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Generation of a neuronal <t>FBXO11</t> 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 <xref ref-type=

antibodies against fbxo11 - by Bioz Stars, 2026-05

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1) Product Images from "Proteasomal activation ameliorates neuronal phenotypes linked to FBXO11 -deficiency"

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

Journal: Human Genetics and Genomics Advances

doi: 10.1016/j.xhgg.2025.100425

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 <xref ref-type=

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). " title="Generation of a neuronal FBXO11 deficient cell model using CRISPR-CAS9 (A) Outline of ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend 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).

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

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 <xref ref-type=

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. " title="Gene expression changes due to loss of FBXO11 in human neurons and fly heads (A) Principal-component ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend 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.

Techniques Used: Gene Expression, Migration, Expressing

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 <xref ref-type=

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. " title="Loss of FBXO11 alters neuronal migration, proliferation, and differentiation (A) Representative ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Climbing Assay, Knockdown, Control, Microscopy

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.

Figure Legend 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.

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

Image Search Results

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.

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.

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.

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.

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

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(***).

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

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(***).

Journal: Clinical and Translational Medicine

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(***).

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

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.

Journal: Clinical and Translational Medicine

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.

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

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.

Journal: Clinical and Translational Medicine

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.

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

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(***).

Journal: Clinical and Translational Medicine

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(***).

Techniques: Derivative Assay, Immunohistochemical staining, Staining

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