Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 7) or HD-PBMCs ( n = 7). ( B ) The concentration of NF-L (pg/mL) in the supernatant when NB-1 cells were cocultured with HAM-PBMCs ( n = 9) and with mogamulizumab (antiCCR4) in a dose-dependent manner for 72 hours. ( C ) The comparison of RGMA mRNA gene expression levels using DNA microarray among normal CD4 + T cells (HD CD4 + : n = 4), HAM patient–derived CD4 + T cells (HAM CD4 + : n = 4), ACs ( n = 2), and smoldering/chronic-type-ATL patient–derived ( n = 3) HTLV-1–infected CD4 + T cells (Non-HAM infected CD4 + T cells: n = 5), and acute-type-ATL patient–derived HTLV-1–infected CD4 + T cells (Acute ATL infected cells: n = 3). ( D ) The comparison of the expression levels of the genes associated with the inhibition of neuroregeneration ( OMG , MAG , RTN4 , and WNT5A ) between HD CD4 + ( n = 4) and HAM CD4 + T cells ( n = 4). ( E ) The enrichment levels of H3K27me3 –2916 bp upstream from the TSS of the RGMA gene locus in HD CD4 + ( n = 3), HAM CD4 + ( n = 4), and acute-ATL infected cells ( n = 4). Data are shown as mean ± SD. ** P < 0.01; *** P < 0.001 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test ( B , C , and E ). NF-L, neurofilament light chain.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Concentration Assay, Comparison, Gene Expression, Microarray, Derivative Assay, Infection, Expressing, Inhibition

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) The validation of RGMA mRNA gene expression levels using qRT-PCR in HD CD4 + ( n = 6) and HAM CD4 + T cells ( n = 6). ( B ) Expression of RGMa protein in CD3 + CD4 + CCR4 + T cells from HAM-PBMCs. Representative dot plots of CCR4 and normal goat IgG (upper) or RGMa expression (bottom) in CD3 + CD4 + gated cells from HD-PBMCs (left) or HAM-PBMCs (right) cultured for 2 days. ( C ) Graph shows the percentage of RGMa protein–expressing cells in CCR4 – cells or CCR4 + cells in CD3 + CD4 + gated cells from HAM-PBMCs ( n = 8) cultured for 2 days, compared with the isotype control, normal goat IgG. ( D ) Graph shows the percentage of RGMa protein–expressing cells among CD3 + CD4 + CCR4 + gated cells from HD-PBMCs ( n = 5) or HAM-PBMCs ( n = 8) cultured for 2 days. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01 by unpaired t test ( A and D ) or 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Expressing, Cell Culture, Control, Comparison

Journal: JCI Insight

Article Title: Virus-induced RGMa expression drives neurodegeneration in HTLV-1–associated myelopathy

doi: 10.1172/jci.insight.184530

Figure Lengend Snippet: ( A ) Tax (left), HBZ (middle), and RGMA (right) gene expression levels in cultured HAM-PBMCs ( n = 7) in a time-dependent manner. RPL19 was used as an internal control. ( B ) Tax-dependent RGMA mRNA gene induction in Jurkat cells, which were infected with lentivirus carrying the Tax gene. Top: Tax expression in the Jurkat cells was confirmed by Western blotting. β-Actin was measured as an internal control. Bottom: The induction levels of the RGMA gene were evaluated by qRT-PCR in a time-dependent manner ( n = 3). ( C ) Tax -dependent RGMA mRNA gene induction in JPX9 cells treated with 20 μM CdCl 2 in a time-dependent manner. Tax mRNA (upper) and RGMA mRNA (bottom) were measured by qRT-PCR ( n = 3). GAPDH was measured as an internal control. ( D ) Tax-dependent RGMa protein induction in JPX9 cells treated with 20 μM CdCl 2 for 3 days. Dot plots of Tax and normal goat IgG (upper) or RGMa expression (bottom) in JPX9 cells. JPX9(-), untreated JPX9 cells; 20 μM CdCl 2 JPX9, CdCl 2 -supplemented JPX9 cells. Data are shown as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test ( A ), 2-sided Student’s t test ( B ), or an unpaired t test ( C ). Experiments were performed in triplicate ( B and C ).

Article Snippet: In the experiment analyzing Tax , HBZ , and RGMA gene expression levels in cultured HAM-PBMCs, mRNA was purified using the Magnetic mRNA isolation kit (New England BioLabs).

Techniques: Gene Expression, Cell Culture, Control, Infection, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Characterization of CAFs and NFs obtained from clinical surgical tissues from patients with head and neck cancer (HNC) ( a ) Morphological comparisons between CAFs and NFs from a representative HNC case showed that CAFs (right panel) consisted of more cytoplasmic protrusions than NFs (left panel). Photographs were captured at 40× magnification. ( b ) Quantitative PCR (left panel) of the culture medium showed a significantly higher expression of vimentin and α-SMA in CAFs than in NFs. Western blot analysis (right panel) also demonstrated that levels of vimentin and α-SMA were significantly higher in CAFs than in NFs. ( c ) Flow cytometric analysis of the cell surface markers, CD10 and GPR77, showed a marked increase in their expression in CAFs compared to NFs. ( d ) A heat map of the gene microarray of NFs and CAFs showed that there were several differences in the expression profile of the secreted genes. The arrow indicates a marked discrepancy of the relative mRNA levels of CCL11 in CAF compared with NF. ( e ) The RT-PCR (left panel) and ELISA (right panel) analysis showed an increased expression of CCL11 in CAFs compared with that in NFs. ( f ) Western blot analysis showed that the protein level of CCL11 was higher in CAFs than in NFs in cell lysates. ( g ) Western blot analysis showed a higher CCL11 expression in CAF-CM compared to NF-CM. The asterisk indicated a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Microarray, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 produced by CAFs causes increased migration and invasion, and the EMT of HNC cells. ( a ) Comparative analysis of the migration and invasion of HNC cells associated with CCL11. Four test groups were classified for comparative analysis of migration and invasive abilities. FaDu and NPC204 cells cultured with medium containing CAF-induced CCL11 presented greater abilities of migration and invasion, with a statistically significant difference, than three other groups: NF, NF with CCL11, and CAFs treated with CCL11 antibody. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) ( b ) Comparative photographs of the infiltrating behavior of FaDu and NPC204 cells in an organotypic culture in four groups seeded onto a mixture layer containing NFs or CAFs with CCL11 or CCL11 antibody. The arrow(s) indicate infiltration buds from the HNC cells seeded above. ( c ) Representative blots of the EMT-associated markers in FaDu and NPC204 cells, as observed upon Western blotting analysis in five groups, showed that treatment with CAF-conditioned medium or the application of rCCL11 decreased the expression of epithelial-type markers (E-cadherin), and increased the expression of mesenchymal-type markers (fibronectin) and EMT regulators (Snail and Twist). In addition, increased expression of invasion-related MMP2 and MMP9 was also seen in those two groups, compared with other groups. The asterisk indicates a significant difference ( p < 0.05) between experimental and control groups. Results are expressed as mean ± SD.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Produced, Migration, Cell Culture, Western Blot, Expressing, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Comparative analysis of induction of CSC properties and drug resistance in HNC cells associated with CCL11. ( a ) Four groups were classified for comparative analysis of the ability of sphere formation. Increased ability of sphere formation in two test groups of HNC cells exposed to a CAF medium and the group with treatment of rCCL11 was noted. ( b ) Flow cytometric analysis showed a significant increase in CD44 and CD44/CD24, as well as in CD133 in HNC cells exposed to rCCL11, compared to control HNC cells ( p < 0.05). ( c ) Flow cytometric analysis showed a marked increase in ALDH-1 activity in HNC cells exposed to rCCL11 compared to control HNC cells. ( d ) Western blot analysis showed that CSC-representative markers, Oct-4, Nanog, and Sox-2, were also overexpressed in addition to the increased expression of two important drug resistance genes, ABCG-2 and MDR-1 , in HNC cells exposed to rCCL11. ( e ) Treatment with Cisplatin at 24 h showed a significant increase in chemoresistance in both FaDu and NPC204 cells exposed to rCCL11 compared with control HNC cells. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Control, Activity Assay, Western Blot, Expressing

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 and CCR3 expression with associated signal pathway in HNC cell lines and their correlation to clinical outcomes in 104 HNC patients. ( a ) Confocal microscopic images showed CCL11 (green) localized to both cell and nuclear membranes, while CCR3 (red) localized only to the cell membrane in FaDu cells; CCL11 and CCR3 co-localized at the cell membrane (yellow). In NPC204 cells, CCL11 (green) and CCR3 (red) were found to co-localize at protrusions polarized to the cells (yellow). ( b ) Using the crisp technique, higher expression of CCR3, MMP2, and MMP3 was found in over-expressed CCL11 cloned-FaDu and NPC204 cells. Cloned CCL11-overexpressed cells were abolished by adding eotaxin siRNA or CCR3 antibody, which reversed the expression of CCR3 and invasion-related MMP2 and MMP9. ( c ) Higher phosphorylation levels of p38 MAPK and ERK were found in cloned CCL11-overexpressed FaDu and NPC204 cells and were reversed by treatment of the p38 MAPK inhibitor (SB203580) and ERK inhibitor (FR180204), respectively. The phosphorylation level of JNK was kept in low condition before and after treatment of the JNK inhibitor (SP600125). ( d ) Photomicrographs of immunohistochemical staining from tissue microarray showing CCL11 and CCR3 expression in three different representative groups of HNC patients (magnification, ×200). ( e ) Kaplan–Meier survival analysis of patients showed that overexpression of CCL11 and CCR3 were statistically associated with poor overall survival).

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Expressing, Membrane, Clone Assay, Phospho-proteomics, Immunohistochemical staining, Staining, Microarray, Over Expression

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: The diagrammatic illustration demonstrates the major mechanism that CAFs secreting CCL11 promotes HNC cell migration and invasion and induces properties of drug resistance and stemness, shown as follows. CAFs in TME secret CCL11 binding to the CCR3 receptors on HNC cells via the paracrine effect. The signal induces overexpression of transcriptional factors, such as Snail and Twist, which regulate EMT and are also responsible for self-induction of CCL11 in an autocrine fashion. As a result, CCL11, via paracrine or autocrine signaling when targeting CCR3 receptors, play a functional role in the induction of EMT and CSC properties, for further tumor progression.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Migration, Binding Assay, Over Expression, Functional Assay

Journal: Frontiers in Microbiology

Article Title: Novel Insights Into Cellular Changes in HPV8-E7 Positive Keratinocytes: A Transcriptomic and Proteomic Analysis

doi: 10.3389/fmicb.2021.672201

Figure Lengend Snippet: Transcriptional changes in HPV8-E7 positive keratinocytes. (A) Venn Diagram comparison of two independent cDNA microarray analyses carried out with PHAK expressing HPV8-E7 in the Akgül lab in Cologne and with PHFK expressing HPV8-E7 in the Alonso lab at the DKFZ in Heidelberg. (B) Gene IDs of 4 genes differentially expressed in both array studies. (C,D) GDF15 mRNA expression in isogenic PM1 keratinocytes cultured either in KGM-Gold or RM+, respectively. (E) GDF15 ELISA performed with cell culture supernatants of HPV8-E7 expressing keratinocytes grown in KGM-Gold medium. (F,G) ATF3 mRNA expression in isogenic PM1 keratinocytes cultured either in KGM-Gold or RM+, respectively. (H) GADD34 mRNA expression in PM1 keratinocytes cultured in KGM-Gold. RTqPCRs were normalized to HPRT1 mRNA levels ( n = 3 independent experiments performed in duplicate). Error bars represent standard deviations. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control conditions (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The Human GDF15 Quantikine ELISA Kit (RnD systems) was used to measure GDF15 in cell culture supernatants.

Techniques: Comparison, Microarray, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Research

Article Title: The BH3 Mimetic ABT-737 Induces Cancer Cell Senescence

doi: 10.1158/0008-5472.can-10-1977

Figure Lengend Snippet: Figure 1. Gene transcription changes after ABT-737 treatment. A, microarray analysis of gene expression induced by ABT-737. PV-10 cells were treated in triplicate with DMSO or 10 mmol/L ABT-737 for 24 hours and gene microarray changes documented. B, gene changes associated with senescence. C, qT-PCR analysis of IL-6 and IL-8 transcripts. PV-10 and 22Rv1 cells were treated in triplicate with DMSO, ABT-737 (10 mmol/L) or enantiomer (En., 10 mmol/ L) for 24 hours (mean SD, n ¼ 3). D, the secretion of IL-6 and IL-8 in PV-10 cells treated with DMSO, ABT-737 or enantiomer for 24 hours was determined by ELISA (n ¼ 6, mean SD).

Article Snippet: Conditioned medium was collected following treatment and used at a 2:1 dilution in the human interleukin-6 (IL-6) or IL-8 ELISA Kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Microarray, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Cancer Research

Article Title: FBXO22 Possesses Both Protumorigenic and Antimetastatic Roles in Breast Cancer Progression

doi: 10.1158/0008-5472.can-17-3647

Figure Lengend Snippet: Figure 1. FBXO22 promotes breast cancer cell proliferation in vitro and tumor growth in vivo. A and B, Immunoblotting analysis of FBXO22 protein expression in 9 pairs of matched primary breast cancer tissues and normal breast tissue (A) and indicated cell lines (B). C–E, MDA-MB-231 and Hs578T cells stably expressing pCDH and HA-FBXO22 (C) were subjected to cell proliferation assays using CCK-8 (D) and colony growth assays (E). Representative images of survival colonies are shown in Supplementary Fig. S1D. F–H, MCF-7 and ZR-75-1 cells stably expressing shNC and shFBXO22 (F) were subjected to cell proliferation assays using CCK-8 (G) and colony growth assays (H). Representative images of survival colonies are shown in Supplementary Fig. S1H. I–K, MDA-MB-231 cells stably expressing pCDH and HA-FBXO22 were injected into mammary fat pads of 6-week-old female BALB/c nude mice (n ¼ 8). After 8 weeks of injections, xenograft tumors were harvested. Tumor growth curves (I), photographs of harvested tumors (J), and tumor weight (K) are shown.

Article Snippet: DNA constructs, transfection, and viral transduction Myc-DDK-FBXO22, Myc-DDK-SNAIL, and short hairpin RNAs (shRNA) targeting human FBXO22 (shFBXO22) were purchased from Origene. siRNA targeting GSK3b (siGSK3b) and nontargeting negative control (siNC) were purchased form GenePharma (Supplementary Table S2).

Techniques: In Vitro, In Vivo, Western Blot, Expressing, Stable Transfection, CCK-8 Assay, Injection

Journal: Cancer Research

Article Title: FBXO22 Possesses Both Protumorigenic and Antimetastatic Roles in Breast Cancer Progression

doi: 10.1158/0008-5472.can-17-3647

Figure Lengend Snippet: Figure 3. FBXO22 expression is associated with the prognosis of patients with breast cancer. A, IHC analysis was carried out on a tissue microarray containing 164 breast cancer samples with clinical follow-up information. Representative IHC images of FBXO22 expression are shown. B and C, Kaplan–Meier curves of OS (B) and DFS (C) of 164 patients with breast cancer with high or low FBXO22 expression.

Article Snippet: DNA constructs, transfection, and viral transduction Myc-DDK-FBXO22, Myc-DDK-SNAIL, and short hairpin RNAs (shRNA) targeting human FBXO22 (shFBXO22) were purchased from Origene. siRNA targeting GSK3b (siGSK3b) and nontargeting negative control (siNC) were purchased form GenePharma (Supplementary Table S2).

Techniques: Expressing, Microarray

Journal: Cancer Research

Article Title: FBXO22 Possesses Both Protumorigenic and Antimetastatic Roles in Breast Cancer Progression

doi: 10.1158/0008-5472.can-17-3647

Figure Lengend Snippet: Figure 2. FBXO22 suppresses breast cancer cell migration, invasion, and metastasis. A–F, MDA-MB-231 and Hs578T cells stably expressing pCDH and HA-FBXO22 were subjected to wound-healing assays (A and B), Boyden's chamber migration assays (C and D), and Matrigel-coated invasion assays (E and F). G–J, MCF-7 and ZR-75-1 cells stably expressing shNC and shFBXO22 were analyzed by wound-healing assays (G and H), Boyden's chamber migration assays (I), and Matrigel-coated invasion assays (J). Representative images of cell migration and invasion are shown in Supplementary Fig. S3E and S3F, respectively. K–M, MDA-MB-231 cells stably expressing pCDH and HA-FBXO22 were injected into 6-week-old female BALB/c nude mice (n ¼ 6) through the tail vein. After 6 weeks of injections, the lungs were harvested. Representative images of lung metastasis (K), quantitative results of lung nodules (L), and representative images of H&E-stained sections of lung tissues (M) are shown.

Article Snippet: DNA constructs, transfection, and viral transduction Myc-DDK-FBXO22, Myc-DDK-SNAIL, and short hairpin RNAs (shRNA) targeting human FBXO22 (shFBXO22) were purchased from Origene. siRNA targeting GSK3b (siGSK3b) and nontargeting negative control (siNC) were purchased form GenePharma (Supplementary Table S2).

Techniques: Migration, Stable Transfection, Expressing, Injection, Staining

Journal: Cancer Research

Article Title: FBXO22 Possesses Both Protumorigenic and Antimetastatic Roles in Breast Cancer Progression

doi: 10.1158/0008-5472.can-17-3647

Figure Lengend Snippet: Figure 4. FBXO22 suppresses EMT and promotes proteasomal degradation of SNAIL. A, Immunoblotting analysis of EMT markers in MCF10A cells stably expressing pCDH and HA-FBXO22. B, Immunofluorescence staining of E-cadherin and vimentin in MCF10A cells stably expressing pCDH and HA-FBXO22. Cell nucleus was counterstained with DAPI. C–D, MDA-MB-231 and Hs578T cells stably expressing pCDH and HA-FBXO22 (C) or MCF-7 and ZR-75-1 cells stably expressing shNC and shFBXO22 (D) were analyzed by immunoblotting. E, Lysates from four pairs of xenograft tumors expressing pCDH and HA-FBXO22 (tumors from Fig. 1J) were analyzed by immunoblotting. F, Lysates from the indicated cell lines were analyzed by immunoblotting. G, Cells stably expressing pCDH and HA-FBXO22 were treated with DMSO or 10 mmol/L MG-132 for 6 hours and analyzed by immunoblotting. H and I, Cells stably expressing shNC and shFBXO22 were treated with 100 mg/mL of CHX for the indicated times and then analyzed by immunoblotting (H). Relative expression levels of SNAIL to vinculin are shown in I. J and K, Cells were transfected with Flag-SNAIL alone or in combination with HA-FBXO22. After 48 hours of transfection, cells were subjected to immunoblotting (J) and immunofluorescent staining (K).

Article Snippet: DNA constructs, transfection, and viral transduction Myc-DDK-FBXO22, Myc-DDK-SNAIL, and short hairpin RNAs (shRNA) targeting human FBXO22 (shFBXO22) were purchased from Origene. siRNA targeting GSK3b (siGSK3b) and nontargeting negative control (siNC) were purchased form GenePharma (Supplementary Table S2).

Techniques: Western Blot, Stable Transfection, Expressing, Staining, Transfection

Journal: Cancer Research

Article Title: FBXO22 Possesses Both Protumorigenic and Antimetastatic Roles in Breast Cancer Progression

doi: 10.1158/0008-5472.can-17-3647

Figure Lengend Snippet: Figure 5. FBXO22 interacts with SNAIL and promotes its polyubiquitination. A, Lysates from HEK293T cells transfected with Flag-SNAIL were incubated with bacterially expressed GST or GST-FBXO22 protein for 2 hours. GST pull-down proteins were analyzed by immunoblotting with an anti-Flag antibody. GST or GST-FBXO22 protein was visualized by Ponceau S staining. B, Lysates from HEK293T cells transfected with HA-FBXO22 were incubated with GST or GST-SNAIL protein for 2 hours. GST pull-down proteins were analyzed by immunoblotting with an anti-HA antibody. GST or GST-SNAIL protein was visualized by Ponceau S staining. C, HEK293T cells were transfected with Flag-SNAIL and HA-FBXO22 alone or in combination. After 48 hours of transfection, cells were treated with 20 mmol/L MG-132 for 6 hours and subjected to sequential immunoprecipitation-immunoblotting analyses. D, Lysates from ZR-75-1 and T47D cells were immunoprecipitated with control IgG or an anti-SNAIL antibody, followed by immunoblotting analysis. E and F, GST pull-down assays were performed using exogenously expressed Flag-SNAIL and purified GST or GST-FBXO22 proteins. G and H, HEK293T cells were transfected with the indicated expression vectors. After 48 hours of transfection, cells were treated with 20 mmol/L MG-132 for 6 hours. Lysates were subjected to immunoprecipitation analysis under the native (G) or denaturing (H) conditions, followed by immunoblotting analysis. I and J, MDA-MB-231 cells stably expressing pCDH and HA-FBXO22 (I) or MCF-7 cells stably expressing shNC and shFBXO22 (J) were treated with 10 mmol/L of MG-132 for 6 hours and subjected to the sequential immunoprecipitation-immunoblotting analysis. K, In vitro ubiquitination assays were performed using the purified intact HA-FBXO22 E3 ligase complex and Flag-SNAIL from HEK293T cells in the presence of ubiquitin, E1, E2 (UbcH5a), and ATP.

Article Snippet: DNA constructs, transfection, and viral transduction Myc-DDK-FBXO22, Myc-DDK-SNAIL, and short hairpin RNAs (shRNA) targeting human FBXO22 (shFBXO22) were purchased from Origene. siRNA targeting GSK3b (siGSK3b) and nontargeting negative control (siNC) were purchased form GenePharma (Supplementary Table S2).

Techniques: Transfection, Incubation, Western Blot, Staining, Immunoprecipitation, Control, Expressing, Stable Transfection, In Vitro, Ubiquitin Proteomics

Journal: Cancer Research

Article Title: FBXO22 Possesses Both Protumorigenic and Antimetastatic Roles in Breast Cancer Progression

doi: 10.1158/0008-5472.can-17-3647

Figure Lengend Snippet: Figure 6. GSK3b phosphorylation is required for ubiquitin-dependent proteasomal degradation of SNAIL by FBXO22. A and B, MDA-MB-231 cells stably expressing pCDH and HA-FBXO22 (A) or MCF-7 cells stably expressing shNC and shFBXO22 (B) were treated with or without 10 mmol/L GSK3b inhibitor CHIR-99021, 10 mmol/L PKA inhibitor H89, and 10 mmol/L PKD1 inhibitor CID755673 for 24 hours and subjected to immunoblotting analysis. C, HEK293T cells were transfected with the indicated expression vectors. After 48 hours of transfection, cells were treated with or without 10 mmol/L CHIR-99021 for 24 hours and subjected to sequential immunoprecipitation-immunoblotting analysis. D, HEK293T cells were transfected with the indicated expression vectors. After 48 hours of transfection, cells were subjected to sequential immunoprecipitation-immunoblotting analysis. E, HEK293T cells were transfected with the indicated expression vectors and analyzed by immunoblotting after 48 hours of transfection. F–G, HEK293T cells were transfected with the indicated expression plasmids. After 48 hours of transfection, cells were treated with 100 mg/mL of CHX for the indicated times and analyzed by immunoblotting (F). The relative expression levels of Flag-SNAIL to vinculin are shown in G. H and I, MDA-MB-231 cells stably expressing pCDH, HA-FBXO22 alone, or in combination with either Flag-SNAIL or Flag-SNAIL S4A were subjected to Boyden's chamber migration assays and Matrigel-coated invasion assays (H). The corresponding quantitative results are shown in I.

Article Snippet: DNA constructs, transfection, and viral transduction Myc-DDK-FBXO22, Myc-DDK-SNAIL, and short hairpin RNAs (shRNA) targeting human FBXO22 (shFBXO22) were purchased from Origene. siRNA targeting GSK3b (siGSK3b) and nontargeting negative control (siNC) were purchased form GenePharma (Supplementary Table S2).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Stable Transfection, Expressing, Western Blot, Transfection, Immunoprecipitation, Migration

Journal: Cancer Research

Article Title: FBXO22 Possesses Both Protumorigenic and Antimetastatic Roles in Breast Cancer Progression

doi: 10.1158/0008-5472.can-17-3647

Figure Lengend Snippet: Figure 7. Patient-derived W52R mutation abrogates the ability of FBXO22 to degrade SNAIL and to suppress cell migration, invasion, and metastasis. A, HEK293T cells were transfected with the indicated express constructs. After 48 hours of transfection, cells were harvested for sequential immunoprecipitation-immunoblotting analysis. B, MDA-MB-231 cells stably expressing pCDH, HA-FBXO22, HA-FBXO22 DF, or HA-FBXO22 W52R were analyzed by immunoblotting. C and D, HEK293T cells were transfected with the indicated expression vectors. After 48 hours of transfection, cells were treated with 10 mmol/L MG-132 for 6 hours and subjected to sequential immunoprecipitation-immunoblotting analysis. E, HEK293T cells were transfected with the indicated expression plasmids. After 48 hours of transfection, cells were treated with 100 mg/mL of CHX for the indicated times and then analyzed by immunoblotting analysis. The relative SNAIL expression levels (SNAIL/vinculin) are shown in Supplementary Fig. S9B. F and G, MDA-MB-231 cells stably expressing pCDH, HA-FBXO22, HA-FBXO22 DF, and HA-FBXO22 W52R were subjected to migration and invasion assays as described above. H–J, MDA-MB-231 cells stably expressing pCDH, HA-FBXO22, HA-FBXO22 DF-box, and HA-FBXO22 W52R were injected into 6-week-old female BALB/c nude mice (n ¼ 6) through the tail vein. The lungs were harvested after 6 weeks of injection. Representative images of lung metastasis (H), quantitative results of lung nodules (I), and representative images of H&E-stained sections of lung tissues (J) are shown.

Article Snippet: DNA constructs, transfection, and viral transduction Myc-DDK-FBXO22, Myc-DDK-SNAIL, and short hairpin RNAs (shRNA) targeting human FBXO22 (shFBXO22) were purchased from Origene. siRNA targeting GSK3b (siGSK3b) and nontargeting negative control (siNC) were purchased form GenePharma (Supplementary Table S2).

Techniques: Derivative Assay, Mutagenesis, Migration, Transfection, Construct, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, Injection, Staining