Journal: Nature cell biology

Article Title: The AMPK-Parkin axis negatively regulates necroptosis and tumorigenesis by inhibiting the necrosome

doi: 10.1038/s41556-019-0356-8

Figure Lengend Snippet: a, Parkin WT and KO MEFs were treated with T/C/Z for 4 h. Ubiquitinated proteins were pull down under denaturing conditions by Ni-NTA agarose and analyzed by immunoblot. b , Cells were transfected with the indicated plasmids (Ubiquitin: WT, K6, K11, K27, K29, K33, K48, or K63 only) and then treated with T/C/Z for 4 h. Cell lysates were then subjected to IP and immunoblotted as indicated. SE, short exposure; LE, long exposure. c-d, Cells were transfected with the indicated plasmids (Ubiquitin: WT, K33R, K48R, K63R, or K33 only) and then treated with T/C/Z for 4 h. Cell lysates were then subjected to IP and immunoblotted as indicated. SE, short exposure; LE, long exposure. e, Cells were transfected with the indicated constructs (Parkin WT, R420H, or H461R) and then treated with T/C/Z. Ubiquitinated proteins were pulled down under denaturing conditions by Ni-NTA agarose and analyzed by immunoblot. f-g, U2OS cells were transfected with the indicated constructs and then treated with T/C/Z for 4 h. The distribution of RIPK3 (Red) was detected using immunofluorescence (f). Quantification of RIPK3 punctation (g). Scale bar, 20 μm (magnification, 200 μm). All experiments were repeated independently at least three times with similar results. Source data are provided in . Uncropped images of blots for (a,b,c,d,e) are shown in . Scale bars, 20 μm (magnification, 200 μm, f).

Article Snippet: HA-tagged ubiquitin and ubiquitin lysine mutants, such as K-6 only, K-11 only, K-27 only, K-29 only, K-33 only, K-48 only, K-63 only, K33R, K48R, and K63R were obtained from Addgene.

Techniques: Western Blot, Transfection, Ubiquitin Proteomics, Construct, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells

doi: 10.1186/1479-5876-11-57

Figure Lengend Snippet: List of target genes of miR-200c, miR-205, and miR-375 identified in breast cancer cell lines

Article Snippet: CFL2 levels in breast tumors and normal breast tissues were evaluated by IHC using anti-CFL2 polyclonal antibody (1:250 dilution, sc-32160, Santa Cruz Biotechnology, Santa Cruz, CA) on commercial tissue arrays (Shanghai Outdo Biotech Co., Shanghai) as previously described [ ].

Techniques: Membrane, Sequencing, Coagulation, Binding Assay, RNA Binding Assay

Journal: Journal of Translational Medicine

Article Title: A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells

doi: 10.1186/1479-5876-11-57

Figure Lengend Snippet: 3 ′ -UTR reporter assays confirming the interaction of individual miRNA with the 3 ′ -UTR of candidate target genes. ( A ) Schema of the pmirGLO dual luciferase vector carrying the 3 ′ -UTR regions of 6 selected genes. The entire 3 ′ -UTR of 6 genes: CDH11 , CFL2 , SEC23A , ZEB-1 , PTPRM , and LDHB were cloned into the pmirGLO dual luciferase vector. ( B ) The alignments of miRNA and their predicted target genes. ( C ) Percent relative luciferase activity 48 hours post-transfection with the indicated reporter vector.

Article Snippet: CFL2 levels in breast tumors and normal breast tissues were evaluated by IHC using anti-CFL2 polyclonal antibody (1:250 dilution, sc-32160, Santa Cruz Biotechnology, Santa Cruz, CA) on commercial tissue arrays (Shanghai Outdo Biotech Co., Shanghai) as previously described [ ].

Techniques: Luciferase, Plasmid Preparation, Clone Assay, Activity Assay, Transfection

Journal: Journal of Translational Medicine

Article Title: A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells

doi: 10.1186/1479-5876-11-57

Figure Lengend Snippet: CFL2 and breast cancer cell migration. ( A ) Bar graph representing the normalized number of migratory or invaded cells after the respective siRNA transfection. ( B ) Overexpression of CFL2 can compensate the effect of exogenous miR-200c. Bar graph represents the normalized number of migratory cells after the respective transfection of miRNA mimic control, miR-200c mimic, and co-transfection of miR-200c and a plasmid encoding CFL2, respectively. *: Student’s T -test, p < 0.05. ( C ). Rhodamin-phalloidin staining of F-actin in the CFL2-siRNA and miR-200c transfected MDA-MB-231 cells. ( D ) Immunohistochemical analysis of TMA of primary breast tumors using antibodies against CFL2. Representative images of TMA cores are presented showing normal breast tissue, grades I, II, and III breast tumors with increasing staining intensities. ( E ) Bar graph representing the quantification of CFL2 immunostaining from blindly scored tissue microarray sections. Staining intensity of each sample was given a modified histochemical score (MH-score) that considers both the intensity and the percentage of cells stained at each intensity level. The intensity of each grade is the average of MH-score of all samples in that grade. Data were analyzed by one-way ANOVA with p-values as noted in the figure.

Article Snippet: CFL2 levels in breast tumors and normal breast tissues were evaluated by IHC using anti-CFL2 polyclonal antibody (1:250 dilution, sc-32160, Santa Cruz Biotechnology, Santa Cruz, CA) on commercial tissue arrays (Shanghai Outdo Biotech Co., Shanghai) as previously described [ ].

Techniques: Migration, Transfection, Over Expression, Control, Cotransfection, Plasmid Preparation, Staining, Immunohistochemical staining, Immunostaining, Microarray, Modification