Journal: The Journal of Biological Chemistry

Article Title: DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA

doi: 10.1074/jbc.RA119.011929

Figure Lengend Snippet: The SAM domain of DLC1 bound to its own RhoGAP domain and the C2 domain of TNS3 or PTEN. A, a schematic depicting the interactions between the DLC1 SAM domain and the TNS3 or PTEN C2 domain and how these interactions regulate cell migration through the RhoGAP-RhoA pathway. B, Western blots (WB) confirming the SAM-C2 domain interaction in cells. HEK293 cells co-expressing GFP-fused PTEN-C2 (left panel) or TNS3-C2 (right panel) and FLAG-tagged full-length DLC1, DLC1-SAM (SAM domain only), or DLC1-ΔSAM (SAM deletion mutant) were subjected to immunoprecipitation (IP) and immunoblotting (IB) using anti-GFP and anti-FLAG antibodies.

Article Snippet: Expression constructs DNA sequence encoding the DLC1-SAM (residues 13–78) was subcloned into the pGEX-2T vector (Addgene) for the expression of GST-SAM in Escherichia coli .

Techniques: Migration, Western Blot, Expressing, Mutagenesis, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA

doi: 10.1074/jbc.RA119.011929

Figure Lengend Snippet: The DLC1 SAM domain bound to peptides derived from PTEN or TNS3 C2 domain. A, peptide-walking arrays of the PTEN-C2 (aa 174–403) or TNS3-C2 domain (aa 166–440) were probed with purified GST-SAM and the bound protein detected by anti-GST Western blotting. Peptides representing phosphorylated peptides, which showed no binding, are identified in white boxes. See Fig. S1 for the peptide array map and peptide identities. B, sequence alignment of the PTEN and TNS3 C2 domains with the SAM-binding peptides/motifs identified in A are shown in black boxes. C, SAM-binding peptides from B mapped onto the structure of the PTEN-C2 domain (PDB code 1D5R, aa 188–351; 230GPTR peptide in green, 257FFHK in red, and 334NRYF in blue). D and E, binding of the SAM domain to peptides from PTEN (230GPTRREDKFMYF) or TNS3 (244CYHKKYRSATRD) C2 domains. Shown are representative binding curves from fluorescence polarization (FP) analysis (n = 3) using the corresponding fluorescein-labeled C2 peptides. ΔFP, difference in fluorescence polarization.

Article Snippet: Expression constructs DNA sequence encoding the DLC1-SAM (residues 13–78) was subcloned into the pGEX-2T vector (Addgene) for the expression of GST-SAM in Escherichia coli .

Techniques: Derivative Assay, Purification, Western Blot, Binding Assay, Peptide Microarray, Sequencing, Fluorescence, Labeling

Journal: The Journal of Biological Chemistry

Article Title: DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA

doi: 10.1074/jbc.RA119.011929

Figure Lengend Snippet: The C2 peptides disrupted the DLC1 SAM-PTEN/TNS3-C2 domain-domain interactions in cells. A and B, effect of the C2 peptides on DLC1-SAM binding to the TNS3-C2 domain in HEK293DLC1 cells without EGF stimulation. The TNS3 C2 domain bound more tightly to the DLC1 SAM domain in the absence of EGF stimulation; accordingly, the TNS3-C2–derived peptide inhibited this interaction more effectively than the PTEN C2–derived peptide. Quantification of the Western blots indicates a significantly different effect between the two C2 peptides at 5 μm (n = 3; *, p < 0.01, Student's t test). C and D, effect of the C2 peptides on DLC1-SAM binding to the PTEN C2 domain in HEK293 cells in the presence of EGF. The PTEN-C2 domain bound to the SAM domain more tightly with EGF stimulation (for 30 min). Accordingly, the PTEN C2–derived peptide blocked this interaction more effectively than the TNS3 C2–derived peptide in HEK293 cells with EGF. Quantification of Western blots (D) indicates a significantly different effect between the two C2 peptides at 5 μm peptide (n = 3; *, p < 0.01, Student's t test). Scr: scrambled TSN3-C2 peptide control, applied at 30 μm.

Article Snippet: Expression constructs DNA sequence encoding the DLC1-SAM (residues 13–78) was subcloned into the pGEX-2T vector (Addgene) for the expression of GST-SAM in Escherichia coli .

Techniques: Binding Assay, Derivative Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA

doi: 10.1074/jbc.RA119.011929

Figure Lengend Snippet: The C2 peptides decreased RhoA activity in multiple cell lines and reduced colony formation by DLC1-expressing HEK293 cells. A, both TNS3 and PTEN C2 peptides were able to inhibit RhoA activation in the DLC1-overexpressing HEK293DLC1 cells in a concentration-dependent manner. B and C, the same RhoA-GTP inhibitory effect was observed for the C2 peptides in EGF-treated MDA-MB-231 cells (B) or HGF-treated HCC78 cells (C). D, colony-formation in soft agar for the HEK293DLC1 cells in the absence (no treatment) or presence of a C2 peptide or the scrambled TNS3-C2 peptide. E, quantify of colony-formation data in D; *, denotes a p value < 0.01 (n = 3), Student's t test; IB, immunoblotting.

Article Snippet: Expression constructs DNA sequence encoding the DLC1-SAM (residues 13–78) was subcloned into the pGEX-2T vector (Addgene) for the expression of GST-SAM in Escherichia coli .

Techniques: Activity Assay, Expressing, Activation Assay, Concentration Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoA

doi: 10.1074/jbc.RA119.011929

Figure Lengend Snippet: C2 peptides reduced anchorage-independent growth and EGF-dependent migration of breast cancer cells. A, colony formation by MDA-MB-231 cells treated with a C2 peptide or scrambled control (of tat-TNS3-C2) or transfected with DLC1-ΔSAM. B, quantification of data in A to show efficiency in colony formation for the above cells compared with the parent cells (set as 100%). C, representative images of wound-healing in peptide-treated MDA-MB-231 cells. D, quantification of the would-healing data in C. For panels B and D, * denotes p < 0.01 (compared with the scramble peptide, n = 3), Student's t test. E, Western blots of total and active RhoA in peptide-treated MDA-MB-231 cells in response to EGF treatment. Peptide concentration in C–E: 18 μm.

Article Snippet: Expression constructs DNA sequence encoding the DLC1-SAM (residues 13–78) was subcloned into the pGEX-2T vector (Addgene) for the expression of GST-SAM in Escherichia coli .

Techniques: Migration, Transfection, Western Blot, Concentration Assay

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Zinc oxide nanoparticles induce murine photoreceptor cell death via mitochondria-related signaling pathway.

doi: 10.1080/21691401.2018.1446018

Figure Lengend Snippet: Figure 5. Determinations of Bax, Bcl-2 and Caspase 3 expressions at mRNA and protein levels after exposure to different concentrations of ZnO nanoparticles for 6 h. (A), (B) and (C) were determined using quantitative PCR, (D), (E) and (F) were determined by ELISA. p < .05 and p < .01 versus relevant controls.

Article Snippet: The supernatant of each sample was collected and the content of cytochrome c released from cells was determined by a Mouse Cytochrome-c (Cyt-C) ELISA Kit (CSB-E08532m) according to the manufacturer’s instructions (CUSABIO, Biotech Co., Ltd., Wuhan, China).

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay