Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , BRCA1 was analyzed using immunoblotting in Cal-27 cells under cisplatin treatment. B , BRCA1 attenuated the cisplatin-induced decrease of miR-593-5p. Cal-27 cells were transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) and then treated with cisplatin for 24h. miR-593-5p were detected using qRT-PCR (upper panel), whereas BRCA1 was analyzed using immunoblotting (lower panel). *P< 0.01 versus cisplatin alone. C , ChIP-qPCR analysis of BRCA1 binding to the promoter of miR-593-5p in the BS3 region. **P< 0.001. D , ChIP-qPCR analysis of the association levels of BRCA1 with the miR-593-5p promoter in the BS3 region under cisplatin treatment. E , A luciferase assay indicated that cisplatin induced a reduction of miR-593-5p promoter activity in the BS3 region. Cal-27 cells were transfected with the wild-type promoter (wt) in the BS3 or empty vector (pGL3-Basic). F , A luciferase assay indicated that BRCA1 activated miR-593-5p promoter activity in the BS3 region. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) were treated with the wild-type promoter (wt) or a promoter with mutations in the BS3 (mut). **P< 0.001.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

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

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , BRCA1 attenuated mitochondrial fission in Cal-27 cells under cisplatin treatment. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or vector control (Vec) were treated with cisplatin for 24h. Scale bar equals 3 μm. **P < 0.001 versus cisplatin alone. B , C and D , Apoptosis was detected using TUNEL, flow cytometry, and caspase-3/7 activity assays. *P < 0.01 versus cisplatin alone; **P < 0.001 versus cisplatin alone. E , The knockdown of miR-593-5p leads to the attenuation of the BRCA1 inhibitory effect on MFF protein levels under cisplatin treatment. Cal-27 cells stably expressing BRCA1 or vector control (Vec) were transfected with miR-593-5p inhibitors or inhibitor-negative control (inhibitor-NC). MFF levels were analyzed using immunoblotting. F , Mitochondrial fission and apoptosis were detected via staining with MitoTracker Red and TUNEL.*P < 0.01.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, TUNEL Assay, Flow Cytometry, Activity Assay, Stable Transfection, Negative Control, Western Blot, Staining

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A, B, C, BALB/c-nu mice bearing Cal-27 cells with the stable expression of MFF shRNA or its scramble form (sc) were treated with saline or cisplatin. ( A ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( B ) Representative photomicrographs of tumors from each group at day 35. ( C ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone. D, E, F, BALB/c-nu mice bearing Cal-27 cells with the stable expression of miR-593-5p or its control (con) were treated with saline or cisplatin. ( D ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( E ) Representative photomicrographs of tumors from each group at day 35. ( F ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone. G, H, I, BALB/c-nu mice bearing Cal-27 cells with the stable expression of BRCA1 or empty vector (Vec) were treated with saline or cisplatin. ( G ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( H ) Representative photomicrographs of tumors from each group at day 35. ( I ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone; *P< 0.01 versus cisplatin alone.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing, shRNA, TUNEL Assay, Plasmid Preparation

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , MFF, miR-593-5p and BRCA1 expression and apoptosis were demonstrated in cisplatin-sensitive versus non-sensitive TSCCs. Left panel: MFF and BRCA1 expression were analyzed via immunohistochemistry; miR-593-5p expression was analyzed using in situ hybridization (×200). Apoptosis was detected using a TUNEL assay. Bar=20 μm. Right panel: Quantification of MFF, miR-593-5p and BRCA1 expression in cisplatin-sensitive versus non-sensitive TSCCs. # P < 0.05; *P < 0.01. B , Associations between MFF, miR-593-5p and BRCA1 expression in TSCCs were analyzed via Spearman order correlation. C , Kaplan-Meier survival curves for TSCCs are plotted for MFF, miR-593-5p and BRCA1 expression, and survival differences were analyzed using a log-rank test. D , Model of the BRCA1–miR-593-5p–MFF axis in regulating mitochondrial fission and cisplatin sensitivity. The dotted line indicates the commonly accepted mechanism of BRCA1 regulation of cisplatin sensitivity, whereas the solid line represents the novel mechanism of BRCA1-mediated cisplatin sensitivity identified in the present study.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing, Immunohistochemistry, In Situ Hybridization, TUNEL Assay

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: Correlation among clinicopathological status and the expression of MFF, miR-593-5p or BRCA1 in TSCC patients

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing