagrammatic representation showing hnRNPA2B1 and its truncated forms. (G) A co‐IP experiment was performed to detect the binding of FBXO11 with hnRNPA2B1 and its truncated forms. (H) The degradation rate of mutant hnRNPA2B1 is not affected by FBXO11. (I) When FBXO11 was overexpressed, ubiquitination levels of wild‐type hnRNPA2B1 and mutant hnRNPA2B1 protein levels were detected. Compared with that of wild‐type hnRNPA2B1, the ubiquitination of mutant hnRNPA2B1 was decreased. (J) FBXO11 specifically promoted the addition of K27‐ and K48‐linked ubiquitin to hnRNPA2B1. (K) K27R and K48R ubiquitin could induce the ubiquitination of hnRNPA2B1. (L) Mutant K27 and K48 ubiquitin does not increase the extent of hnRNPA2B1 ubiquitination. The experiments were dependently repeated three times.

Article Snippet: Antibodies against FBXO11 (27610‐1‐AP), CUL1 (12895‐1‐AP), FBXO5 (EPR15320‐103), FBW7 (28424‐1‐AP), FBX15 (13024‐1‐AP), hnRNPA2B1 (14813‐1‐AP), Flag (80010‐1‐RR), HA (51064‐2‐AP), His (66005‐1‐Ig), FASN (10624‐2‐AP), ACC (21923‐1‐AP), PPARD (60193‐1‐Ig), PPARG (66936‐1‐Ig), NR2F2 (24573‐1‐AP), TNFR1 (21574‐1‐AP), TRAF2 (26846‐1‐AP), P65 (80979‐1‐RR), Caspase8 (66093‐1‐Ig) and β‐ actin (66009‐1‐Ig) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Mutagenesis, Co-Immunoprecipitation Assay, Binding Assay

Journal: Clinical and Translational Medicine

Article Title: Cullin‐associated and neddylation‐dissociated 1 regulate reprogramming of lipid metabolism through SKP1‐Cullin‐1‐F‐box FBXO11 ‐mediated heterogeneous nuclear ribonucleoprotein A2/B1 ubiquitination and promote hepatocellular carcinoma

doi: 10.1002/ctm2.1443

Figure Lengend Snippet: AAV‐shCAND1 effectively inhibits hepatocellular carcinoma (HCC) as a gene therapy. (A) Images of the patient‐derived xenograft (PDX) mice. (B) Plot showing tumour volume over time in the PDX mouse model. (C) Statistics of body weights of the PDX mice. (D) Interstitial fluid pressure (IFP) of tumours in PDX models. (E) Survival curve of the mice bearing PDX tumours. (F) Images of tumours in the PDX models. (G) Statistics of tumour weight of PDX mice. (H) Immunohistochemical staining of CAND1, hnRNPA2B1, FASN, ACC1 and ACLY, and oil red O staining. (I) Representative images of mouse livers with tumours induced by myr‐AKT/NRASV12 and immunohistochemical staining of fatty acid synthesis‐related proteins and oil red O staining of HCC mouse model tumour tissue. (J) IHC of tumour tissue from HCC patients. (K) Diagram of the molecular mechanisms underlying the CAND1‐SCF FBXO11 ‐hnRNPA2B1 axis. p < .05(*), p < .01(**) or p < .001(***).

Article Snippet: Antibodies against FBXO11 (27610‐1‐AP), CUL1 (12895‐1‐AP), FBXO5 (EPR15320‐103), FBW7 (28424‐1‐AP), FBX15 (13024‐1‐AP), hnRNPA2B1 (14813‐1‐AP), Flag (80010‐1‐RR), HA (51064‐2‐AP), His (66005‐1‐Ig), FASN (10624‐2‐AP), ACC (21923‐1‐AP), PPARD (60193‐1‐Ig), PPARG (66936‐1‐Ig), NR2F2 (24573‐1‐AP), TNFR1 (21574‐1‐AP), TRAF2 (26846‐1‐AP), P65 (80979‐1‐RR), Caspase8 (66093‐1‐Ig) and β‐ actin (66009‐1‐Ig) were purchased from Proteintech.

Techniques: Derivative Assay, Immunohistochemical staining, Staining