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Figure 2. MARCH5 regulates SMAD2 and <t>ATG5</t> mRNA and protein levels. (A and B) MARCH5 mRNA and protein levels were downregulated by infection with LV3–1 and LV3–2. (C) Ovarian cancer SKOV3 cells were infected with LV3-NC and LV3–1. SMAD2 and ATG5 mRNA and protein levels were downregulated by infection with LV3–1 and LV3–2. (D) Ovarian cancer SKOV3 cells were transfected with MARCH5 30 UTR. SMAD2 and ATG5 mRNA and protein levels were upregulated. (E) Putative binding sites targeted by MIR30A were predicted to be located in the 30 UTR of MARCH5, ATG5, and SMAD2 mRNA. (F) SKOV3 cells were cotransfected with MIR30A mimics or control RNA (NC) with luciferase reporter plasmids containing either wild-type (pMIR-MARCH5–3UTR, pMIR-ATG5–3UTR, and pMIR-SMAD2–3UTR) or mutant 30 UTR (pMIR- MARCH5–3UTRm, pMIR-ATG5–3UTRm, and pMIR-SMAD2–3UTRm) of MARCH5, ATG5, and SMAD2 genes. Luciferase expression was measured. The fold changes of the rel- ative luciferase activity in MIR30A mimics with the indicated plasmids transfected cells were normalized to NC with the corresponding indicated plasmid-transfected cells. (G) Ovarian cancer SKOV3 cells were transfected with MIR30 mimics or control RNA (NC). (H) Ovarian cancer SKOV3 cells were transfected with MIR30 inhibitor or control RNA (NC). (I) Ovarian cancer SKOV3 cells were infected with LV3-NC and LV3–1. One group was infected with LV3–1-transfected MIR30A inhibitors. Error bars represent standard error. The symbols and indicate p < 0.05 and 0.01, respectively.
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Figure 2. MARCH5 regulates SMAD2 and ATG5 mRNA and protein levels. (A and B) MARCH5 mRNA and protein levels were downregulated by infection with LV3–1 and LV3–2. (C) Ovarian cancer SKOV3 cells were infected with LV3-NC and LV3–1. SMAD2 and ATG5 mRNA and protein levels were downregulated by infection with LV3–1 and LV3–2. (D) Ovarian cancer SKOV3 cells were transfected with MARCH5 30 UTR. SMAD2 and ATG5 mRNA and protein levels were upregulated. (E) Putative binding sites targeted by MIR30A were predicted to be located in the 30 UTR of MARCH5, ATG5, and SMAD2 mRNA. (F) SKOV3 cells were cotransfected with MIR30A mimics or control RNA (NC) with luciferase reporter plasmids containing either wild-type (pMIR-MARCH5–3UTR, pMIR-ATG5–3UTR, and pMIR-SMAD2–3UTR) or mutant 30 UTR (pMIR- MARCH5–3UTRm, pMIR-ATG5–3UTRm, and pMIR-SMAD2–3UTRm) of MARCH5, ATG5, and SMAD2 genes. Luciferase expression was measured. The fold changes of the rel- ative luciferase activity in MIR30A mimics with the indicated plasmids transfected cells were normalized to NC with the corresponding indicated plasmid-transfected cells. (G) Ovarian cancer SKOV3 cells were transfected with MIR30 mimics or control RNA (NC). (H) Ovarian cancer SKOV3 cells were transfected with MIR30 inhibitor or control RNA (NC). (I) Ovarian cancer SKOV3 cells were infected with LV3-NC and LV3–1. One group was infected with LV3–1-transfected MIR30A inhibitors. Error bars represent standard error. The symbols and indicate p < 0.05 and 0.01, respectively.

Journal: Autophagy

Article Title: MARCH5 RNA promotes autophagy, migration, and invasion of ovarian cancer cells.

doi: 10.1080/15548627.2016.1256520

Figure Lengend Snippet: Figure 2. MARCH5 regulates SMAD2 and ATG5 mRNA and protein levels. (A and B) MARCH5 mRNA and protein levels were downregulated by infection with LV3–1 and LV3–2. (C) Ovarian cancer SKOV3 cells were infected with LV3-NC and LV3–1. SMAD2 and ATG5 mRNA and protein levels were downregulated by infection with LV3–1 and LV3–2. (D) Ovarian cancer SKOV3 cells were transfected with MARCH5 30 UTR. SMAD2 and ATG5 mRNA and protein levels were upregulated. (E) Putative binding sites targeted by MIR30A were predicted to be located in the 30 UTR of MARCH5, ATG5, and SMAD2 mRNA. (F) SKOV3 cells were cotransfected with MIR30A mimics or control RNA (NC) with luciferase reporter plasmids containing either wild-type (pMIR-MARCH5–3UTR, pMIR-ATG5–3UTR, and pMIR-SMAD2–3UTR) or mutant 30 UTR (pMIR- MARCH5–3UTRm, pMIR-ATG5–3UTRm, and pMIR-SMAD2–3UTRm) of MARCH5, ATG5, and SMAD2 genes. Luciferase expression was measured. The fold changes of the rel- ative luciferase activity in MIR30A mimics with the indicated plasmids transfected cells were normalized to NC with the corresponding indicated plasmid-transfected cells. (G) Ovarian cancer SKOV3 cells were transfected with MIR30 mimics or control RNA (NC). (H) Ovarian cancer SKOV3 cells were transfected with MIR30 inhibitor or control RNA (NC). (I) Ovarian cancer SKOV3 cells were infected with LV3-NC and LV3–1. One group was infected with LV3–1-transfected MIR30A inhibitors. Error bars represent standard error. The symbols and indicate p < 0.05 and 0.01, respectively.

Article Snippet: Detection of protein expression by western blotting The expressions of SMAD2, p-SMAD2, ATG5, MARCH5, and GAPDH proteins were analyzed by western blot.23 The primary antibodies used include polyclonal rabbit anti-MARCH5 (Bioss Biotechnology Company, bs-9339R); rabbit monoclonal to SMAD2 (Abcam, ab40855); rabbit monoclonal to p-SMAD2 (Abcam, ab188334); and polyclonal rabbit anti-GAPDH (Santa Cruz Biotechnology, AB10016).

Techniques: Infection, Transfection, Binding Assay, Control, Luciferase, Mutagenesis, Expressing, Activity Assay, Plasmid Preparation

Figure 3. ATG5 and SMAD2 modulation of MARCH5 levels are MIR30A dependent. (A) Ovarian cancer SKOV3 cells were transfected with control RNA (NC), ATG5 siRNA, and ATG5 siRNA C MIR30A inhibitor. (B) Ovarian cancer SKOV3 cells were transfected with control RNA (NC), SMAD2 siRNA, and SMAD2 siRNAC MIR30A inhibitor. (C) Ovarian cancer SKOV3 cells were transfected with control RNA (NC), ATG5 siRNA, and ATG5 siRNA C MIR30A inhibitor. (D) Ovarian cancer SKOV3 cells were transfected with control RNA (NC), SMAD2 siRNA, and SMAD2 siRNA C MIR30A inhibitor. Error bars represent standard error. The symbols and indicate p < 0.05 and 0.01, respectively.

Journal: Autophagy

Article Title: MARCH5 RNA promotes autophagy, migration, and invasion of ovarian cancer cells.

doi: 10.1080/15548627.2016.1256520

Figure Lengend Snippet: Figure 3. ATG5 and SMAD2 modulation of MARCH5 levels are MIR30A dependent. (A) Ovarian cancer SKOV3 cells were transfected with control RNA (NC), ATG5 siRNA, and ATG5 siRNA C MIR30A inhibitor. (B) Ovarian cancer SKOV3 cells were transfected with control RNA (NC), SMAD2 siRNA, and SMAD2 siRNAC MIR30A inhibitor. (C) Ovarian cancer SKOV3 cells were transfected with control RNA (NC), ATG5 siRNA, and ATG5 siRNA C MIR30A inhibitor. (D) Ovarian cancer SKOV3 cells were transfected with control RNA (NC), SMAD2 siRNA, and SMAD2 siRNA C MIR30A inhibitor. Error bars represent standard error. The symbols and indicate p < 0.05 and 0.01, respectively.

Article Snippet: Detection of protein expression by western blotting The expressions of SMAD2, p-SMAD2, ATG5, MARCH5, and GAPDH proteins were analyzed by western blot.23 The primary antibodies used include polyclonal rabbit anti-MARCH5 (Bioss Biotechnology Company, bs-9339R); rabbit monoclonal to SMAD2 (Abcam, ab40855); rabbit monoclonal to p-SMAD2 (Abcam, ab188334); and polyclonal rabbit anti-GAPDH (Santa Cruz Biotechnology, AB10016).

Techniques: Transfection, Control

Figure 5. Silencing MARCH5 inhibited pelvic peritoneal metastasis in a nude mice model. (A) Mean tumor weight on d 35 after tumor cell injection. LV3–1- and LV3-NC- infected SKOV3 cells were implanted by IP injection. The arrow indicates the lesion in the abdominal cavity. (B) Immunohistochemical analysis of MARCH5, ATG5, SMAD2, and p-SMAD2 expression was performed on tumor xenografts. Representative images are shown (original magnification £200). Error bars represent standard error. The symbols and indicate p < 0.05 and 0.01, respectively. Scale bar: 100 mm.

Journal: Autophagy

Article Title: MARCH5 RNA promotes autophagy, migration, and invasion of ovarian cancer cells.

doi: 10.1080/15548627.2016.1256520

Figure Lengend Snippet: Figure 5. Silencing MARCH5 inhibited pelvic peritoneal metastasis in a nude mice model. (A) Mean tumor weight on d 35 after tumor cell injection. LV3–1- and LV3-NC- infected SKOV3 cells were implanted by IP injection. The arrow indicates the lesion in the abdominal cavity. (B) Immunohistochemical analysis of MARCH5, ATG5, SMAD2, and p-SMAD2 expression was performed on tumor xenografts. Representative images are shown (original magnification £200). Error bars represent standard error. The symbols and indicate p < 0.05 and 0.01, respectively. Scale bar: 100 mm.

Article Snippet: Detection of protein expression by western blotting The expressions of SMAD2, p-SMAD2, ATG5, MARCH5, and GAPDH proteins were analyzed by western blot.23 The primary antibodies used include polyclonal rabbit anti-MARCH5 (Bioss Biotechnology Company, bs-9339R); rabbit monoclonal to SMAD2 (Abcam, ab40855); rabbit monoclonal to p-SMAD2 (Abcam, ab188334); and polyclonal rabbit anti-GAPDH (Santa Cruz Biotechnology, AB10016).

Techniques: Injection, Infection, Immunohistochemical staining, Expressing

Figure 6. MARCH5 promotion of SKOV3 cell autophagy, migration, and invasion involves ATG5, SMAD2 and the TGFB1-SMAD2/3 pathway. (A) A2780 cells infected with LV5-GFP and LV5-MARCH5. After 48 h, puromycin was added at a concentration of 2.5 mg/ml. The cells were transfected with ATG5 siRNA, SMAD2 siRNA or LY2109761 was added (10 mM) for 48 h. The migration and invasion assays were performed in the presence of TGFB1 (10 ng/ml). (B) SKOV3 cells transfected with a plasmid encoding mRFP-GFP-LC3 and pCMV5 empty vector, pCMV5-MARCH5, pCMV5-MARCH5 C siATG5, pCMV5-MARCH5 C siSMAD2, and pCMV5-MARCH5 C LY2109761. After 48 h, TGFB1 was added at a concentration of 10 ng/ml. mRFP-GF-LC3 distribution in SKOV3 was analyzed by confocal microscopy after TGFB1 treatment for 24 h. The LC3 dots were quantified using image pro-plus 6.0 software. All experiments were repeated 3 times and the representative results are shown. The right panel indicates the quanti- fication of LC3 puncta numbers. Error bars represented standard error. The symbols and indicate p < 0.05 and 0.01, respectively. Scale bar: 100 mm.

Journal: Autophagy

Article Title: MARCH5 RNA promotes autophagy, migration, and invasion of ovarian cancer cells.

doi: 10.1080/15548627.2016.1256520

Figure Lengend Snippet: Figure 6. MARCH5 promotion of SKOV3 cell autophagy, migration, and invasion involves ATG5, SMAD2 and the TGFB1-SMAD2/3 pathway. (A) A2780 cells infected with LV5-GFP and LV5-MARCH5. After 48 h, puromycin was added at a concentration of 2.5 mg/ml. The cells were transfected with ATG5 siRNA, SMAD2 siRNA or LY2109761 was added (10 mM) for 48 h. The migration and invasion assays were performed in the presence of TGFB1 (10 ng/ml). (B) SKOV3 cells transfected with a plasmid encoding mRFP-GFP-LC3 and pCMV5 empty vector, pCMV5-MARCH5, pCMV5-MARCH5 C siATG5, pCMV5-MARCH5 C siSMAD2, and pCMV5-MARCH5 C LY2109761. After 48 h, TGFB1 was added at a concentration of 10 ng/ml. mRFP-GF-LC3 distribution in SKOV3 was analyzed by confocal microscopy after TGFB1 treatment for 24 h. The LC3 dots were quantified using image pro-plus 6.0 software. All experiments were repeated 3 times and the representative results are shown. The right panel indicates the quanti- fication of LC3 puncta numbers. Error bars represented standard error. The symbols and indicate p < 0.05 and 0.01, respectively. Scale bar: 100 mm.

Article Snippet: Detection of protein expression by western blotting The expressions of SMAD2, p-SMAD2, ATG5, MARCH5, and GAPDH proteins were analyzed by western blot.23 The primary antibodies used include polyclonal rabbit anti-MARCH5 (Bioss Biotechnology Company, bs-9339R); rabbit monoclonal to SMAD2 (Abcam, ab40855); rabbit monoclonal to p-SMAD2 (Abcam, ab188334); and polyclonal rabbit anti-GAPDH (Santa Cruz Biotechnology, AB10016).

Techniques: Migration, Infection, Concentration Assay, Transfection, Plasmid Preparation, Confocal Microscopy, Software

Figure 8. The TGFB1-SMAD2/3 pathway regulates MARCH5, ATG5, and SMAD2 through MIR30A. (A) The TGFB1–SMAD2/3 signal pathway luciferase reporter activity was detected in ectopically expressing MARCH5 ovarian cancer A2780 cells infected with LV5-MARCH5. (B) The TGFB1–SMAD2/3 signal pathway luciferase reporter activity was detected in LV3–1-, LV3–2-, and LV3-NC-infected SKOV3 cells. (C) Ovarian cancer SKOV3 cells were transfected with LV5-GFP, LV5-MARCH5, LV5-MARCH5 C ATG5 siRNA, or LV5-MARCH5 C SMAD2 siRNA. The protein expression of p-SAMD2 was detected by western blot. (Di) The expression of MARCH5, ATG5, and SMAD2 mRNA in SKOV3 cells was regulated by TGFB1 and MIR30A mimics. (Dii) The expression of MIR30A in SKOV3 cells was regulated by TGFB1. (Diii) The expression of MARCH5, ATG5, and SMAD2 mRNA in SKOV3 cells was regulated by LY2109761 and MIR30A inhibitor. (Div) The expression of MIR30A in SKOV3 cells was regulated by LY2109761. (E) The putative binding sites between MIR30A and SMAD2/3. (F) A graphic abstract for the significance of the MARCH5 pathway (MIR30A, SMAD2/3, MARCH5, ATG5, and SMAD2). Data are expressed as mean § SD from 3 independent experiments. p < 0.05, and p < 0.01.

Journal: Autophagy

Article Title: MARCH5 RNA promotes autophagy, migration, and invasion of ovarian cancer cells.

doi: 10.1080/15548627.2016.1256520

Figure Lengend Snippet: Figure 8. The TGFB1-SMAD2/3 pathway regulates MARCH5, ATG5, and SMAD2 through MIR30A. (A) The TGFB1–SMAD2/3 signal pathway luciferase reporter activity was detected in ectopically expressing MARCH5 ovarian cancer A2780 cells infected with LV5-MARCH5. (B) The TGFB1–SMAD2/3 signal pathway luciferase reporter activity was detected in LV3–1-, LV3–2-, and LV3-NC-infected SKOV3 cells. (C) Ovarian cancer SKOV3 cells were transfected with LV5-GFP, LV5-MARCH5, LV5-MARCH5 C ATG5 siRNA, or LV5-MARCH5 C SMAD2 siRNA. The protein expression of p-SAMD2 was detected by western blot. (Di) The expression of MARCH5, ATG5, and SMAD2 mRNA in SKOV3 cells was regulated by TGFB1 and MIR30A mimics. (Dii) The expression of MIR30A in SKOV3 cells was regulated by TGFB1. (Diii) The expression of MARCH5, ATG5, and SMAD2 mRNA in SKOV3 cells was regulated by LY2109761 and MIR30A inhibitor. (Div) The expression of MIR30A in SKOV3 cells was regulated by LY2109761. (E) The putative binding sites between MIR30A and SMAD2/3. (F) A graphic abstract for the significance of the MARCH5 pathway (MIR30A, SMAD2/3, MARCH5, ATG5, and SMAD2). Data are expressed as mean § SD from 3 independent experiments. p < 0.05, and p < 0.01.

Article Snippet: Detection of protein expression by western blotting The expressions of SMAD2, p-SMAD2, ATG5, MARCH5, and GAPDH proteins were analyzed by western blot.23 The primary antibodies used include polyclonal rabbit anti-MARCH5 (Bioss Biotechnology Company, bs-9339R); rabbit monoclonal to SMAD2 (Abcam, ab40855); rabbit monoclonal to p-SMAD2 (Abcam, ab188334); and polyclonal rabbit anti-GAPDH (Santa Cruz Biotechnology, AB10016).

Techniques: Luciferase, Activity Assay, Expressing, Infection, Transfection, Western Blot, Binding Assay