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

Journal: Scientific Reports

Article Title: Genome-wide analysis suggests a differential microRNA signature associated with normal and diabetic human corneal limbus

doi: 10.1038/s41598-017-03449-7

Figure Lengend Snippet: List of antibodies used in this study.

Article Snippet: Ki-67 , Rabbit mAb ab92742 , Abcam , 359 , 1:200.

Techniques:

Journal: ImmunoHorizons

Article Title: SRF617 Is a Potent Inhibitor of CD39 with Immunomodulatory and Antitumor Properties

doi: 10.4049/immunohorizons.2200089

Figure Lengend Snippet: SRF617 does not modulate proliferation or apoptosis in MOLP-8 xenograft tumors. SCID mice were injected s.c. with MOLP-8 cells. When tumors reached 100 mm 3 , mice ( n = 2–4/group) were treated i.p. with 20 mg/kg SRF617, 60 mg/kg SRF617, or isotype control antibody twice per week for 2 wk. Tumors were collected on day 14 and formalin fixed. Fixed samples were stained for ( A ) Ki-67 or ( B ) CC3. The proliferation index (%) was calculated from Ki-67–stained images, and the CC3 H-index was quantitated. Data are shown as mean ± SEM. ( C ) Representative images from the treatment groups are shown (original magnification ×10).

Article Snippet: DAKO protein block (Agilent Technologies) was applied to slides and incubated for 15 min. Rabbit anti-human Ki67 primary mAb (Abcam) or rabbit anti-CC3 primary mAb (Cell Signaling Technology) was diluted at 0.4 μg/ml concentration (for Ki-67) or 1:400 (for CC3) in Bond Ab diluent (Leica Biosystems), and 150 μl diluted Ab solution was dispensed on each slide and incubated for 60 min at room temperature.

Techniques: Injection, Control, Staining

Journal: Cancer Research Communications

Article Title: Novel Syngeneic Cell Lines for Studying High-Risk BRAF V600E -Driven Colorectal Cancer In Vivo

doi: 10.1158/2767-9764.CRC-25-0599

Figure Lengend Snippet: All NaJa cell lines successfully engraft into syngeneic immunocompetent C57BL/6N mice and recapitulate their individual 2D morphologies in vivo . A, Schematic overview of the PVI of NaJa cells ( n = 2 NaJa-D, n = 10 NaJa-F, and n = 3 NaJa-G). B, Left, livers showing metastatic nodules 4 weeks after injection (black arrows) and invasive growth in the pancreatic tissue (blue circles). Scale bar, 1 cm. Hematoxylin and eosin: Histology showing that all NaJa cell lines establish tumors (pale blue) in the liver parenchyma (pink) with a differentiation state matching their epithelial or mesenchymal in vitro morphology. Right, IHC and immunofluorescence (IF) staining of proliferation marker Ki-67 (left) and intestinal differentiation markers CDX2 (middle) and E-cadherin (right). L, liver; T, tumor. Scale bar (overview), 500 μm, scale bar (zoom, IHC, and IF), 50 μm. C, Quantification of Ki-67 and CDX2 IHC staining in NaJa cell–induced liver metastases. Each dot represents an individual tumor; different shapes indicate individual tissue sections, and colors denote individual mice. Data are shown as the median ±95% confidence interval (CI).Statistical significance was assessed using the Kruskal–Wallis test followed by Dunn multiple comparison test. **, P < 0.01; ****, P < 0.0001. D, IHC staining of ERK and its downstream target DUSP6. Red arrows indicate examples of nuclear ERK staining. L, liver; T, tumor. Scale bar, 50 μm. [ A, Created by J. Traichel in BioRender. Brummer, T. (2025) https://BioRender.com/8ogg64c .]

Article Snippet: The formalin-fixed, paraffin-embedded (FFPE) sections underwent standard processing and staining procedures using the following primary antibodies directed against E-cadherin (1:50, #610181, BD Laboratories; RRID: AB_397580) and Ki-67 (1:400, #9129, Cell Signaling Technology; RRID: AB_2687446).

Techniques: In Vivo, Injection, In Vitro, Immunofluorescence, Staining, Marker, Immunohistochemistry, Comparison