Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: Overexpression of RBX2 in colorectal carcinoma tissues and cell lines. A. Relative expression of RBX2 mRNA in 56 CRC cancer and paired adjacent normal tissues as determined by qPCR. Increased expression of RBX2 mRNA as compared with normal tissue was observed. B. Expression of RBX2 protein was determined by IHC analysis. Representative images of IHC staining of RBX2 in CRC cancer tissues and adjacent normal tissues. Scale bar represents 100 μm. C. Box plots show increased levels of RBX2 in CRC (right) compared with normal tissues in Skrzypczak colorectal microarray data set. **P < 0.01, compared with normal colon tissues was determined by the Student’s t test. D. Western blotting analysis (left panel) of the level of RBX2 in various CRC cell lines. β-actin was used as loading controls. qPCR analysis (right panel) of RBX2 mRNA level in CRC cells. PCR values were normalized to the levels of β-actin. Data were presented as the mean ± SD from three independent measurements. E. Representative fluorescence activated cell sorter (FACS) dot plots of HCT116, SW480, LOVO, and HT29 cells stained with the anti-RBX2 antibody. Histograms reported the percentage of RBX2 positive cells as assessed by FACS. Mean ± SD of three independent experiments. Quantitative analysis demonstrates expression of RBX2 positive CRC cells range from 40%-60%.

Article Snippet: HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Over Expression, Expressing, Immunohistochemistry, Microarray, Western Blot, Fluorescence, Staining

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: RBX2 depletion inhibits growth in colorectal cancer cells. A. Western blot to test the efficiency and specificity of RBX2 knockout plasmid transfection was shown completely loss of RBX2 relative to parental HCT116 and SW480 cells (left panel). qPCR analysis of RBX2 mRNA levels in both CRC cell lines. PCR values were normalized to the levels of β-actin. Data are presented as the mean ± SD from three independent measurements. B. Cell growth rate from parental and RBX2 knockout HCT116 cells (left) and SW480 cells (right) at indicated time point. C. Colony formation assay of parental and RBX2 knockout HCT116 and SW480 cells. 1000 cells were plated in 6-well plate in complete medium and cultured for 14 days and colonies were stained and counted. D. Representative images of xenografts. Tumor growth curve for HCT116 (left panel) and SW480 (right panel) xenogaft tumor models. Immunohistochemistry analysis of RBX2 and Ki67 in xenografts of the indicated groups. Scale bar represents 100 μm.

Article Snippet: HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Western Blot, Knock-Out, Plasmid Preparation, Transfection, Colony Assay, Cell Culture, Staining, Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: RBX2 loss suppresses cancer metastasis. A. In vitro wound healing assay with human HCT116 and SW480 cells after knock out with RBX2 expression. Image was acquired at 0, 24 h time points after scratching (left panel). Scale bar represents 100 μm. Quantification of wound closure was calculated (right panel). B. Representative staining of invasive potentials of human HCT116 and SW480 cells from Transwell assay in vitro (left panel). Quantification of invasive cells per field was analyzed (right panel). Scale bar: 100 μm. Data were compared using the two-tailed students t-test, for indicated comparisons, **P < 0.01. C. RBX2 loss significant compromised tumor metastasis in melanoma B16F10 cells lung metastasis model in vivo. Quantification of lung metastatic was calculated (right panel).

Article Snippet: HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: In Vitro, Wound Healing Assay, Knock-Out, Expressing, Staining, Transwell Assay, Two Tailed Test, In Vivo

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: The effects of RBX2 modulation on the growth of xenografts in nude mice. A. Western blot analysis of RBX2 in HCT116 and SW480 cells transfected with vector or RBX2 (left panel). RBX2 mRNA level in HCT116 and SW480 cell lines are evaluated by qPCR analysis (right panel). PCR values were normalized to the levels of β-actin. Data are presented as the mean ± SD from three independent measurements. B. MTT analysis of CRC cancer cells infected with the indicated lentivirus. 3 × 103 cells were seeded in 96 well plates and cultured for the indicated hours. C. Colony formation assay of parental and RBX2 over-expressing CRC cells. Cells were plated in 6-well plate in complete medium and cultured for 14 days and colonies were stained and counted. D. HCT116 or SW480 cells infected with RBX2 or vector and then were implanted subcutaneously into Balb/c-nude mice to form xenografts. Representative images of tumor xenografts and immunohistochemistry analysis of Ki67 in xenografts of the indicated groups. Scale bar: 100 μm.

Article Snippet: HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Plasmid Preparation, Infection, Cell Culture, Colony Assay, Expressing, Staining, Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: RBX2 overexpression in CRC cancer cells increase the migration and invasion. A. HCT116 and SW480 CRC cells with high RBX2 expression exhibited stronger migration abilities in wound healing assay (left panel). Scale bar represents 200 μm. Quantification of wound healing was calculated (right panel). Scale bar: 100 μm. B. HCT116 and SW480 CRC cells with high RBX2 expression exhibited stronger invasion abilities in Transwell assay. Scale bar: 200 μm. Data were compared using the two-tailed students t-test, for indicated comparisons, **P < 0.01 compared with the cells transfected with vector. Scale bar represents 100 μm. C. More metastatic foci in lung were visible in mouse injected with B16F10-RBX2 cells (left panel). Data were compared using the two-tailed Students t-test, **P < 0.01 compared with B16F10 cells transfected with vector. Quantification of lung metastasis loci was calculated (right panel).

Article Snippet: HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Over Expression, Migration, Expressing, Wound Healing Assay, Transwell Assay, Two Tailed Test, Transfection, Plasmid Preparation, Injection

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: RBX2 enhances chemotherapeutic sensitivity in colon cancer cells. A. RBX2 knock-out (KO) significantly enhanced the sensitivity of HCT116 to paclitaxel and significantly reduced their IC50 based on the MTT assay. The data were presented as mean ± SD. The values were expressed as percentage of viable cells normalized to percentage of viable cells in 0.5% DMSO-treated cells. The concentration of paclitaxel resulting in 50% inhibition of control growth (IC50) was calculated by SPSS statistics software using Probit model. B. Colony formation assay was performed to detect the chemotherapeutic effects of paclitaxel on RBX2 KO HCT116 cells and control cells growth in vitro. 1000 RBX2 KO HCT116 cells or parental cells were plated in 6-well plate in complete medium and cultured with paclitaxel. After two weeks, colonies were stained and counted. C. RBX2 knock-out HCT116 cells showed greater sensitivity towards paclitaxel. Values were presented as the mean ± SD for three independent experiments. **P < 0.01 compared with control cells, ##P < 0.01 compared to control cells treated with paclitaxel.

Article Snippet: HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Knock-Out, MTT Assay, Concentration Assay, Inhibition, Control, Software, Colony Assay, In Vitro, Cell Culture, Staining

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: Inhibition of mTOR significantly suppresses RBX2 over-expression tumor cell growth. A. Enrichment scores of signaling pathways among RBX2 targets. B. Heat-map of genes differentially expressed in parental cells and RBX2 KO HCT116 cells. C. Western blot analysis of the expression of p-mTORC1S2448, p-mTORC2S2481, t-mTOR, p-S6K1T389 and S6K1 in the HCT116 cells in response to RBX2 up-regulation. β-actin was used as loading control. D. Whole cell lysates were prepared from everolimus and vehicle treated HCT116-RBX2 cells and examined for p-mTORC1S2448, p-mTORC2S2481, t-mTOR, p-S6K1T389 and S6K1 by immunoblotting. E. Cell growth rate from everolimus and vehicle treated HCT116-RBX2. F. Representative pictures (left panel) and statistical analysis (right panel) shown colony formation from everolimus and vehicle treated HCT116-RBX2 cells. G. In vitro wound closure of everolimus and vehicle treated HCT116-RBX2 cells from 24 h after scratch assay. Scale bar: 100 μm. H. Transwell invasion assay of everolimus and vehicle treated HCT116-RBX2 cells (left panel). Quantification of invasive cells per field was analyzed (right panel). Scale bar: 100 μm. Data were compared using the two-tailed students t-test, for indicated comparisons, **P < 0.01 compared to the cells treatment with vehicle. I. Mice were injected with HCT116-RBX2 cells and then were treated with everolimus and vehicle (n = 6). Tumor volume was analyzed at indicated time point. J. Histochemistry staining of Ki67, p-mTORC1S2448, p-mTORC2S2481 and p-S6K1T389 shown decreased positive cells from everolimus treated tumors originally from injection of HCT116-RBX2 cells compare to vehicle-treated group. Scale bar: 100 μm.

Article Snippet: HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Inhibition, Over Expression, Protein-Protein interactions, Western Blot, Expressing, Control, In Vitro, Wound Healing Assay, Transwell Invasion Assay, Two Tailed Test, Injection, Staining

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: Proposed model of RBX2 promotes colon cancer metastasis via mTOR/S6K1 signaling pathway.

Article Snippet: HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques:

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: Overexpression of RBX2 in colorectal carcinoma tissues and cell lines. A. Relative expression of RBX2 mRNA in 56 CRC cancer and paired adjacent normal tissues as determined by qPCR. Increased expression of RBX2 mRNA as compared with normal tissue was observed. B. Expression of RBX2 protein was determined by IHC analysis. Representative images of IHC staining of RBX2 in CRC cancer tissues and adjacent normal tissues. Scale bar represents 100 μm. C. Box plots show increased levels of RBX2 in CRC (right) compared with normal tissues in Skrzypczak colorectal microarray data set. **P < 0.01, compared with normal colon tissues was determined by the Student’s t test. D. Western blotting analysis (left panel) of the level of RBX2 in various CRC cell lines. β-actin was used as loading controls. qPCR analysis (right panel) of RBX2 mRNA level in CRC cells. PCR values were normalized to the levels of β-actin. Data were presented as the mean ± SD from three independent measurements. E. Representative fluorescence activated cell sorter (FACS) dot plots of HCT116, SW480, LOVO, and HT29 cells stained with the anti-RBX2 antibody. Histograms reported the percentage of RBX2 positive cells as assessed by FACS. Mean ± SD of three independent experiments. Quantitative analysis demonstrates expression of RBX2 positive CRC cells range from 40%-60%.

Article Snippet: Establishment of RBX2 knockout and stable expression cells HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Over Expression, Expressing, Immunohistochemistry, Microarray, Western Blot, Fluorescence, Staining

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: RBX2 depletion inhibits growth in colorectal cancer cells. A. Western blot to test the efficiency and specificity of RBX2 knockout plasmid transfection was shown completely loss of RBX2 relative to parental HCT116 and SW480 cells (left panel). qPCR analysis of RBX2 mRNA levels in both CRC cell lines. PCR values were normalized to the levels of β-actin. Data are presented as the mean ± SD from three independent measurements. B. Cell growth rate from parental and RBX2 knockout HCT116 cells (left) and SW480 cells (right) at indicated time point. C. Colony formation assay of parental and RBX2 knockout HCT116 and SW480 cells. 1000 cells were plated in 6-well plate in complete medium and cultured for 14 days and colonies were stained and counted. D. Representative images of xenografts. Tumor growth curve for HCT116 (left panel) and SW480 (right panel) xenogaft tumor models. Immunohistochemistry analysis of RBX2 and Ki67 in xenografts of the indicated groups. Scale bar represents 100 μm.

Article Snippet: Establishment of RBX2 knockout and stable expression cells HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Western Blot, Knock-Out, Plasmid Preparation, Transfection, Colony Assay, Cell Culture, Staining, Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: RBX2 loss suppresses cancer metastasis. A. In vitro wound healing assay with human HCT116 and SW480 cells after knock out with RBX2 expression. Image was acquired at 0, 24 h time points after scratching (left panel). Scale bar represents 100 μm. Quantification of wound closure was calculated (right panel). B. Representative staining of invasive potentials of human HCT116 and SW480 cells from Transwell assay in vitro (left panel). Quantification of invasive cells per field was analyzed (right panel). Scale bar: 100 μm. Data were compared using the two-tailed students t-test, for indicated comparisons, **P < 0.01. C. RBX2 loss significant compromised tumor metastasis in melanoma B16F10 cells lung metastasis model in vivo. Quantification of lung metastatic was calculated (right panel).

Article Snippet: Establishment of RBX2 knockout and stable expression cells HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: In Vitro, Wound Healing Assay, Knock-Out, Expressing, Staining, Transwell Assay, Two Tailed Test, In Vivo

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: The effects of RBX2 modulation on the growth of xenografts in nude mice. A. Western blot analysis of RBX2 in HCT116 and SW480 cells transfected with vector or RBX2 (left panel). RBX2 mRNA level in HCT116 and SW480 cell lines are evaluated by qPCR analysis (right panel). PCR values were normalized to the levels of β-actin. Data are presented as the mean ± SD from three independent measurements. B. MTT analysis of CRC cancer cells infected with the indicated lentivirus. 3 × 103 cells were seeded in 96 well plates and cultured for the indicated hours. C. Colony formation assay of parental and RBX2 over-expressing CRC cells. Cells were plated in 6-well plate in complete medium and cultured for 14 days and colonies were stained and counted. D. HCT116 or SW480 cells infected with RBX2 or vector and then were implanted subcutaneously into Balb/c-nude mice to form xenografts. Representative images of tumor xenografts and immunohistochemistry analysis of Ki67 in xenografts of the indicated groups. Scale bar: 100 μm.

Article Snippet: Establishment of RBX2 knockout and stable expression cells HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Plasmid Preparation, Infection, Cell Culture, Colony Assay, Expressing, Staining, Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: RBX2 overexpression in CRC cancer cells increase the migration and invasion. A. HCT116 and SW480 CRC cells with high RBX2 expression exhibited stronger migration abilities in wound healing assay (left panel). Scale bar represents 200 μm. Quantification of wound healing was calculated (right panel). Scale bar: 100 μm. B. HCT116 and SW480 CRC cells with high RBX2 expression exhibited stronger invasion abilities in Transwell assay. Scale bar: 200 μm. Data were compared using the two-tailed students t-test, for indicated comparisons, **P < 0.01 compared with the cells transfected with vector. Scale bar represents 100 μm. C. More metastatic foci in lung were visible in mouse injected with B16F10-RBX2 cells (left panel). Data were compared using the two-tailed Students t-test, **P < 0.01 compared with B16F10 cells transfected with vector. Quantification of lung metastasis loci was calculated (right panel).

Article Snippet: Establishment of RBX2 knockout and stable expression cells HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Over Expression, Migration, Expressing, Wound Healing Assay, Transwell Assay, Two Tailed Test, Transfection, Plasmid Preparation, Injection

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: RBX2 enhances chemotherapeutic sensitivity in colon cancer cells. A. RBX2 knock-out (KO) significantly enhanced the sensitivity of HCT116 to paclitaxel and significantly reduced their IC50 based on the MTT assay. The data were presented as mean ± SD. The values were expressed as percentage of viable cells normalized to percentage of viable cells in 0.5% DMSO-treated cells. The concentration of paclitaxel resulting in 50% inhibition of control growth (IC50) was calculated by SPSS statistics software using Probit model. B. Colony formation assay was performed to detect the chemotherapeutic effects of paclitaxel on RBX2 KO HCT116 cells and control cells growth in vitro. 1000 RBX2 KO HCT116 cells or parental cells were plated in 6-well plate in complete medium and cultured with paclitaxel. After two weeks, colonies were stained and counted. C. RBX2 knock-out HCT116 cells showed greater sensitivity towards paclitaxel. Values were presented as the mean ± SD for three independent experiments. **P < 0.01 compared with control cells, ##P < 0.01 compared to control cells treated with paclitaxel.

Article Snippet: Establishment of RBX2 knockout and stable expression cells HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Knock-Out, MTT Assay, Concentration Assay, Inhibition, Control, Software, Colony Assay, In Vitro, Cell Culture, Staining

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: Inhibition of mTOR significantly suppresses RBX2 over-expression tumor cell growth. A. Enrichment scores of signaling pathways among RBX2 targets. B. Heat-map of genes differentially expressed in parental cells and RBX2 KO HCT116 cells. C. Western blot analysis of the expression of p-mTORC1S2448, p-mTORC2S2481, t-mTOR, p-S6K1T389 and S6K1 in the HCT116 cells in response to RBX2 up-regulation. β-actin was used as loading control. D. Whole cell lysates were prepared from everolimus and vehicle treated HCT116-RBX2 cells and examined for p-mTORC1S2448, p-mTORC2S2481, t-mTOR, p-S6K1T389 and S6K1 by immunoblotting. E. Cell growth rate from everolimus and vehicle treated HCT116-RBX2. F. Representative pictures (left panel) and statistical analysis (right panel) shown colony formation from everolimus and vehicle treated HCT116-RBX2 cells. G. In vitro wound closure of everolimus and vehicle treated HCT116-RBX2 cells from 24 h after scratch assay. Scale bar: 100 μm. H. Transwell invasion assay of everolimus and vehicle treated HCT116-RBX2 cells (left panel). Quantification of invasive cells per field was analyzed (right panel). Scale bar: 100 μm. Data were compared using the two-tailed students t-test, for indicated comparisons, **P < 0.01 compared to the cells treatment with vehicle. I. Mice were injected with HCT116-RBX2 cells and then were treated with everolimus and vehicle (n = 6). Tumor volume was analyzed at indicated time point. J. Histochemistry staining of Ki67, p-mTORC1S2448, p-mTORC2S2481 and p-S6K1T389 shown decreased positive cells from everolimus treated tumors originally from injection of HCT116-RBX2 cells compare to vehicle-treated group. Scale bar: 100 μm.

Article Snippet: Establishment of RBX2 knockout and stable expression cells HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques: Inhibition, Over Expression, Protein-Protein interactions, Western Blot, Expressing, Control, In Vitro, Wound Healing Assay, Transwell Invasion Assay, Two Tailed Test, Injection, Staining

Journal: American Journal of Cancer Research

Article Title: Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis

doi:

Figure Lengend Snippet: Proposed model of RBX2 promotes colon cancer metastasis via mTOR/S6K1 signaling pathway.

Article Snippet: Establishment of RBX2 knockout and stable expression cells HCT116 and SW480 cells were transfected with RBX2 CRISPR/Cas9 knock out (KO) Plasmid (Santa Cruz, sc-417138) using lipofectamine 2000 (Invitrogene) according to the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Schematic of APEX2-catalyzed biotinylation at the mitochondrial outer membrane (MOM) via fusion to a mitochondrial targeted peptide derived from MAVS (mitochondrial antiviral-signaling protein). Overlap of APEX2-MOM data with the ESI (E3-substrate interaction) network reveals the association of CRLs with mitochondria. B , Representative Western blot of biotinylated proteins in neonatal rat ventricular cardiomyocytes (NRVCs) with adenoviral (Ad) expression of APEX2-MOM. NRVCs were treated with CCCP (10 µM) before H 2 O 2 activation. The resultant biotinylated proteins were enriched by streptavidin beads. C , Representative Western blot of cytosol and mitochondrial fractions from NRVCs treated with CCCP (10 µM) for the indicated times. Arrowhead, neddylated CUL5. Tubulin and VDAC serve as cytosol and mitochondrial markers, respectively. D , Immuno-gold electron microscopic images showing the localization of RBX2 on mitochondrial membranes (arrowheads) in NRVCs expressing HA-RBX2. NRVCs infected with Ad-GFP and Ad-HA-OMP25 (MOM protein) serve as negative and positive controls, respectively. Scale bars, 0.5 µm. E , Representative Western blot of mitochondria with or without proteinase K (PK) treatment for 30 min on ice after isolation from NRVCs. F , Confocal images showing the colocalization (arrowhead) of RBX2 with mitochondria in CCCP-treated cardiomyocytes. NRVCs with adenoviral expression of HA-RBX2 were treated with CCCP for 3 hours and stained or immunostained as indicated. HA, green. Mitotracker, red. TOMM20, blue. Line scan co-localization analysis was done for all channels. Scale bars, 50 µm.

Article Snippet: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

Techniques: Membrane, Derivative Assay, Western Blot, Expressing, Activation Assay, Infection, Isolation, Staining

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Schematics of creation of tamoxifen-inducible, cardiac-specific RBX2 knockout (iCKO) mice. B , Western blot of indicated proteins in mouse hearts at 12 days after tamoxifen injection. C , Quantification of B . D , Survival curve. E , Gross morphology of mouse heart (top) and hematoxylin and eosin staining of myocardium section (bottom) at 12 days after tamoxifen injection. F , Heart weight to tibial length ratio and lung weight to tibial length ratio. F/F: n=13, MCM: n=5, iCKO: n=18. G , Representative B-mode images. H , Quantification of echocardiographic parameters before (F/F: n=17, MCM: n=6, iCKO: n=23) and after (F/F: n=12, MCM: n=6, iCKO: n=12) tamoxifen treatment. I , Wheat germ agglutinin (WGA) staining (left) of myocardium sections and quantification of cardiomyocyte (CM) cross-sectional area (right). More than 100 cells/heart and 3 and 12 hearts from F/F and iCKO mice, respectively, were quantified. J , Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of myocardium sections (left) and quantification (right). Three fields per heart, and 3 hearts per group, were quantified. K , qPCR analysis of the indicated genes. F/F: n=4, iCKO: n=4. One-way ANOVA followed by post hoc Tukey test was used in C , F and H . Log-rank (Mantel-Cox) test in D . Nested t test in I and J . Student t test in K . * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

Techniques: Knock-Out, Western Blot, Injection, Staining, TUNEL Assay

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Scheme of procedures for identification Rbx2-regulated proteome in NRVCs. NRVCs were transfected with indicated siRNAs, followed by CCCP (10 µM) treatment for 6 hours. Cell lysates were collected for trypsin digestion. The resultant peptides were labeled by TMT before mass spectrometry analysis. B , Principal component analysis (PCA) of normalized protein expression in whole cell proteome. CTL, siLuci. KD, siRBX2. C , Analysis of the percentage of differential expression of proteins (DEPs) in each group. D, Volcano plot of differentially expressed proteins (Sig., blue and red) in CCCP-treated RBX2-deficient CMs (KD) compared with CCCP-treated CTL. E , Venn diagram showing overlap of RBX2-regulated proteome and mitochondrial proteins annotated in MitoCarta 3.0. F , Venn diagram showing the identification of RBX2-regulated MOMs. G , Heatmap showing the relative expression of MOM proteins amongst the groups of cells. H , Western blot of MOM proteins in control and RBX2-deficient cardiomyocytes. N=4 for each group. Multiple t test was used. ** P <0.01, *** P <0.001, and **** P <0.0001. I , Western blot of WT and RBX2KO Hela cells treated with cycloheximide (CHX, 100 nM) for indicated times. RBX2 was deleted in Hela cells via CRISPR/Cas9-mediated gene editing. J , WT and RBX2KO Hela cells were transfected with plasmids expressing HA-Ub (pCDNA3-HA-Ub), treated with a proteasome inhibitor Bortezomib (BZM, 100 nM) for 6 hours, and subjected to immunoprecipitation followed by Western blot.

Article Snippet: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

Techniques: Transfection, Labeling, Mass Spectrometry, Expressing, Western Blot, CRISPR, Immunoprecipitation

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: Adult MCM and RBX2 iCKO (iCKO) mice were administered with tamoxifen (50 mg/kg/d for 5 days). Tissues were collected for indicated analyses ( A - H ) at 12 days after tamoxifen injections. A , Representative confocal images (left) of MCM and RBX2 iCKO myocardium sections immunostained with pUb (green), HSP60 (red, mitochondrial marker) and DAPI. Scale bars, 10 µm. B , Quantification of pUb+ foci normalized by mitochondria (HSP60+) area. A total of 16 views from two hearts per group were quantified. C - D , Western blot ( C ) and quantification ( D ) of mitochondrial (mito) and cytosolic (cyto) P62 in mouse hearts. E - F , Western blot ( E ) and quantification ( F ) of mitochondrial (mito) and cytosolic (cyto) LC3-II in mouse hearts. Mice at 12 days after tamoxifen administration were intraperitoneally injected with bafilomyocin A1 (BFA, 3 µmol/kg) for 3 hours before tissue harvest. G , Representative confocal images of mt-Keima at 488 nm and 568 nm, respectively, in epicardial cardiomyocytes and the derived heatmaps. Neonatal MCM and RBX2 iCKO mice were transduced with AAV9-mt-Keima (1X10 GC/pup). At 10 weeks of age, mice were treated with tamoxifen and intact mouse hearts were excised 12 days later and scanned for mt-Keima signals in epicardial cardiomyocytes in situ with confocal microscope. Scale bars, 20 µm. H , Quantification of relative 568/488 ratio. 20-40 views per heart, 3 hearts per group were quantified. I , Western blot of indicated proteins in adult cardiomyocytes isolated from 2-month-old CTL (RBX2 F/F ) or RBX2 CKO (CKO) mouse hearts. Results from two different batches of cells are shown. Nested t test was was used in B and H , Mann-Whitney test in D , and One-way ANOVA in F. * P < 0.05, ** P < 0.01, **** P <0.0001.

Article Snippet: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

Techniques: Marker, Western Blot, Injection, Derivative Assay, Transduction, In Situ, Microscopy, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Western blot of Parkin in NRVCs. Cells were infected with Ad-Parkin and transfected with indicated siRNAs. B , Western blots (left) and quantification (right) of pS65-Ub. Neonatal mouse ventricular CMs (NMVCs) were isolated from WT or Parkin -/- mouse hearts, transfected with siRNA, and treated with CCCP (10 µM) for 12 hours. B , Quantification of phosphorylated Ub. C , Western blots of cell lysates from NRVCs transfected with siRNAs and treated with CCCP (10 µM). D , Western blots of cell lysates from NRVCs infected with Ad-Parkin, transfected with siRNAs, and treated with CCCP (10 µM). E , Schematics of generation of RBX2 and Parkin double knockout (RBX2 CKO /Parkin -/- ) mice. F , Ejection fraction and fractional shortening at 5 (Parkin -/- : n=5, RBX2 Het /Parkin +/- : n=8, RBX2 CKO : n=12. RBX2 CKO /Parkin -/- , n=8) and 8 (Parkin -/- : n=7, RBX2 Het /Parkin +/- : n=7, RBX2 CKO : n=11. RBX2 CKO /Parkin -/- , n=8) months of age. G , Survival curves of indicated mice. K , A proposed model showing the role of RBX2-CRL5 in regulation of physiological mitophagy and cardiac homeostasis. Student t test was used in A and B . One-way ANOVA followed by post hoc Tukey test in F . Log-rank (Mantel-Cox) test in G . ** P <0.01. ns, not significant.

Article Snippet: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

Techniques: Western Blot, Infection, Transfection, Isolation, Double Knockout

Journal: bioRxiv

Article Title: The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

doi: 10.1101/2024.02.24.581168

Figure Lengend Snippet: A , Western blots of indicated proteins in NRVCs. Cells were infected with Ad-PINK1, transfected with indicated siRNAs and treated with or without CCCP (10 µM) for 12 hours. B , Western blots. RBX2 was deleted in Hela cells via CRISPR/Cas9 using a single guided RNA against RBX2 (gRBX2). WT and RBX2KO cells with treated with CCCP (10 µM) for indicated times before harvest. C , Analysis of Pink1 transcript levels in NRVCs transfected with indicated siRNAs by qPCR. D , Western blots. NRVCs were infected with Ad-PINK1, transfected with siRNAs, and treated with Bortezomib (BZM, 100 nM) for 6 hours. E , Cycloheximide-based pulse chase assay. NRVCs were transfected with siRNAs, treated with CCCP (10 µM) for 3 hours, followed by removal of CCCP, and then chased for the indicated time in the presence of cycloheximide (CHX, 100 nM). F , Immunoprecipitation of PINK1, followed by Western blots. NRVCs were transfected with indicated siRNAs and treated with or without Bortezomib (BZM, 100 nM) for 6 hours. G , Western blots of cell lysates from NRVCs transfected with indicated siRNAs and treated with CCCP (10 µM) for 3 hours. H , A proposed model showing the role of RBX2-CRL5 in regulation of physiological mitophagy and cardiac homeostasis.

Article Snippet: Inducible cardiomyocyte-specific RBX2 knockout (RBX2 iCKO ) mice were generated by crossing Rbx2 F/+ mice with αMHC-MerCreMer mice (MCM, the Jackson Laboratory, strain # 005657).

Techniques: Western Blot, Infection, Transfection, CRISPR, Pulse Chase, Immunoprecipitation

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis

doi: 10.1002/dvdy.24675

Figure Lengend Snippet: Disruption of cul5 and rbx2 causes embryonic lethality at different developmental stages. Diagram of cul5GT (A) and rbx2GT (B) alleles indicating the LacZ cassette insertion in the first intron in both genes. Notice that whereas cul5GT and rbx2GT generate null alleles, rbx2GT can be converted to a conditional allele (rbx2fl) via FLPe recombination and to a knockout allele (rbx2KO) via CRE recombination. White boxes in exons indicate untranslated regions and gray boxes indicate coding sequences. Genotyping primers and their relative positions are indicated. Detailed explanation of genotyping strategy can be found in the Material and Methods section. C: No homozygous cul5GT embryos were collected from n = 6 litters at E3.5 and n = 5 litters analyzed at P0. On the contrary, homozygous rbx2KO embryos were collected at E3.5 (n = 15 litters) but failed to survive until birth (n = 6 litters) (D). The number of embryos obtained per genotype and age is indicated in each case. Differences from expected Mendelian ratio were tested using the Chi-square test (χ2), and P values are indicated in each case. ß-gal, beta-galactosidase; ß-geo, beta-galactosidase + Neomycin resistance gene; Chr9, chromosome 9; En2 intr1, partial Engrailed 2 Intron 1; En2 SA, Engrail 2 splicing acceptor; NeoR, Neomycin-resistance gene; pA, polyadenylation site; SA, splicing acceptor; T2A, thosea asigna virus 2A peptide.

Article Snippet: Similar to cul5GT , no rbx2 mutant pups (postnatal day P0) were recovered from heterozygous rbx2 KO intercrossing (Chi-square test P = 0.00228) ( ).

Techniques: Disruption, Knock-Out, Virus

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis

doi: 10.1002/dvdy.24675

Figure Lengend Snippet: Co-expression of Cul5 and Rbx2 in the adult brain. Comparative analysis of Cul5 and Rbx2 by immunofluorescence in consecutive wild-type adult brain sections. In the cerebellum, both Cul5 and Rbx2 were detected in the nuclei of Purkinje cells (purple arrowheads) and broadly distributed in the internal granular layer (A). In the hippocampus, Cul5 was detected principally in the cytoplasm of the pyramidal cells of the cornu ammonis and in mossy fibers. Rbx2 was detected in the nuclei of pyramidal cells and in mossy fibers (B). In the neocortical neurons, Cul5 was detected in the cytoplasm and Rbx2 in the nucleus. On the contrary, in the caudate-putamen, both Cul5 and Rbx2 have a nuclear localization. D-D’“: Immunofluorescence against ß-galactosidase and Cul5 in adult rbx2GT tissue shows ubiquitous colocalization, including the neocortex (D), caudate-putamen (D”), hippocampus (D“), and cerebellum (D”’). MF, mossy fibers; sl, stratum lucidum. Scale bars A,B = 100 μm. Scale bar C = 500 μm. Scale bar D = 25 μm.

Article Snippet: Similar to cul5GT , no rbx2 mutant pups (postnatal day P0) were recovered from heterozygous rbx2 KO intercrossing (Chi-square test P = 0.00228) ( ).

Techniques: Expressing, Immunofluorescence

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis

doi: 10.1002/dvdy.24675

Figure Lengend Snippet: Expression of rbx2 during development and in the adult brain. rbx2 expression parallels cux5 expression in all the areas analyzed. In the olfactory bulb, rbx2 was strongly expressed in the mitral layer at all ages analyzed, and expression of rbx2 was detected in granule cells at postnatal stages only (A). rbx2 expression in the neocortex was detected in proliferative zones as well as in somatic areas (B) (E16.5 and P0). In the adult neocortex, strong LacZ staining was observed in all cortical layers (AD). Similar to cu/5, rbx2 was detected in proliferative areas of the hippocampus and in the stratum pyramidale at early stages (C) (E16.5). LacZ signal was detected in the dentate gyrus starting at postnatal stages and reaching maximum expression in the adult (P0 and AD). Comparative analysis of rbx2 expression in the forebrain indicated that rbx2 was ubiquitously expressed during development and in the adult (D). In the hindbrain, rbx2 expression was detected principally in Purkinje cells, deep cerebellar nuclei, and medulla (E14.5 and P0). In the adult cerebellum, a strong LacZ staining was observed in Purkinje cells, but especially in granule cells of the internal granular layer, similar to cu/5. th, thalamus; 6N, abducens nucleus; 7N, facial nucleus. Scale bars A,C = 100 μm. Scale bars D,E = 500 μm.

Article Snippet: Similar to cul5GT , no rbx2 mutant pups (postnatal day P0) were recovered from heterozygous rbx2 KO intercrossing (Chi-square test P = 0.00228) ( ).

Techniques: Expressing, Staining

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis

doi: 10.1002/dvdy.24675

Figure Lengend Snippet: Disruption of cul5 and rbx2 causes embryonic lethality at different developmental stages. Diagram of cul5GT (A) and rbx2GT (B) alleles indicating the LacZ cassette insertion in the first intron in both genes. Notice that whereas cul5GT and rbx2GT generate null alleles, rbx2GT can be converted to a conditional allele (rbx2fl) via FLPe recombination and to a knockout allele (rbx2KO) via CRE recombination. White boxes in exons indicate untranslated regions and gray boxes indicate coding sequences. Genotyping primers and their relative positions are indicated. Detailed explanation of genotyping strategy can be found in the Material and Methods section. C: No homozygous cul5GT embryos were collected from n = 6 litters at E3.5 and n = 5 litters analyzed at P0. On the contrary, homozygous rbx2KO embryos were collected at E3.5 (n = 15 litters) but failed to survive until birth (n = 6 litters) (D). The number of embryos obtained per genotype and age is indicated in each case. Differences from expected Mendelian ratio were tested using the Chi-square test (χ2), and P values are indicated in each case. ß-gal, beta-galactosidase; ß-geo, beta-galactosidase + Neomycin resistance gene; Chr9, chromosome 9; En2 intr1, partial Engrailed 2 Intron 1; En2 SA, Engrail 2 splicing acceptor; NeoR, Neomycin-resistance gene; pA, polyadenylation site; SA, splicing acceptor; T2A, thosea asigna virus 2A peptide.

Article Snippet: The rbx2 knockout first allele ( rbx2 GT) mouse embryos (Rnf7tm1a(EUCOMM)Wtsi) were obtained from the International Mouse Phenotyping Consortium (IMPC) ( Skarnes et al., 2011 ) and maintained in a mixed 129Sv/C57BL6 strain background.

Techniques: Disruption, Knock-Out, Virus

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis

doi: 10.1002/dvdy.24675

Figure Lengend Snippet: Co-expression of Cul5 and Rbx2 in the adult brain. Comparative analysis of Cul5 and Rbx2 by immunofluorescence in consecutive wild-type adult brain sections. In the cerebellum, both Cul5 and Rbx2 were detected in the nuclei of Purkinje cells (purple arrowheads) and broadly distributed in the internal granular layer (A). In the hippocampus, Cul5 was detected principally in the cytoplasm of the pyramidal cells of the cornu ammonis and in mossy fibers. Rbx2 was detected in the nuclei of pyramidal cells and in mossy fibers (B). In the neocortical neurons, Cul5 was detected in the cytoplasm and Rbx2 in the nucleus. On the contrary, in the caudate-putamen, both Cul5 and Rbx2 have a nuclear localization. D-D’“: Immunofluorescence against ß-galactosidase and Cul5 in adult rbx2GT tissue shows ubiquitous colocalization, including the neocortex (D), caudate-putamen (D”), hippocampus (D“), and cerebellum (D”’). MF, mossy fibers; sl, stratum lucidum. Scale bars A,B = 100 μm. Scale bar C = 500 μm. Scale bar D = 25 μm.

Article Snippet: The rbx2 knockout first allele ( rbx2 GT) mouse embryos (Rnf7tm1a(EUCOMM)Wtsi) were obtained from the International Mouse Phenotyping Consortium (IMPC) ( Skarnes et al., 2011 ) and maintained in a mixed 129Sv/C57BL6 strain background.

Techniques: Expressing, Immunofluorescence

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis

doi: 10.1002/dvdy.24675

Figure Lengend Snippet: Expression of rbx2 during development and in the adult brain. rbx2 expression parallels cux5 expression in all the areas analyzed. In the olfactory bulb, rbx2 was strongly expressed in the mitral layer at all ages analyzed, and expression of rbx2 was detected in granule cells at postnatal stages only (A). rbx2 expression in the neocortex was detected in proliferative zones as well as in somatic areas (B) (E16.5 and P0). In the adult neocortex, strong LacZ staining was observed in all cortical layers (AD). Similar to cu/5, rbx2 was detected in proliferative areas of the hippocampus and in the stratum pyramidale at early stages (C) (E16.5). LacZ signal was detected in the dentate gyrus starting at postnatal stages and reaching maximum expression in the adult (P0 and AD). Comparative analysis of rbx2 expression in the forebrain indicated that rbx2 was ubiquitously expressed during development and in the adult (D). In the hindbrain, rbx2 expression was detected principally in Purkinje cells, deep cerebellar nuclei, and medulla (E14.5 and P0). In the adult cerebellum, a strong LacZ staining was observed in Purkinje cells, but especially in granule cells of the internal granular layer, similar to cu/5. th, thalamus; 6N, abducens nucleus; 7N, facial nucleus. Scale bars A,C = 100 μm. Scale bars D,E = 500 μm.

Article Snippet: The rbx2 knockout first allele ( rbx2 GT) mouse embryos (Rnf7tm1a(EUCOMM)Wtsi) were obtained from the International Mouse Phenotyping Consortium (IMPC) ( Skarnes et al., 2011 ) and maintained in a mixed 129Sv/C57BL6 strain background.

Techniques: Expressing, Staining