Journal: Molecular carcinogenesis

Article Title: IL-33 Promotes Growth and Liver Metastasis of Colorectal Cancer in Mice by Remodeling the Tumor Microenvironment and Inducing Angiogenesis

doi: 10.1002/mc.22491

Figure Lengend Snippet: IL-33 enhanced tumor growth. (A) Proliferation of MC38-Vector and MC38-IL33 cells in tissue culture. (B and C) Weights (grams) of primary tumors derived from MC38-Vector or MC38-IL33 cells 6 weeks after cecal implantation (B) or subcutaneous injection (C). ***P <0.001. (D) Incidence of primary tumor growth in cecum of C57Bl/6 mice implanted with MC38-Vector or MC38-IL33 cells (n =20). (E) H&E images (400×) and primary cecal tumor sections stained with anti Ki67 and cleaved caspase-3 antibodies. (F) Immunoblot analysis of total protein extracts from MC38-Vector and MC38-IL33 clones with antibodies against PCNA and cleaved caspase-3. (G) Serum levels of IL-33 in mice bearing MC38-Vector or MC38-IL33 tumors analyzed by ELISA.

Article Snippet: Endogenous peroxidases were blocked with 3% H 2 O 2 for 15 min. Nonspecific epitopes were blocked with normal horse serum (Jackson ImmunoResearch, West Grove, PA) for 1 h. The sections were incubated overnight at 4°C with antibodies against one of the following proteins: LOX, MMP9, MMP2, LYVE1, VEGF, and S100A8 (all from Abcam, Cambridge, MA), S100A9, IL-33 (R&D Systems, Minneapolis, MN), cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA), Ki67 (OriGene Technologies, Rockville, MD).

Techniques: Plasmid Preparation, Derivative Assay, Injection, Staining, Western Blot, Clone Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Medical Sciences

Article Title: LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2

doi: 10.7150/ijms.114095

Figure Lengend Snippet: Characterisation of H9C2 proliferating clones whose genome was modified within the Tmpo gene sequence by CRISPR-Cas9 to reduce or extinguish LAP2a protein expression. ( A ) Schema of the Tmpo gene and LAP2a protein domains. The following are indicated: the number of base pairs targeted by one of the SgRNAs within the Tmpo gene and the amino acid position; the mutations induced by our SgRNA (at protein level) for the four H9C2 clones selected as effectively edited to trigger a premature stop codon; the names of the WT +/+ (unedited CRISPR clones) and of the LAP2a +/- and LAP2a -/- clones considered throughout this study. ( B ) Western blots are shown for whole protein extracts of naive H9C2 and CRISPR clones (WT (21E10, 21B1, 22A11); LAP2a +/- (21H4, 22G2, 22G3) and LAP2a -/- (22B3)), as indicated. Proteins of interest were detected using anti LAP2a Ab (middle panel) and anti TMPO Ab (lower panel). Red Ponceau staining (upper panel) was used to normalize ECL signals. ( C ) The graphs show the ECL signal quantification of western blots (arbitrary units; a.u) after revelation with anti TMPO Ab as shown in 1B. The graphs present the individual values and means ± s.e.m. (N= 2 to 5 independent samples per clone). * p<0.05, ** p<0.01, **** p<0.0001 (Mann Whitney test). ( D ) Immunofluorescence of CRISPR clones (WT (22A11, 21B1), LAP2a -/- (22B3) and LAP2a +/- (21H4)), using a rabbit Ab to detect LAP2a (red), phalloidin to label cytoplasmic actin (green) and DAPI to label nuclear DNA (blue). Scale bar = 50 um. ( E ) The graphs represent the % of cells with either a negative (-) or relatively higher or lower (+, ++, +++) mean signal (a.u) as detected by immunofluorescence when using a rabbit anti LAP2a Ab, as shown in 1D. For the analysed experiment (N = 1), the total n (numbers of cell nuclei) were 184, 321, 174 and 216 for the clones 22B3, 22A11, 21H4 and 21B1, respectively. ( F ) The graph represents the cell doubling time (mean value ± s.e.m.) calculated for naive H9C2 cells, WT CRISPR clones, LAP2a +/- and LAP2a -/- CRISPR edited clones, as indicated. The individual values and means ± s.e.m are given. (N = 4 to 8 independent experiments per clone). * p<0.05; **** p<0.0001. (Mann Whitney test) ( G ) The graph represents the amount of cells that were either positively (green) or negatively (black) stained in situ by immunofluorescence for the proliferation marker Ki67. For the analysed experiment (N = 1), the total n (numbers of cells) were 499, 562 and 647 for the WT, LAP2a +/- and LAP2a -/- clones, respectively. * p<0.05. *** p<0.001 (Chi square test for a contingency table).

Article Snippet: We used the following primary antibodies, according to the manufacturer's instructions: mouse anti-Actin alpha 1 cardiac muscle antibody (Ab) (33-32R, Novus Biological, BioTechne France), mouse anti-Histone H3 Ab (1G1, sc-517576; Santa Cruz Biotechnology, Germany), rabbit anti-Ki67 Ab (NB500-170; Novus Biological, BioTechne France), mouse anti-Lamin A/C Ab (4C11; Cell Signaling Technology, USA); rabbit anti-TMPO/LAP2 Ab (14651-1-AP; Proteintech Europe, United Kingdom) which recognizes all TMPO isoforms (a,b,g); mouse anti-Myosin-2 Ab (MF20, 14-6503-80, Life Technologies Europe, United Kingdom), rabbit anti-LAP2 alpha Ab (IQ175; Immuquest, Europe, United Kingdom); rabbit anti-Lamin B1 Ab ; rabbit anti-LEMD2 Ab (HPA017340, Merck Sigma, France), mouse anti-cardiac troponin T monoclonal Ab (13-11; MA512960 , Life Technologies, France) and phalloidin (P2141-Sigma).

Techniques: Clone Assay, Modification, Sequencing, CRISPR, Expressing, Western Blot, Staining, MANN-WHITNEY, Immunofluorescence, In Situ, Marker