Journal: Cancer Science

Article Title: Synthetic disulfide-bridged cyclic peptides mimic the anti-angiogenic actions of chondromodulin-I

doi: 10.1111/j.1349-7006.2012.02276.x

Figure Lengend Snippet: Effects of the synthetic and tailed chondromodulin-I (ChM-I) cyclic peptide on tumor angiogenesis and growth in an animal model in which OUMS-27 cells (5 × 10 6 cells) were subcutaneously inoculated in the backs of 4 week-old nude mice. (A) Time-course of tumor volume changes. When the tumor volume reached about 45 mm 3 , each mouse was injected around the tumor mass each day for the initial 5 days with PBS (50 μL) alone (♢), PBS containing 4.3 nmol (20 μg) ChM-I cyclic peptide with a tail (○), or PBS containing 0.2 nmol (5 μg) recombinant human ChM-I (rhChM-I) (●) (arrows). The tumor volumes were determined by the width 2 × length × 0.52. Values are the means ± SD from at least six animals per group. (B) Gross appearance of tumors excised on day 21. Bar, 10 mm. (C) Immunohistochemical staining of the CD31-positive vasculature. On day 21, tumor tissues were excised, fixed, and cross-sectioned. The sections were then stained with toluidine blue (left panels), and semi-serial sections were stained with an anti-type II collagen antibody ( green signal in the right panels) and an anti-CD31 antibody ( red signal in the right panels), respectively. Bars, 100 μm. (D) The CD31-positive area was measured as described in the methods section. Values are the means ± SD of five tumors per group. * P < 0.05, ** P < 0.01.

Article Snippet: Anti-CD31 antibody and anti-collagen type II antibody were obtained from BD PharMingen (San Diego, CA, USA) and Rockland (Gilvertsville, PA, USA), respectively.

Techniques: Animal Model, Injection, Recombinant, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells.

doi: 10.18632/oncotarget.4373

Figure Lengend Snippet: Figure 1: Doxorubicin induced apoptosis and reduced Madcam1 expression. A.-B. Doxo induced dose-dependent cleavage of Caspase substrates (CCS), as analyzed by WB using an anti-cleaved caspase substrate antibodies, in SMMC-7721 and Bel-7402 cells treated with DMSO or Doxo (final concentration 0.5-2.0 µg/ml) for 24 h. The representative western blots are shown in panel A. The CCS was calculated as the ratio between the levels of cleaved Caspase substrate and GAPDH, and the data are shown in panel B. The groups treated with 0.5 µg/ml Doxo were arbitrarily set to 100%. C. Doxo induced Caspase 3/7 activity, as measured by a Caspase 3/7 Glo luciferase kit from Promega, in cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h. The “ DMSO” group was arbitrarily set to 100%. D. Doxo reduced cell proliferation, as measured by an MTT-based assay, in cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h. The “ DMSO” group was arbitrarily set to 100%. E. Potential proteins that were regulated by Doxo. Endogenous expression of the indicated proteins were tested by WB in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h. F. The relative levels of Madcam1 protein expression in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h were calculated as the ratio between Madcam1 and GAPDH. The “DMSO” group was arbitrarily set to 100 %. G.-H. Exogenous Madcam1-Myc was measured in Bel-7402 transfected with GFP and Madcam1-Myc expressing plasmids and treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h. Representative WB images of are shown in panel G, the relative expression levels of exogenous Myc were calculated as the ratio to GFP, and the data are shown in panel H. The “DMSO” group was arbitrarily set to 100 %. I. Subcellular localization of Madcam1 in Bel-7402 and SMMC-7721 cells, as measured by the immunofluorescence assay. Scale bar, 5 µM. J. Madcam1 mRNA levels in Bel-7402 and SMMC-7721 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h, as measured by qPCR assays. The Madcam1 levels were normalized to GAPDH. The “DMSO” group was arbitrarily set to 100 %. K. Bel-7402 cells were pretreated with DMSO or Doxo (final concentration 2.0 µg/ml) for 2 h before adding DMSO or actinomycin D (final concentration 10 μg/ml) for further treatment for 24 h. Then, the RNA was extracted and measured by qPCR assays. The Madcam1 levels were normalized to GAPDH. The group treated with DMSO only was arbitrarily set to 100 %. L.-M. Doxo had no influence on Madcam1 protein stability. Bel-7402 cells were pretreated with DMSO or Doxo (final concentration 2.0 g/ml) for 2 h before being treated with CHX (final concentration 50 μg/ml) for the indicated times. Then, the cells were harvested for WB using anti-Madcam1 and anti-GAPDH antibodies L.. The Madcam1 protein levels were normalized to GAPDH, and the data are shown in panel M.. The group treated with CHX at the 0 h point was arbitrarily set to 100 %. N.-O. Time-dependent enrichment of eIF4E on the Madcam1 mRNA. Bel-7402 N. and SMMC- 7721 O. cells were treated with same amount of DMSO or Doxo (final concentration 2.0 µg/ml) for the indicated times. Then, the RNA was extracted and subjected into RNA-IP assays using an anti-eIF4E antibody. The “0h” point was arbitrarily set to 100 %. **, Indicates statistical significance between “DMSO” and “Doxo” at specific time points. The data are shown as the means + SD from three independent experiments (including WB). **, p < 0.01 using the Student’s t test.

Article Snippet: The cDNA fragments encoding human Madcam1 were purchased from Origene (Beijing, China) and subcloned into the pcDNA3.1 (+) vector with a C-terminal FLAG tag or into the pLJM-based lentiviral vector with a C-terminal Myc tag.

Techniques: Expressing, Concentration Assay, Western Blot, Activity Assay, Luciferase, MTT Assay, Transfection, Immunofluorescence

Journal: Oncotarget

Article Title: Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells.

doi: 10.18632/oncotarget.4373

Figure Lengend Snippet: Figure 2: Madcam1 suppressed Doxorubicin-induced apoptosis in HCC cells. A.-B. CCS and Madcam1 in different cells that were treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h before harvest for WB using an anti-cleaved Caspase substrate antibody. Representative images of the WB are shown in panel A. The levels of CCS and Madcam1 were normalized to GAPDH, and the data are shown in panel B. The data from the “DMSO-treated” HL-7702 cells was arbitrarily set to 100 %. C.-D. Madcam1 overexpression reduced Doxo-induced apoptosis. Bel-7402 and SMMC-7721 cells infected with empty or Madcam1-Myc expressing lentiviral plasmids were treated with same amount of DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h before harvest for WB analysis using anti- cleaved Caspase substrate antibodies. Representative images of the WB are shown in panel C. The CCS levels were normalized to GAPDH, and the data are shown in panel D. The SMMC-7721 cells infected with the empty vector and treated with DMSO were arbitrarily set to 100 %. E.-F. Madcam1 knockdown reinforced Doxo-induced apoptosis. Control (infected with GFP-sh) and Madcam1 knockdown (infected with Madcam1-sh1 and –sh2, respectively) Bel-7402 or SMMC-7721 cells were treated with DMSO or Doxo (final concentration 2.0 µg/ ml) for 24 h before harvest for WB analysis using anti-cleaved Caspase substrate antibodies. Representative images of the WB are shown in panel E. The CCS levels were normalized to GAPDH, and the data are shown in panel F. The cells infected with Mad-sh1 and treated with DMSO were arbitrarily set to 100 %. G. Madcam1 prevented Doxo-induced apoptosis, as measured by a flow cytometer using an Annexin V-FITC early apoptosis detection kit, in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h. H. Madcam1 reduced cleaved Caspase 3 expression, as measured by immunofluorescence assay, in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h. Scale bar, 20 µM. I. Madcam1 reduced Doxo-induced and Caspase 3/7 activity. Control (Mock) and Madcam1 overexpressing or knockdown Bel-7402 and SMMC-7721 cells were treated with DMSO or Doxo (final concentration 2.0 µg/ ml) for 24 h before harvest for testing the Caspase 3/7 activity using a Caspase 3/7 Glo luciferase kit. Data from the “Mock treated with DMSO” group were arbitrarily set to 100 %. J.-K. Time-dependent Caspase 3/7 activities after Doxo (final concentration 0.5 µg/ml) treatment in the indicated Bel-7402 J. and SMMC-7721 K. cells was measured by a Caspase 3/7 Glo luciferase kit from Promega. The data from the “Mock-0h” group were arbitrarily set to 100 %. **, p < 0.01 versus the Mock group at the same time point using the Student’s t test. L. Madcam1 knockdown reduced cell proliferation, as measured by an MTT-based assay, in Bel-7402 and SMMC-7721 cells. The initial cell number is 5,000, and the cells were cultured for another 5 days before the OD value was determined. M. Measurement of xenografts generated by Bel-7402 cells in vivo. The tumor size formed from the subcutaneous injection of Bel-7402 cells with the different indicated treatments was measured with calipers at 30 days after injection and calculated as 0.5 × L × W2 (n = 5 per group). The data are shown as the means + SD from three independent experiments (including WB). **, p < 0.01 using the Student’s t test.

Article Snippet: The cDNA fragments encoding human Madcam1 were purchased from Origene (Beijing, China) and subcloned into the pcDNA3.1 (+) vector with a C-terminal FLAG tag or into the pLJM-based lentiviral vector with a C-terminal Myc tag.

Techniques: Concentration Assay, Over Expression, Infection, Expressing, Plasmid Preparation, Knockdown, Control, Flow Cytometry, Immunofluorescence, Activity Assay, Luciferase, MTT Assay, Cell Culture, Generated, In Vivo, Injection

Journal: Oncotarget

Article Title: Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells.

doi: 10.18632/oncotarget.4373

Figure Lengend Snippet: Figure 3: Madcam1 suppressed Doxorubicin in translation initiation A. Screening for possible Doxo target proteins, as analyzed by WB using a human apoptosis antibody array (containing 43 Targets) from Abcam. Bel-7402 cells were treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h before being subjected to the array analysis. The images of the array membrane are shown on the left. Shorter, shorter exposure; Longer, longer exposure. The spot positions of protein on the membrane are listed on the right. B. The same samples from panel A were subjected to WB for the confirmation of Doxo-induced apoptosis using anti-cleaved Caspase substrate antibodies. C. Screening targets before and after Madcam1 knockdown. Bel-7402 cells infected with GFP-sh or Madcam1-sh1 were harvested for WB using the array described in panel A. Shorter, shorter exposure; Longer, longer exposure. D. Madcam1-sh1knockdown efficiency in the samples from panel C. E. Representative western blots for the indicated proteins in Bel-7402 and HL-7702 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h. F. Representative western blots of the indicated proteins in Bel-7402 cells infected with GFP-sh, Madcam1-sh1 or Madcam1–sh2. G.-H. mRNA levels in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h G. or in Bel-7402 cells infected with GFP-sh or Madcam1-sh1 H.. The mRNA levels were measured by qPCR assays. The levels in the “DMSO” or “GFP-sh” groups were arbitrarily set to 100 %. I. Doxo had no impact on protein stability. Bel- 7402 cells were pretreated with DMSO or Doxo (final concentration 2.0 µg/ml) for 2 h before adding CHX for the indicated times. Then, the cell lysates were prepared for WB. J. Madcam1 had no effect on protein stability. Bel-7402 cells stably transfected with the empty or Madcam1-Myc expressing plasmids were treated with CHX for the indicated times. Then, the cell lysates were prepared for WB. K. Doxo and Madcam1 had opposite influences on eIF4E binding. The RNA was extracted from Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 4 h (upper panel) or Bel-7402 cells infected with GFP-sh or Madcam1-sh1 (lower panel), and subjected to RNA-IP assays using anti-eIF4E antibodies. The data from the “DMSO” or “GFP-sh” groups were arbitrarily set to 100 %. L.-M. Inhibiting protein synthesis induced apoptosis. Bel-7402, HL-7702 and SMMC-7721 cells were treated with DMSO or CHX (final concentration 50 μg/ml) for 24 h before harvest for WB using anti-cleaved Caspase substrate antibodies. Representative images of the WB are shown in panel L. The relative CCS levels were calculated as the ratio to GAPDH, and the data are shown in the panel M. The data are shown as the means + SD from three independent experiments. **, p < 0.01 using the Student’s t test.

Article Snippet: The cDNA fragments encoding human Madcam1 were purchased from Origene (Beijing, China) and subcloned into the pcDNA3.1 (+) vector with a C-terminal FLAG tag or into the pLJM-based lentiviral vector with a C-terminal Myc tag.

Techniques: Ab Array, Concentration Assay, Membrane, Knockdown, Infection, Western Blot, Stable Transfection, Transfection, Expressing, Binding Assay

Journal: Oncotarget

Article Title: Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells.

doi: 10.18632/oncotarget.4373

Figure Lengend Snippet: Figure 4: Doxorubicin and Madcam1 had opposite effects on eIF4E. A. Doxo reduced 4EBP1 phosphorylation. Phosphorylation of translation-associated proteins was detected in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 4 h before harvest for WB using the indicated antibodies. B. p-4EBP1 and 4EBP1 were detected by WB in Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 4 h before harvest. C. Madcam1 overexpression induced 4EBP1 phosphorylation, as detected by WB, in Bel-7402 cells transfected with empty or Madcam1-Myc expressing plasmids. D. Madcam1 knockdown reduced 4EBP1 phosphorylation, as detected by WB, in Bel-7402 cells infected with GFP-sh, Madcam1-sh1 or Madcam1–sh2. E. Doxo induced 4EBP1 binding to eIF4E. Bel-7402 cells were treated with DMSO or increasing concentrations of Doxo (final concentration from 0.5-2.5 µg/ml) for 5 h before harvest in IP lysis buffer. Endogenous eIF4E was then immunoprecipitated with an anti-eIF4E antibody and 4EBP1 co-immunoprecipitation was detected by WB. F. Madcam1 reduced 4EBP1 binding to eIF4E. Endogenous eIF4E was immunoprecipitated with an anti-eIF4E antibody and 4EBP1 co-immunoprecipitation was detected by WB for control (transfected with Empty vector) and Madcam1-Myc transfected Bel-7402 cells, using increasing amounts of ectopically expressed Madcam1-Myc. G. Identification of the subcellular localization of Madcam1. Co-localization of Madcam1 and possible organelles was analyzed in Bel-7402 cells by immunofluorescence assays using a combination of anti-Madcam1 (red) and anti-LC3B (autophagosome marker, green), anti-COX IV (mitochondria marker, green), anti- Calnexin (endoplasmic reticulum (ER) marker, green) or anti-NUP98 (nuclear envelop marker, green) antibodies. The area indicated by the square was enlarged on the right side. Scale bar, 10 µM. H. Madcam1 reversed Doxo-induced dephosphorylation of 4EBP1. Bel-7402 cells were treated with or without Doxo (final concentration 2.0 µg/ml) for 4 h in the presence or absence of ectopically expressed Madcam1- Myc before harvest for WB using anti-p-4EBP1 and 4EBP1 antibodies. I. Madcam1 prevented Doxo-induced protein down-regulation. Bel-7402 cells were treated as described in Figure 4H and were harvested for WB using the indicated antibodies. J. Madcam1 inhibited Doxo-induced dissociation of eIF4E from the mRNA. The RNA was extracted from Bel-7402 cells that were treated as in Figure 4h for RNA-IP assays using anti-eIF4E antibodies. The data from the “Mock” group were arbitrarily set to 100 %. The data are shown as the means + SD from three independent experiments (including WB). *, p < 0.05 using the Student’s t test.

Article Snippet: The cDNA fragments encoding human Madcam1 were purchased from Origene (Beijing, China) and subcloned into the pcDNA3.1 (+) vector with a C-terminal FLAG tag or into the pLJM-based lentiviral vector with a C-terminal Myc tag.

Techniques: Phospho-proteomics, Concentration Assay, Over Expression, Transfection, Expressing, Knockdown, Infection, Binding Assay, Lysis, Immunoprecipitation, Control, Plasmid Preparation, Immunofluorescence, Marker, De-Phosphorylation Assay

Journal: Oncotarget

Article Title: Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells.

doi: 10.18632/oncotarget.4373

Figure Lengend Snippet: Figure 5: Doxorubicin inhibited while Madcam1 stimulated AKT. A. Screening for possible signaling nodes that were regulated by both Doxo and Madcam1. Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 4 h as well as Bel-7402 cells infected with GFP-sh or Madcam1-sh1 were harvested and subjected to the Pathscan™ antibody array (CST). The array images are shown on the left side, and the spots representing the positive control and p-AKT (Thr308) are indicated on the right side by the square and rectangle, respectively. B.-C. Verification of Doxo-induced AKT de-phosphorylation. Western blots of p-AKT and AKT from Bel-7402 cells treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 4 h B.. The levels of p-AKT in Bel-7402 and SMMC-7721 cells were normalized to AKT C.. The data from the “DMSO” group were arbitrarily set to 100%. D.-E. Madcam1 overexpression induced AKT phosphorylation. Representative western blots of p-AKT and AKT from Bel-7402 cells transfected with the empty or Madcam1- Myc expressing plasmids D.. The p-AKT levels from Bel-7402 and SMMC-7721 cells were normalized to f AKT, and the data from the “Empty” group were arbitrarily set to 100% E..F.-G. Madcam1 knockdown reduced AKT phosphorylation. Representative western blots of p-AKT and AKT from Bel-7402 cells infected with GFP-sh, Madcam1-sh1 or Madcam1–sh2 F.. The p-AKT levels from Bel-7402 and SMMC-7721 cells were normalized to AKT, and the data from the “GFP-sh” group were arbitrarily set to 100% G..H.-I. Representative images of TMA stained with anti-Madcam1 or anti-p-AKT antibodies. Scale bar, 200 M H.. The TMA data were analyzed using the χ2 test I.. J.-K. Blockage of PI3K/AKT inhibited 4EBP1 phosphorylation. Representative western blots of the indicated proteins from Bel-7402 cells treated with DMSO or Wortmannin (final concentration 50 μM) for 4 h J.. The ratio between p-4EBP1 and 4EBP1 are shown in panel K.. The data from the “DMSO” group were arbitrarily set to 100%. L. Blockage of PI3K/AKT inhibited eIF4E binding to the mRNA, as measured by RNA-IP followed by qPCR using anti-eIF4E antibodies and the indicated primer sets, in Bel-7402 cells treated with DMSO or Wortmannin (final concentration 50 μM) for 4 h. The data from the “DMSO” group were arbitrarily set to 100%. M.-N. Blockage of PI3K/AKT reduced protein expression. Western blots of the indicated proteins from Bel-7402 cells treated with same amount of DMSO or Wortmannin (final concentration 50 μM) for 24 h. Representative images of the WB are shown in panel M. The relative levels of the indicated proteins were calculated as the ratio to GAPDH, and the data are shown in panel N. The data from the “DMSO” group were arbitrarily set to 100%. O. Blockage of PI3K/AKT induced apoptosis, as measured by a Caspase 3/7 Glo-luciferase kit, in Bel-7402 and SMMC-7721 cells treated with DMSO or Wortmannin (final concentration 50 μM) for 24 h. The data are shown as the means + SD from three independent experiments (including WB). **, p < 0.01 using the Student’s t test.

Article Snippet: The cDNA fragments encoding human Madcam1 were purchased from Origene (Beijing, China) and subcloned into the pcDNA3.1 (+) vector with a C-terminal FLAG tag or into the pLJM-based lentiviral vector with a C-terminal Myc tag.

Techniques: Concentration Assay, Infection, Ab Array, Positive Control, De-Phosphorylation Assay, Western Blot, Over Expression, Phospho-proteomics, Transfection, Expressing, Knockdown, Staining, Binding Assay, Luciferase

Journal: Oncotarget

Article Title: Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells.

doi: 10.18632/oncotarget.4373

Figure Lengend Snippet: Figure 6: The interaction between Madcam1 and AKT was critical for the regulation of apoptosis. A. Madcam1 bound AKT. Madcam1-FLAG was co-transfected with AKT-Myc into Bel-7402 cells, as indicated. Madcam1 and AKT associations were examined by reciprocal co-IP, as indicated. B. The interaction between endogenous AKT and Madcam1 was measured by reciprocal co-IP using the indicated antibodies. A control immunoglobulin G (IgG) was used as the negative control for IP. C. Red Fluorescent emissions in Bel-7402 and SMMC-7721 cells co-transfected with exogenous AKT-Myc and Madcam1-FLAG were detected by a Duolink™ proximity ligation (PLA) kit. The areas with red signals were indicated and enlarged by a square. Scale bar, 20 μM. The arrow indicates cells co- transfected with AKT-Myc and Madcam1-FLAG, while the asterisk indicates cells without successful transfection or with transfection of either AKT-Myc or Madcam1-FLAG. D. The interactions between endogenous AKT and Madcam1 in Bel-7402 and SMMC-7721 cells were detected by a Duolink™ proximity ligation (PLA) kit using anti-AKT and anti-Madcam1 antibodies, as indicated. Parallel negative controls were also performed using no antibody (Ab) or only one antibody as indicated. The areas with red signals were indicated and enlarged by a square. Scale bar, 100 μM. The asterisk indicates strong interaction signals. Ms, mouse origin; Rb, rabbit origin. E.-F. AKT acted as a downstream effecter of Madcam1. Apoptosis was evaluated by a Caspase 3/7 Glo-luciferase kit E. in Bel-7402 and SMMC-7721 cells with or without AKT or Madcam1 knockdown in the presence or absence of ectopically expressed Madcam1-FLAG or AKT-Myc. Cell proliferation was measured by an MTT-based assay F.. The data from cells without any treatment were arbitrarily set to 100 %. The data are shown as the means + SD from three independent experiments. **, p < 0.01 using the Student’s t test.

Article Snippet: The cDNA fragments encoding human Madcam1 were purchased from Origene (Beijing, China) and subcloned into the pcDNA3.1 (+) vector with a C-terminal FLAG tag or into the pLJM-based lentiviral vector with a C-terminal Myc tag.

Techniques: Transfection, Co-Immunoprecipitation Assay, Control, Negative Control, Ligation, Luciferase, Knockdown, MTT Assay

Journal: Oncotarget

Article Title: Doxorubicin induces apoptosis by targeting Madcam1 and AKT and inhibiting protein translation initiation in hepatocellular carcinoma cells.

doi: 10.18632/oncotarget.4373

Figure Lengend Snippet: Figure 7: Doxorubicin sensitivity depended on the p-AKT level. A. Western blots of p-AKT, AKT and Madcam1 in HCC tissues and adjacent normal liver tissues. Normal samples #1, 2, and 3 were paired with the HCC samples #1, 2, and 3, while HCC samples #4, 5, 6, and 7 had no paired normal samples. B.-C. Sequential overexpression of AKT and Madcam1 increased AKT phosphorylation, as measured by WB, in HL-7702 cells transfected with AKT-Myc and increasing concentrations (0.5- 1.5 µg) of Madcam1-Myc expressing plasmids. Endogenous protein expression in Bel-7402 cells was also examined B.. The data were shown as the p-AKT to AKT ratio in panel C., and the data from the “Mock (without treatment)” group were arbitrarily set to 100 %. D.-E. Sequential overexpression of AKT and Madcam1 increased protein expression. Western blots of the indicated proteins from HL-7702 cells with the same treatment as in Figures 7B and 7C. Protein expression in the Bel-7402 cells served as the control. Representative images of the WB are shown in panel D. The relative levels of the indicated proteins were calculated as the ratio to GAPDH, and the data are shown in panel E. The data from the “HL-7702 Mock (without treatment)” group were arbitrarily set to 100%. F. Sequential overexpression of AKT and Madcam1 recruited eIF4E binding to the mRNAs, as measured by RNA-IP followed by RT-qPCR using anti-eiF4E antibodies and the indicated primer sets, in HL-7702 cells with the same treatment as in Figure 7B-7C. Bel-7402 cells served as the control. The data from the “HL-7702 Mock (without treatment)” group were arbitrarily set to 100 %. G. Sequential overexpression of AKT and Madcam1 increased Doxo sensitivity. HL-7702 cells with the same treatment as in Figure 7B-7C were treated with DMSO or Doxo (final concentration 2.0 µg/ml) for 24 h before harvest for Caspase 3/7 activity measurements using a Caspase Glo-luciferase kit. Bel-7402 cells served as the control. The data from the “HL-7702 Mock-DMSO” group were arbitrarily set to 100 %. H.-I. Sequential knockdown of Madcam1 and AKT reduced AKT phosphorylation. Bel-7402 cells infected with Madcam-sh1 and AKT-sh1 and treated with or without Wortmannin (final concentration 50 μM) for 24 h were subjected to WB to detect p-AKT, AKT and Madcam1 H.. The data were shown as the p-AKT to AKT ratio in panel I., and the data from the untreated cells were arbitrarily set to 100 %. J. Sequential knockdown of Madcam1 and AKT reduced Doxo-induced Caspase 3/7 activity, as measured by a Caspase 3/7 Glo-luciferase kit, in Bel-7402 cells with the same treatment as in Figure 7G-7H. The data from the control DMSO-treated cells were arbitrarily set to 100 %. K. Illustration of a possible mechanism underlying the critical interaction between Madcam1 and AKT in the Doxorubicin-mediated regulation of apoptosis in HCC cells. The data are shown as the means + SD from three independent experiments (including WB, except panel A). *, p < 0.05 and **, p < 0.01 using the Student’s t test.

Article Snippet: The cDNA fragments encoding human Madcam1 were purchased from Origene (Beijing, China) and subcloned into the pcDNA3.1 (+) vector with a C-terminal FLAG tag or into the pLJM-based lentiviral vector with a C-terminal Myc tag.

Techniques: Western Blot, Over Expression, Phospho-proteomics, Transfection, Expressing, Control, Binding Assay, Quantitative RT-PCR, Concentration Assay, Activity Assay, Luciferase, Knockdown, Infection

Journal: Biomedicines

Article Title: Functional Characterization of the SCN5A p.D372H Variant Associated with Brugada Syndrome

doi: 10.3390/biomedicines14030582

Figure Lengend Snippet: The SCN5A p.D372H variant reduces Nav1.5 expression through decreased mRNA abundance and stability, independent of plasmid DNA levels. ( A ) Relative SCN5A mRNA expression measured by RT-qPCR. Transcript levels were significantly reduced in the D372H group compared with WT (**** p < 0.0001). Co-expression of WT and D372H (WT+MUT) also resulted in a significant decrease in SCN5A mRNA expression compared with WT (**** p < 0.0001). Data were normalized to WT. ( B ) Quantification of intracellular SCN5A plasmid DNA levels. No significant differences were observed among WT, WT+MUT, and MUT groups (ns), indicating comparable transfection efficiency. ( C ) SCN5A mRNA decay following transcriptional inhibition with actinomycin D. Relative mRNA remaining was normalized to the 0-h time point. The D372H group exhibited accelerated mRNA decay compared with WT, indicating reduced transcript stability. ( D ) Representative Western blot images showing total Nav1.5 protein expression in BLANK, CONTROL, WT, WT+MUT, and MUT groups. Molecular weight markers are indicated in kilodaltons (kDa). The predicted molecular weight of native Nav1.5 is approximately 227 kDa; however, in this study SCN5A was expressed as a GFP-tagged fusion protein, resulting in an apparent band migrating above 245 kDa. Tubulin (50 kDa) was used as a loading control. ( E ) Quantitative analysis of Nav1.5 protein expression normalized to Tubulin. Protein levels were significantly reduced in the D372H group compared with WT (* p < 0.05). The WT+MUT group also showed decreased expression compared with WT (** p < 0.01). Data are presented as mean ± SD from at least three independent experiments.

Article Snippet: The membrane was then incubated with primary antibodies against Nav1.5 (1:1000, Cat# 23016-1-AP, Proteintech, Wuhan, China) and Tubulin (1:000, Cat# A03989-1, Boster, Wuhan, China) to detect the target protein and serve as a loading control, respectively.

Techniques: Variant Assay, Expressing, Plasmid Preparation, Quantitative RT-PCR, Transfection, Inhibition, Western Blot, Control, Molecular Weight