Journal: Integrative Cancer Therapies

Article Title: Antitumor Activity of the Chinese Medicine JC-001 Is Mediated by Immunomodulation in a Murine Model of Hepatocellular Carcinoma

doi: 10.1177/1534735416664173

Figure Lengend Snippet: In vitro effect on viability of JC-001 treatment on normal liver epithelial cell and cancer cell strains. MTT assay was used for analysis of the cell viability of human normal liver epithelial cell (THLE-3), human hepatoma cell (HepG2, Hep3B, SK-Hep-1), and murine hepatoma cell (Hepa 1-6) for 96 hours JC-001 treatment. Each bar represents mean ± SD (n = 4). * P < .05 compared with the 0-µg/mL group.

Article Snippet: A murine hepatoma cell line, Hepa 1-6 (ATCC number: CRL-1830), and 2 human hepatocellular carcinoma cell lines, HepG2 (ATCC number: HB-8065) and Hep3B (ATCC number: HB-8064), were obtained from the Bioresource Collection and Research Center, Taiwan.

Techniques: In Vitro, MTT Assay

Journal: PLoS ONE

Article Title: Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

doi: 10.1371/journal.pone.0044802

Figure Lengend Snippet: ( A ) Schematic description of the structures of AdCN103, AdCN205-GFP and AdCN205-IL12. In AdCN103, the E1A promoter was replaced by hTERT promoter and deletion of the adenoviral genome 923 to 946 nucleotides, which enables viral replication within malignant cells with abnormal RB functions. In AdCN205-GFP and AdCN205-IL12, E3 6.7 K/gp19K genes were substituted by GFP reporter gene and hIL-12 therapeutic gene, respectively. ( B ) Expression of transgene in the cells after infection with oncolytic adenoviruses. Tumor cells (HuH-7) and normal cells (AKN-1) were infected with AdCN205-IL12 at the multiplicity of infection (MOI) of 10. At different time points, the culture supernatant was collected for determination of hIL-12 levels by ELISA. ( C ) hIL-12 level in the cells infected with Ad-IL12 at the MOI of 10. The data was presented as the mean ± SD of three independent experiments. ( D ) Representative photomicrographs were obtained from BEL7404 and AKN-1 infected with AdCN205-EGFP at the MOI of 10. Original magnification, 200×. ( E ) Selective replication of oncolytic adenoviral vectors in vitro . Tumor cells (BEL7404, SMMC7721, MHCC-97H, HuH-7, Hep3B) and normal cells (AKN-1) were infected with AdCN205-GFP, AdCN205-IL12 or Ad-IL12 at the MOI of 10 respectively. At 48 hours after viral infection, cells and medium were harvested, and viral particles were released by three cycles of freezing and thawing. The viral titers were measured by using plaque assay of QuickTiter™ Adenovirus Titer Immunoassay Kit on HEK293 cells. The data was presented as the mean ± SD of three independent experiments.

Article Snippet: Human hepatocellular carcinoma (HCC) cell lines Hep3B (HBs pos , undetected Rb), were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA), BEL7404 (HBs neg , Rb wt ), SMMC7721 (HBs neg , Rb wt ), MHCC97H (HBs pos , Rb wt hyperphosphorylated) and HuH-7 (HBs neg , Rb wt hyperphosphorylated) , , were purchased from the Shanghai Cell Collection, China.

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, In Vitro, Plaque Assay

Journal: PLoS ONE

Article Title: Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

doi: 10.1371/journal.pone.0044802

Figure Lengend Snippet: Liver tumor cells (BEL7404, Hep3B and HuH-7) and normal cells (AKN-1) were infected with AdCN205-GFP, AdCN205-IL12 or Ad-IL12, at the MOI of 10. At different time points after infection, the cell survival was measured by CCK8 assay. Results were expressed as percentage of untreated control. The data was presented as the mean ± SD of three independent experiments. p = 0.035357 (AdCN205-GFP) and 0.033118 (AdCN205-IL12) compared with Ad-IL12 in figure A; P = 0.02159 (AdCN205-GFP) and 0.042144 (AdCN205-IL12) compared with Ad-IL12 in figure B; P = 0.045401 (AdCN205-GFP) and 0.048508 (AdCN205-IL12) compared with Ad-IL12 in figure C.

Article Snippet: Human hepatocellular carcinoma (HCC) cell lines Hep3B (HBs pos , undetected Rb), were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA), BEL7404 (HBs neg , Rb wt ), SMMC7721 (HBs neg , Rb wt ), MHCC97H (HBs pos , Rb wt hyperphosphorylated) and HuH-7 (HBs neg , Rb wt hyperphosphorylated) , , were purchased from the Shanghai Cell Collection, China.

Techniques: Infection, CCK-8 Assay, Control

Journal: PLoS ONE

Article Title: Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

doi: 10.1371/journal.pone.0044802

Figure Lengend Snippet: Liver tumor cells (HuH-7 and Hep3B) were treated with CIK cells alone at effector:target ratio of 10∶1, or AdCN205-GFP, AdCN205-IL12 at the MOI of 10, or treated with both CIK at effector:target ratio of 10∶1 and AdCN205-GFP or AdCN205-IL12 at the MOI of 10. The cell survival was determined by measuring luciferase activity. Results were expressed as percentage of untreated control. The data was presented as the mean ± SD of three independent experiments.

Article Snippet: Human hepatocellular carcinoma (HCC) cell lines Hep3B (HBs pos , undetected Rb), were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA), BEL7404 (HBs neg , Rb wt ), SMMC7721 (HBs neg , Rb wt ), MHCC97H (HBs pos , Rb wt hyperphosphorylated) and HuH-7 (HBs neg , Rb wt hyperphosphorylated) , , were purchased from the Shanghai Cell Collection, China.

Techniques: Luciferase, Activity Assay, Control

Journal: International journal of oncology

Article Title: The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

doi: 10.3892/ijo.2015.3087

Figure Lengend Snippet: Figure 1. Effect of panobinostat (LBH589) on gene expression of connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF) and fms-like tyrosine kinase-1 (FLT-1) in vitro in HepG2 and Hep3B cells. (A) Quantitative real-time RT-PCR analysis after 24, 48 and 72 h treatment with 0.01 µM panobinostat (A) and 0.1 µM panobinostat (B) in HepG2 cells, with 0.01 µM panobinostat (C) and 0.1 µM panobinostat (D) in Hep3B cells. Gene expression was normalized to β-actin and expressed relative to untreated controls (set at 1.0). Asterisks indicate statistically significant differences in gene expression vs. the untreated controls (p<0.01). The symbol (x) indicates that very few viable cells remained after 72 h of treatment in these cells.

Article Snippet: The human hepatoma cell lines HepG2 (p53wt) and Hep3B (p53null) were obtained from the German collection of microorganisms and cell cultures (DSMZ, braunschweig, Germany) and maintained under standard conditions as described previously (41).

Techniques: Gene Expression, In Vitro, Quantitative RT-PCR

Journal: International journal of oncology

Article Title: The pan-deacetylase inhibitor panobinostat affects angiogenesis in hepatocellular carcinoma models via modulation of CTGF expression.

doi: 10.3892/ijo.2015.3087

Figure Lengend Snippet: Figure 2. Western blot analysis of expression of connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), fms-like tyrosine kinase-1 (FLT-1), kinase insert domain containing receptor (KDR), MAPK, p-MAPK in vitro and densitometric quantification. HepG2 and Hep3B cells were incubated with 0.1 and 0.01 µM panobinostat for 12, 24 and 48 h. Western blot results show representative examples for expression of CTGF, VEGF, FLT-1, KDR, MAPK and p-MAPK as well as β-actin, which served as loading control (A). Protein expression analyzed using western blot analysis was quantified by densitometry (B). Shown are expression values for CTGF, MAPK, p-MAPK, KDR and VEGF after treatment of HepG2 and Hep3B cell lines with 0.01 and 0.1 µM of panobinostat for 12-48 h.

Article Snippet: The human hepatoma cell lines HepG2 (p53wt) and Hep3B (p53null) were obtained from the German collection of microorganisms and cell cultures (DSMZ, braunschweig, Germany) and maintained under standard conditions as described previously (41).

Techniques: Western Blot, Expressing, In Vitro, Incubation, Control

Journal: Oncology Reports

Article Title: Long non-coding RNA H19 is involved in sorafenib resistance in hepatocellular carcinoma by upregulating miR-675

doi: 10.3892/or.2020.7608

Figure Lengend Snippet: H19 expression is negatively related to sorafenib sensitivity in HCC cell linws (Huh7, Hep3B, SNU-449, SNU-387). (A) Cell viability was examined by CCK-8 assay in four HCC cell lines subjected to sorafenib treatment. (B) Statistical analysis of the IC 50 in four HCC cell lines subjected to sorafenib treatment. (C) Relative mRNA levels of H19 as measured by RT-qPCR and normalized to β-actin in four HCC cell lines. (D) Cell proliferation was examined by EdU assay in four HCC cell lines subjected to sorafenib treatment. **P<0.01, ***P<0.001, compared to the control. HCC, hepatocellular carcinoma; IC 50 , half maximal inhibitory concentration.

Article Snippet: Human HCC cell lines (Huh7, Hep3B, SNU-449, SNU-387) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, CCK-8 Assay, Quantitative RT-PCR, EdU Assay, Control, Concentration Assay

Journal: Oncology Reports

Article Title: Long non-coding RNA H19 is involved in sorafenib resistance in hepatocellular carcinoma by upregulating miR-675

doi: 10.3892/or.2020.7608

Figure Lengend Snippet: H19 knockdown sensitizes HCC cells to sorafenib in vitro . (A) Sorafenib sensitivity of HCC cells transfected with NC siRNA or H19 siRNA. (B) Proliferative capacity of HCC cells transfected with NC siRNA or H19 siRNA and subjected to sorafenib treatment. **P<0.01, ***P<0.001, compared with the sorafenib group. (C) H19 expression in HCC cells transfected with NC siRNA or H19 siRNA and subjected to sorafenib treatment. *P<0.05, **P<0.01, ***P<0.001, compared to the control. HCC, hepatocellular carcinoma; NC, negative control.

Article Snippet: Human HCC cell lines (Huh7, Hep3B, SNU-449, SNU-387) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Knockdown, In Vitro, Transfection, Expressing, Control, Negative Control

Journal: Oncology Reports

Article Title: Long non-coding RNA H19 is involved in sorafenib resistance in hepatocellular carcinoma by upregulating miR-675

doi: 10.3892/or.2020.7608

Figure Lengend Snippet: H19 knockdown inhibits EMT and H19 positively regulates miR-675. (A) Immunofluorescence staining showed that E-cadherin (green) and vimentin (green) levels were altered after transfection of NC siRNA or H19 siRNA in the SNU-449 and SNU-387 cells. (B) E-cadherin and vimentin as assessed by western blot analysis. GAPDH was used as loading control. (C) Relative mRNA levels of miR-675 were measured by RT-qPCR and normalized to U6 after transfection of NC siRNA or H19 siRNA in four HCC cell lines. *P<0.05, **P<0.01, ***P<0.001, compared to the control. HCC, hepatocellular carcinoma; NC, negative control; EMT, epithelial-mesenchymal transition.

Article Snippet: Human HCC cell lines (Huh7, Hep3B, SNU-449, SNU-387) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Knockdown, Immunofluorescence, Staining, Transfection, Western Blot, Control, Quantitative RT-PCR, Negative Control

Journal: Oncology Reports

Article Title: Long non-coding RNA H19 is involved in sorafenib resistance in hepatocellular carcinoma by upregulating miR-675

doi: 10.3892/or.2020.7608

Figure Lengend Snippet: miR-675 regulates sensitivity of HCC cells to sorafenib. (A) Sorafenib sensitivity of HCC cells transfected with NC siRNA, miR-675 mimic, or miR-675 inhibitor. (B) Proliferation of HCC cells transfected with NC siRNA, miR-675 mimic, or miR-675 inhibitor in the presence of sorafenib. ***P<0.001, compared with the sorafenib group. (C) Expression of miR-675 in HCC cells transfected with NC siRNA, miR-675 mimic, or miR-675 inhibitor in the presence of sorafenib. *P<0.05, **P<0.01, ***P<0.001. HCC, hepatocellular carcinoma; NC, negative control.

Article Snippet: Human HCC cell lines (Huh7, Hep3B, SNU-449, SNU-387) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Transfection, Expressing, Negative Control

Journal: Oncology Reports

Article Title: Long non-coding RNA H19 is involved in sorafenib resistance in hepatocellular carcinoma by upregulating miR-675

doi: 10.3892/or.2020.7608

Figure Lengend Snippet: miR-675 alters EMT. (A) Immunofluorescence showed that the staining of E-cadherin (green) and vimentin (green) was altered after transfection with the miR-675 mimic, inhibitor, or NC siRNA in SNU-449 and SNU-387 cells. (B) E-cadherin and vimentin levels in HCC cells transfected with NC siRNA, miR-675 mimic, or miR-675 inhibitor in the presence of sorafenib. HCC, hepatocellular carcinoma; NC, negative control; EMT, epithelial-mesenchymal transition.

Article Snippet: Human HCC cell lines (Huh7, Hep3B, SNU-449, SNU-387) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Immunofluorescence, Staining, Transfection, Negative Control

Journal: Oncology Reports

Article Title: Long non-coding RNA H19 is involved in sorafenib resistance in hepatocellular carcinoma by upregulating miR-675

doi: 10.3892/or.2020.7608

Figure Lengend Snippet: miR-675 mediates the regulatory effect of lncRNA H19 on sorafenib sensitivity. Sorafenib sensitivity of HCC cells transfected with NC siRNA or H19 siRNA was evaluated by CCK-8 assay in the presence of the miR-675 mimic for 48 h. HCC, hepatocellular carcinoma; NC, negative control.

Article Snippet: Human HCC cell lines (Huh7, Hep3B, SNU-449, SNU-387) were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Transfection, CCK-8 Assay, Negative Control

Journal: Disease Markers

Article Title: hsa_circ_0129047 Upregulates LYVE1 to Inhibit Hepatocellular Carcinoma Progression by Sponging miR-492

doi: 10.1155/2023/6978234

Figure Lengend Snippet: Expression of circ_0129047 decreases during HCC progression. (a) RT-qPCR analysis of circ_0129047 in HCC cells (Huh7, Hep 3B, and SNU-182) and human liver cell line THLE2. ∗∗ P < 0.001, vs. THLE2. (b) RT-qPCR analysis of circ_0129047 in HCC and normal tissues. (c) RT-qPCR analysis of circ_0129047 in the nuclear-cytoplasmic fractionation of Huh7 and Hep 3B cells. (d) RT-qPCR analysis of circ_0129047 and LIFR (the linear gene of circ_0129047) in Huh7 and Hep 3B cells with and without RNAse R treatment. ∗∗ P < 0.001, vs. RNAse R.

Article Snippet: A human liver cell line (THLE2) and two HCC cell lines (Hep 3B and SNU-182) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA.

Techniques: Expressing, Quantitative RT-PCR, Fractionation

Journal: Disease Markers

Article Title: hsa_circ_0129047 Upregulates LYVE1 to Inhibit Hepatocellular Carcinoma Progression by Sponging miR-492

doi: 10.1155/2023/6978234

Figure Lengend Snippet: circ_0129047 overexpression delays in vitro and in vivo HCC growth. Overexpression of circ_0129047 (OE-circ) and empty vectors (OE-NC) was induced in Hep 3B and Huh7 cells. (a) RT-qPCR was verifying the exogenous expression of circ_0129047 in Hep 3B and Huh7 cells. (b) CCK8 assays examining the proliferation of Hep 3B and Huh7 cells transfected with OE-circ and OE-NC. (c) Scratch migration assay for examining HCC cell migration. (d) HCC cell invasion ability was detected using a transwell invasion assay. (e) Hep 3B cells overexpressing circ_0129047 or NC were introduced into nude mice through subcutaneous flank injection. Tumor volumes were recorded every 7 days, and xenograft tumors were excised and weighed at 5 weeks. The xenograft tumors were photographed, measured, and weighed. ∗∗ P < 0.001, vs. OE-NC.

Article Snippet: A human liver cell line (THLE2) and two HCC cell lines (Hep 3B and SNU-182) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA.

Techniques: Over Expression, In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Transfection, Migration, Transwell Invasion Assay, Injection

Journal: Disease Markers

Article Title: hsa_circ_0129047 Upregulates LYVE1 to Inhibit Hepatocellular Carcinoma Progression by Sponging miR-492

doi: 10.1155/2023/6978234

Figure Lengend Snippet: Circ_0129047 targets miR-492. (a) The binding pairs of miR-492 and circ_0129047 were predicted by CircInteractome. (b) Hep 3B and Huh7 cells were cotransfected with a miR-492 mimic or mimic NC and circ_0129047-WT or circ_0129047-MUT luciferase vectors. Luciferase activities were determined using the dual-luciferase system. ∗∗ P < 0.001, vs. miR-NC. (c) RT-qPCR analysis of immunoprecipitated circ_0129047 and miR-492 using an anti-Ago2 antibody. ∗∗ P < 0.001, vs. Anti-lgG. (d) RT-qPCR analysis of miR-492 expression in HCC tissues. (e) RT-qPCR analysis of miR-492 expression in HCC (Hep 3B and Huh7) and THLE2 cells. ∗∗ P < 0.001, vs. THLE2. (f) Pearson analysis of the correlation between miR-492 and circ_0129047. (g) Hep 3B and Huh7 cells transfected with OE-NC, OE-circ (OE-circ_0129047), miR-492 mimic, mimic NC, and OE-circ + mimic. The expression of miR-492 was determined by RT-qPCR at 48 hr. ∗∗ P < 0.001, vs. OE-NC; ## P < 0.001, vs.mimic-NC; && P < 0.001, vs.OE + mimic.

Article Snippet: A human liver cell line (THLE2) and two HCC cell lines (Hep 3B and SNU-182) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA.

Techniques: Binding Assay, Luciferase, Quantitative RT-PCR, Immunoprecipitation, Expressing, Transfection

Journal: Disease Markers

Article Title: hsa_circ_0129047 Upregulates LYVE1 to Inhibit Hepatocellular Carcinoma Progression by Sponging miR-492

doi: 10.1155/2023/6978234

Figure Lengend Snippet: miR-492 downregulation is critical for the inhibitory effect of circ_0129047 on HCC cell survival. Hep 3B and Huh7 cells were transfected with OE-NC, OE-circ (OE-circ_0129047), miR-492 mimic, NC mimic, or OE-circ+mimic. (a) CCK8 assays were conducted to determine HCC cell proliferation. (b) Scratch migration assays were conducted to evaluate the migration rate of HCC cells after 24 hr of transfection. (c) Transwell migration assays were conducted to examine HCC cell invasion after 48 hr of transfection. ∗ P < 0.05; ∗∗ P < 0.001, vs. mimic; ## P < 0.001, vs.mimic-NC; & P < 0.05; && P < 0.001, vs.OE + mimic.

Article Snippet: A human liver cell line (THLE2) and two HCC cell lines (Hep 3B and SNU-182) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA.

Techniques: Transfection, Migration

Journal: Disease Markers

Article Title: hsa_circ_0129047 Upregulates LYVE1 to Inhibit Hepatocellular Carcinoma Progression by Sponging miR-492

doi: 10.1155/2023/6978234

Figure Lengend Snippet: MiR-492 targets LYVE1. (a) TargetScan was used to identify the target of miR-492. (b) Luciferase activity driven by 3′UTR LYVE1 MUT and WT was assessed in Hep 3B and Huh7 cells cotransfected with miR-492 mimic or mimic NC. ∗∗ P < 0.001, vs. miR-NC. (c) RT-qPCR analysis of LYVE1 expression in HCC and normal tissues. (d) RT-qPCR analysis of LYVE1 in THLE2 and HCC cells. ∗∗ P < 0.001, vs. THLE2. (e) Pearson analysis of the association between LYVE1 and miR-492 in HCC tissues. (f) Western blotting was conducted to examine the LYVE1 expression in Hep 3B and Huh7 cells transfected with OE-NC, mimic NC, OE-circ mimic, or OE-circ + mimic. ∗∗ P < 0.001, vs. OE-NC; ## P < 0.001, vs.mimic-NC; && P < 0.001, vs.OE- circ + mimic.

Article Snippet: A human liver cell line (THLE2) and two HCC cell lines (Hep 3B and SNU-182) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA.

Techniques: Luciferase, Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Transfection

Journal: Disease Markers

Article Title: hsa_circ_0129047 Upregulates LYVE1 to Inhibit Hepatocellular Carcinoma Progression by Sponging miR-492

doi: 10.1155/2023/6978234

Figure Lengend Snippet: LYVE1 downregulation contributes to the oncogenic behaviors of miR-492. Hep 3B and Huh7 cells were transfected with OE-NC, mimic NC, OE-LYVE1 mimic, or OE-LYVE1+mimic. (a) Western blot conducted to examine LYVE1 expression in Hep 3B and Huh7 cells. (b) The CCK8 assay was conducted to determine HCC cell proliferation. (c) Scratch migration assays were conducted to evaluate the migration rate of HCC cells after 24 hr of transfection. (d) Transwell migration assays were conducted to examine HCC cell invasion after 48 hr of transfection. ∗∗ P < 0.001, vs.OE-NC; # P < 0.05; ## P < 0.001, vs. mimic-NC; & P < 0.05; && P < 0.001, vs. OE + mimic.

Article Snippet: A human liver cell line (THLE2) and two HCC cell lines (Hep 3B and SNU-182) were purchased from the American Type Culture Collection (ATCC), Manassas, VA, USA.

Techniques: Transfection, Western Blot, Expressing, CCK-8 Assay, Migration

Journal: BMC Biochemistry

Article Title: Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

doi: 10.1186/1471-2091-9-9

Figure Lengend Snippet: A pro-protein convertase inhibitor prevents accumulation of soluble 40 kDa RGMc . A. Map of mouse RGMc with RGD motif, von Willebrandt factor (vWF) type D domain, GPI anchor, and intra-molecular cleavage site (arrow) indicated. The conserved PC site ( underlined , arginines bolded ) is aligned below the map for 7 mammalian species. B. Absence of putative PC sites in RGMa or b. Alignment of human RGMa, b, and c; vertical lines or asterisks denote identical residues between paralogous proteins. C. Immunoblot of affinity-purified RGMc from mouse serum and muscle cell conditioned medium, with 50 and 40 kDa isoforms indicated. D. Immunoblot of affinity-purified RGMc from blood from two humans (a, b) and two mice (c, d), with 50 and 40 kDa isoforms indicated. Soluble HA-RGMc is included as a reference. E. Immunoblot of RGMc from muscle cell conditioned medium (50 μl) incubated without or with the small molecule PC inhibitor, RVKR. F. Immunoblot of RGMc from conditioned medium (25 μl) of C2 myoblasts or Hep3B cells infected with Ad-HA-RGMc and incubated ± RVKR. For C – F , black arrows indicate 50 kDa RGMc and white arrows 40 kDa.

Article Snippet: Cos-7 (ATCC #CRL-1651) and Hep3B cells (ATCC #HB-8064) were grown in DMEM and 10% FCS.

Techniques: Western Blot, Affinity Purification, Incubation, Infection

Journal: BMC Biochemistry

Article Title: Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

doi: 10.1186/1471-2091-9-9

Figure Lengend Snippet: Altered processing of newly synthesized RGMc by PC inhibition . A. Time course of accumulation of RGMc on the cell surface and in conditioned medium after infection of Hep3B cells with Ad-HA-RGMc ± RVKR. Cell-surface proteins were labeled with non-permeable biotin (EZ-link), followed by incubation ± RVKR, and streptavidin pull-down, as described in 'Methods'. Immunoblot for cadherin measures sample loading. B. PC inhibition does not prevent acute release of RGMc from the cell surface. Membrane-associated RGMc was labeled with EZ-link, followed by incubation ± RVKR, and detection of soluble RGMc after streptavidin pull-down by immunoblotting. For A – B , arrows are described in legend to Fig. 1. Similar results were observed with Cos-7 cells.

Article Snippet: Cos-7 (ATCC #CRL-1651) and Hep3B cells (ATCC #HB-8064) were grown in DMEM and 10% FCS.

Techniques: Synthesized, Inhibition, Infection, Labeling, Incubation, Western Blot, Membrane

Journal: BMC Biochemistry

Article Title: Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

doi: 10.1186/1471-2091-9-9

Figure Lengend Snippet: Furin cleaves soluble RGMc to produce the 40 kDa isoform . A. Only 50 kDa RGMc accumulates in culture medium of cells expressing a protein with a modified PC cleavage site. Immunoblot of soluble RGMc from conditioned medium of Cos-7 cells transfected with either wild-type (WT) RGMc or a derivative containing a mutated PC site (RXXR mut ), after incubation ± RVKR. Similar results were observed with Hep3B cells. B. Hep3B cells cleave 50 kDa RGMc. RGMc was added to medium of Hep3B cells for 24 h ± RVKR, followed by immunoblotting (nt = non-treated). Identical results were seen with Cos-7 cells. C. Furin cleaves 50 kDa RGMc. Immunoblot shows results of in vitro incubation of soluble 50 kDa HA-RGMc ± recombinant furin. Antibodies are indicated. For A – C , arrows are as in legend to Fig. 1.

Article Snippet: Cos-7 (ATCC #CRL-1651) and Hep3B cells (ATCC #HB-8064) were grown in DMEM and 10% FCS.

Techniques: Expressing, Modification, Western Blot, Transfection, Incubation, In Vitro, Recombinant

Journal: BMC Biochemistry

Article Title: Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

doi: 10.1186/1471-2091-9-9

Figure Lengend Snippet: A. Iron loading increases expression of RGMc on the cell membrane and diminishes accumulation in extra-cellular fluid . A. Hep3B cells infected with Ad-HA-RGMc were incubated for 24 h with the concentrations of ferric ammonium citrate (FAC) indicated, followed by cell-surface biotin labeling, and detection of RGMc by immunoblotting in cell lysates (left panel), on the membrane after surface biotin labeling and streptavidin pull-down (middle panel), and in the medium (right panel). Similar results were observed with Cos-7 cells. B. Detection of RGMc by immunoblotting of conditioned medium from transiently transfected Cos-7 cells following incubation for 24 h with the concentrations of FAC or deferoxamine (DFO) indicated. Identical results were seen with Hep3B cells. For A – B , arrows are as in legend to Fig. 2.

Article Snippet: Cos-7 (ATCC #CRL-1651) and Hep3B cells (ATCC #HB-8064) were grown in DMEM and 10% FCS.

Techniques: Expressing, Membrane, Infection, Incubation, Labeling, Western Blot, Transfection