Journal: Human Molecular Genetics

Article Title: De novo missense variants in FBXO11 alter its protein expression and subcellular localization

doi: 10.1093/hmg/ddab265

Figure Lengend Snippet: Overview of the clinical and mutational spectrum in individuals with FBXO11 aberrations. ( A ) Clinical images of affected individuals. ( B ) Schematic drawing of FBXO11 (NM_001190274.1, NP_001177203.1) with annotation of novel [top, blue (missense variants) and purple (LGD variants)] and previously published (bottom, black) aberrations. Domains are color-coded according to InterPro . The spectrum encompasses large multi-gene and intragenic deletions, LGD variants, single amino acid deletions and missense variants. Most variants are unique, recurrent variants are underlined. All variants included in the functional assays are marked with the symbol of hash. LGD variants predicted to escape nonsense mediated decay are marked with a black box. Single letter amino acid codes were used due to space constraints.

Article Snippet: Staining was performed with antibodies against FBXO11 (NB100-59826, Novus Biologicals, Centennial, CO, USA 1:200), Myc (M4439, Sigma-Aldrich, St. Louis, MO, USA 1:500), EEA1 (610 456, BD Biosciences, Franklin Lakes, NJ, USA 1:200), GOLGIN-97 (A-21270, Thermo Scientific, Waltham, MA, USA 1:100), LAMP-1 (sc-20 011, Santa Cruz Biotechnology, CA, USA 1:500), SEC31A (612 350, BD Biosciences, Franklin Lakes, NJ, USA 1:1000), SKP1 (2156, Cell Signaling, Cambridge, UK; Millipore, Burlington, MA, USA 1:50), CUL1 (sc-17 775, Santa Cruz Biotechnology, CA, USA 1:50), FK2 (04–263, Millipore, Burlington, MA, USA 1:500) at indicated dilutions.

Techniques: Functional Assay

Journal: Human Molecular Genetics

Article Title: De novo missense variants in FBXO11 alter its protein expression and subcellular localization

doi: 10.1093/hmg/ddab265

Figure Lengend Snippet: FBXO11 variants affect stability of the protein. Results of mutational modelling for three novel [p.(Gly421 Arg), p.(Gly549His) and p.(Asn679Ser)] and one previously published missense variants [p.(Tyr506Cys; ]are shown. ( A ) Vicinity of G421 (dark grey) within the β-helix (β-strands are indicated as yellow arrows and key residues are shown in space-filled presentation). ( B ) The R421 sidechain (dark grey) points towards the interior of the β-helix and forms steric clashes (red arrows) with the sidechains of I400, I415 and H418. ( C ) Vicinity of G549 within the β-helix. ( D ) The bulkier R549 sidechain forms steric clashes (red arrows) with the sidechains of H594 and Y598. ( E ) Y506 forms hydrophobic interactions (green arrows) with H508 and W529. ( F ) These interactions cannot be formed by the shorter cysteine sidechain in the Y506C variant. ( G ) N679 forms a sidechain hydrogen bond (green line) to N656. ( H ) This interaction cannot be formed by the shorter serine sidechain in the N679S variant.

Article Snippet: Staining was performed with antibodies against FBXO11 (NB100-59826, Novus Biologicals, Centennial, CO, USA 1:200), Myc (M4439, Sigma-Aldrich, St. Louis, MO, USA 1:500), EEA1 (610 456, BD Biosciences, Franklin Lakes, NJ, USA 1:200), GOLGIN-97 (A-21270, Thermo Scientific, Waltham, MA, USA 1:100), LAMP-1 (sc-20 011, Santa Cruz Biotechnology, CA, USA 1:500), SEC31A (612 350, BD Biosciences, Franklin Lakes, NJ, USA 1:1000), SKP1 (2156, Cell Signaling, Cambridge, UK; Millipore, Burlington, MA, USA 1:50), CUL1 (sc-17 775, Santa Cruz Biotechnology, CA, USA 1:50), FK2 (04–263, Millipore, Burlington, MA, USA 1:500) at indicated dilutions.

Techniques: Variant Assay

Journal: Human Molecular Genetics

Article Title: De novo missense variants in FBXO11 alter its protein expression and subcellular localization

doi: 10.1093/hmg/ddab265

Figure Lengend Snippet: Overview of functional testing results of FBXO11 missense variants

Article Snippet: Staining was performed with antibodies against FBXO11 (NB100-59826, Novus Biologicals, Centennial, CO, USA 1:200), Myc (M4439, Sigma-Aldrich, St. Louis, MO, USA 1:500), EEA1 (610 456, BD Biosciences, Franklin Lakes, NJ, USA 1:200), GOLGIN-97 (A-21270, Thermo Scientific, Waltham, MA, USA 1:100), LAMP-1 (sc-20 011, Santa Cruz Biotechnology, CA, USA 1:500), SEC31A (612 350, BD Biosciences, Franklin Lakes, NJ, USA 1:1000), SKP1 (2156, Cell Signaling, Cambridge, UK; Millipore, Burlington, MA, USA 1:50), CUL1 (sc-17 775, Santa Cruz Biotechnology, CA, USA 1:50), FK2 (04–263, Millipore, Burlington, MA, USA 1:500) at indicated dilutions.

Techniques: Functional Assay, Expressing, Variant Assay

Journal: Human Molecular Genetics

Article Title: De novo missense variants in FBXO11 alter its protein expression and subcellular localization

doi: 10.1093/hmg/ddab265

Figure Lengend Snippet: FBXO11 variants affect subcellular localization. Schematic drawing of FBXO11 on top left indicates variants with altered subcellular localization in different colors (nuclear exclusion: purple, cytoplasmic aggregates: blue). For immunofluorescence analysis HeLa cells were transiently transfected with 400 ng per 12 well of wildtype or mutant Myc-FBXO11. Cells were fixed 48 h post transfection and stained with an anti-Myc antibody. Images were taken on a Axioimager Z2 with Apotome. Scale bar 20 μM. Arrows point to subcellular aggregates present in several mutants.

Article Snippet: Staining was performed with antibodies against FBXO11 (NB100-59826, Novus Biologicals, Centennial, CO, USA 1:200), Myc (M4439, Sigma-Aldrich, St. Louis, MO, USA 1:500), EEA1 (610 456, BD Biosciences, Franklin Lakes, NJ, USA 1:200), GOLGIN-97 (A-21270, Thermo Scientific, Waltham, MA, USA 1:100), LAMP-1 (sc-20 011, Santa Cruz Biotechnology, CA, USA 1:500), SEC31A (612 350, BD Biosciences, Franklin Lakes, NJ, USA 1:1000), SKP1 (2156, Cell Signaling, Cambridge, UK; Millipore, Burlington, MA, USA 1:50), CUL1 (sc-17 775, Santa Cruz Biotechnology, CA, USA 1:50), FK2 (04–263, Millipore, Burlington, MA, USA 1:500) at indicated dilutions.

Techniques: Immunofluorescence, Transfection, Mutagenesis, Staining

Journal: Human Molecular Genetics

Article Title: De novo missense variants in FBXO11 alter its protein expression and subcellular localization

doi: 10.1093/hmg/ddab265

Figure Lengend Snippet: FBXO11 variants affect protein expression levels. ( A ) Representative image of western blots used for quantification of FBXO11 expression levels is shown. ( B ) Quantification of FBXO11 protein levels from western blotting. Value of wildtype FBXO11 was set to 100. Experiments were repeated at least 4 times. Individual values are shown as dots with mean values shown as bars with SEM. P -values were calculated using a one sample t-test with the hypothetical mean set to 100 and a significance threshold of < 0.05 ( * < 0.05, * * < 0.01). ( C ) Schematic overview of different functional consequences observed for tested missense variants with schematic drawing of protein with mutations color-coded according to results of functional assays (red: mislocalization and reduced expression, green: mislocalization, blue: reduced protein expression, black: no functional defect observed).

Article Snippet: Staining was performed with antibodies against FBXO11 (NB100-59826, Novus Biologicals, Centennial, CO, USA 1:200), Myc (M4439, Sigma-Aldrich, St. Louis, MO, USA 1:500), EEA1 (610 456, BD Biosciences, Franklin Lakes, NJ, USA 1:200), GOLGIN-97 (A-21270, Thermo Scientific, Waltham, MA, USA 1:100), LAMP-1 (sc-20 011, Santa Cruz Biotechnology, CA, USA 1:500), SEC31A (612 350, BD Biosciences, Franklin Lakes, NJ, USA 1:1000), SKP1 (2156, Cell Signaling, Cambridge, UK; Millipore, Burlington, MA, USA 1:50), CUL1 (sc-17 775, Santa Cruz Biotechnology, CA, USA 1:50), FK2 (04–263, Millipore, Burlington, MA, USA 1:500) at indicated dilutions.

Techniques: Expressing, Western Blot, Functional Assay

Journal: The Journal of Clinical Investigation

Article Title: PRMT5-mediated FUBP1 methylation accelerates prostate cancer progression

doi: 10.1172/JCI175023

Figure Lengend Snippet: ( A ) FUBP1 methylation status after PRMT5 or PRMT9 knockdown in HEK293T cells. Scr, scrambled control siRNA. ( B ) Re-expression of PRMT5 rescues FUBP1 methylation in HEK293T cells. DM1, a PRMT5 enzyme-dead mutant with G365A/R368A mutations; DM2, a PRMT5 enzyme-dead mutant with an E444Q mutation. ( C ) Status of FUBP1 methylation in different cell lines after treatment with the PRMT5 inhibitor GSK591, 100 nM. ( D ) PRMT5 inhibition specifically affects FUBP1 methylation at R359/R361/R363 in LNCaP cells. FUBP1 and FUBP1 3K , a mutant with R359/R361/R363K mutations, were transiently expressed in LNCaP cells. ( E ) FUBP1 methylation in PRMT5-depleted prostate cancer cell lines. ( F ) Expression levels of FUBP1 target genes in LNCaP and VCaP cells with PRMT5 depletion. ( G ) FUBP1 methylation in MTAP-depleted prostate cancer cells. ( H ) Expression levels of FUBP1 target genes in MTAP-depleted LNCaP and VCaP cells. ( I ) Endogenous interaction of FUBP1 with PRMT5 in various cell lines. ( J ) Direct binding of PRMT5 to FUBP1 in vitro. His-FUBP1 was purified from E. coli , and FLAG-PRMT5 was enriched from HEK293T cell lysate. ( K ) In vitro methylation of FUBP1 by PRMT5. ( L ) Endogenous FUBP1 methylation and PRMT5 in different cell lines. Results are shown as mean ± SD. ** P < 0.01; 1-way ANOVA with Dunnett’s (T3) multiple-comparison test.

Article Snippet: The following antibodies were used: FUBP1 (Abcam, ab192867), PRMT5 (Cell Signaling Technology, 79998), PRMT9 (Proteintech, 67365-1-Ig), PDK1 (Proteintech, 18262-1-AP), SLC7A11 (Proteintech, 26864-1-AP), FLAG (ABClonal, AE063), monomethyl arginine (Cell Signaling Technology, 8015), symmetric dimethyl arginine (Cell Signaling Technology, 13222), asymmetric dimethyl arginine (Cell Signaling Technology, 13522), BRD4 (Cell Signaling Technology, E2A7X), Myc (c#006-549), MTAP (Abcam, ab126770), SV40T (Abcam, 16879), α-SMA (Sigma-Aldrich, 2547), and actin (ABClonal, AC006).

Techniques: Methylation, Knockdown, Control, Expressing, Mutagenesis, Inhibition, Binding Assay, In Vitro, Purification, Comparison

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