Review



rbx2 knockout  (Santa Cruz Biotechnology)


Bioz Verified Symbol Santa Cruz Biotechnology is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
    • 86
      Buy from Supplier

    Structured Review

    Santa Cruz Biotechnology rbx2 knockout
    Overexpression of <t>RBX2</t> 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%.
    Rbx2 Knockout, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rbx2 knockout/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    rbx2 knockout - by Bioz Stars, 2026-05
    86/100 stars

    Images

    1) Product Images from "Identification of RING-box 2 as a potential target for combating colorectal cancer growth and metastasis"

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

    Journal: American Journal of Cancer Research

    doi:

    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%.
    Figure Legend 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%.

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

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

    Techniques Used:



    Similar Products

    rbx2 knockout first allele ( rbx2 gt) mouse embryos (rnf7tm1a(eucomm)wtsi  (International Mouse Phenotyping Consortium)
    90
    International Mouse Phenotyping Consortium rbx2 knockout first allele ( rbx2 gt) mouse embryos (rnf7tm1a(eucomm)wtsi
    Disruption of cul5 and <t>rbx2</t> 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.
    Rbx2 Knockout First Allele ( Rbx2 Gt) Mouse Embryos (Rnf7tm1a(eucomm)wtsi, supplied by International Mouse Phenotyping Consortium, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rbx2 knockout first allele ( rbx2 gt) mouse embryos (rnf7tm1a(eucomm)wtsi/product/International Mouse Phenotyping Consortium
    Average 90 stars, based on 1 article reviews
    rbx2 knockout first allele ( rbx2 gt) mouse embryos (rnf7tm1a(eucomm)wtsi - by Bioz Stars, 2026-05
    90/100 stars
    		  Buy from Supplier
    rbx2 knockout  (Santa Cruz Biotechnology)
    86
    Santa Cruz Biotechnology rbx2 knockout
    Overexpression of <t>RBX2</t> 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%.
    Rbx2 Knockout, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rbx2 knockout/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    rbx2 knockout - by Bioz Stars, 2026-05
    86/100 stars
    		  Buy from Supplier

    Image Search Results


    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.

    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

    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.

    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

    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.

    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

    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%.

    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

    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.

    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

    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).

    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

    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.

    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

    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).

    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

    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.

    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

    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.

    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

    Proposed model of RBX2 promotes colon cancer metastasis via mTOR/S6K1 signaling pathway.

    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: