Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: Expressed genes with significant overall mutation burden in sun-exposed melanoma

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: Mutagenesis

Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: Statistical analysis of RAC1 P29S in the Yale tumor cohort

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: Mutagenesis

Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: Crystal structure of RAC1 P29S . ( a ) Overall schematic of RAC1 P29S showing the P-loop (pink), switch I (light green), switch II (purple), Mg 2+ (cyan) and the slowly hydrolyzing GTP analog, GMP-PNP (stick format). The red sphere indicates the location of p.Pro29Ser. The dashed box indicates the regions shown in b – e . ( b ) Close-up view showing close contacts of ribose hydroxyls with the switch I region in RAC1 P29S . ( c ) RAC1 WT bound to GMP-PNP. ( d ) HRAS in complex with GMP-PNP (PDB 5P21) . Direct hydrogen bonding between the switch I backbone and ribose are commonly observed in activated HRAS but are less frequently observed in Rho family GTPases, including RAC1. A RAS-like hydrogen bonding pattern is observed in activated RAC1 P29S (dashed red lines). ( e ) Superposition of the switch I region and GMP-PNP. RAC1 P29S is shown in light green, RAC1 WT is shown in gray, and HRAS is shown in light blue. The red sphere indicates the location of p.Pro29Ser. The residues discussed in the text are labeled. The figure was made using CCP4mg .

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: Labeling

Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: In vitro RAC1 P29S binding to downstream effectors. ( a ) PAK1 pulldown assay showing that recombinant histidine-tagged RAC1 P29S (His-RAC1 P29S ) has higher binding to its effector PAK1 compared to His-RAC1 WT for samples that include GTP and GTPγS. NC, PAK1-PBD beads alone as a negative control. The His-RAC1 WT and His-RAC P29S lanes show unbound wild-type and mutant protein, as indicated. The multiple bands in all lanes are RAC1; all bands are observed when RAC1 crystals are run on SDS-PAGE. ( b ) RAC1 pulldown assay showing that recombinant His-GST-RAC1 P29S has higher binding to endogenous MLK3 expressed in melanoma cells compared to His-GST-RAC1 WT in all reaction samples. The immobilized recombinant RAC1 proteins were incubated with cell lysates from two independent melanomas (YULAC that is RAC1 WT and BRAF V600K and YUHEF that is RAC1 P29S and BRAF WT ). The figure shows western blot of bound proteins with antibodies to MLK3. As negative controls, we used lysates incubated with GST-bound beads or recombinant RAC1 proteins that were not incubated with cell extracts. The western blot analysis also shows the expression of the relevant proteins in whole-cell lysate (WCL). The reaction mixtures were likely to contain nucleotides contributed by the cell lysates in addition to those added in vitro , as indicated. ( c ) PAK1 pulldown assay comparing the binding activity of RAC1 WT , RAC1 P29S and RAC1 F28L proteins. The numbers under each lane indicate the band density as determined by scanning the membranes with ImageJ.

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: In Vitro, Binding Assay, Recombinant, Negative Control, Mutagenesis, SDS Page, Incubation, Western Blot, Expressing, Activity Assay

Journal: Nature genetics

Article Title: Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

doi: 10.1038/ng.2359

Figure Lengend Snippet: Cellular function of RAC1 P29S compared to RAC1 WT . ( a ) Western blot analysis of RAC1 in mouse melanocytes infected with wild-type (WT) or mutant RAC P29S retroviral expression vectors compared to nontransfected cells (parental). Actin indicates protein loading in each lane. ( b ) RAC1 P29S enhances melanocyte proliferation. The proliferation of melanocytes transiently expressing RAC1 WT (green) or RAC1 P29S (red) compared to noninfected parental cells (blue). Error bars indicate standard errors. ( c ) RAC1 P29S enhances cell migration. The graphs show the rate of migration of parental, RAC1 WT - and RAC1 P29S -expressing melanocytes at daily intervals. ( d ) RAC1 P29S enhances ERK activation. Western blots of lysates from mouse melanocytes expressing RAC1 WT or RAC1 P29S probed with RAC1, pERK, ERK and actin antibodies, as indicated on the side of the gel. ( e ) Localization of GFP-tagged RAC1 WT (GFP-RAC1 WT ) and GFP-RAC1 P29S in COS-7 cells. COS-7 cells transiently expressing GFP-RAC1 WT (i, ii) or GFP-RAC1 P29S (iii, iv) were fixed with paraformaldehyde and imaged with a spinning-disk confocal-based inverted Olympus microscope. Insets (yellow boxes) illustrate the localization of RAC1 P29S , but not RAC1 WT , in membrane ruffles (compare i and ii to the yellow arrows in iii and iv, respectively). pERK, phosphorylated ERK. Scale bars, 10 μm.

Article Snippet: The pBabe-CyPet- RAC1 retroviral expression vector and the pcDNA3- eGFP - RAC1 plasmid were purchased from Addgene.

Techniques: Cell Function Assay, Western Blot, Infection, Mutagenesis, Expressing, Migration, Activation Assay, Microscopy

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