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: Expression levels of cardiac markers and cardiac transcription factors in LAP2a CRISPR clones after 7 days in differentiation medium . ( A ) Whole cell protein extracts were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (Prolif.) or incubated for 7 days in differentiation medium (Diff.) and analysed by western blot. The latter were revealed either with mouse anti Actin alpha 1 cardiac muscle Ab and successively with mouse anti cardiac troponin T monoclonal Ab, or with mouse anti Myosin-2 Ab. Red Ponceau staining prior to incubation with antibodies is also shown. ( B ) The graphs depict the relative protein amount (mean ± s.e.m normalized to Red Ponceau) for a1 cardiac Actin, cardiac troponin T2 (TNNT2) and Myosin-2 in cells grown in proliferation (P) or differentiation (D) medium. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). The graphs present the individual values and means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 or 5 independent differentiation experiments for the LAP2a -/- clone). * p<0.05, ** p<0.01 (Mann Whitney test). ( C ) The graphs depict relative mRNA levels normalized to Hmbs for Actc1 , Tnnt2 and Myh7 in cells grown in proliferation (P) or differentiation (D) medium, as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 or 5 independent differentiation experiments for the LAP2a -/- clone). ** p<0.01, *** p<0.001 (Mann Whitney test). ( D ) The graphs depict relative mRNA levels normalized to Hmbs for Gata4 , Mef2c and P300 in cells grown in proliferation (P) or differentiation (D) medium, as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 3 independent differentiation experiments for each WT +/+ clone, N = 1 or 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 independent differentiation experiments for the LAP2a -/- clone). * p<0.05, ** p<0.01, *** p<0.001 (Mann Whitney test).

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: Expressing, CRISPR, Clone Assay, Incubation, Western Blot, Staining, MANN-WHITNEY, Quantitative RT-PCR

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: Changes in LAP2a and LAP2b expression levels in vitro upon cardiac differentiation. ( A ) mRNAs were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (P) or incubated for 7 days in differentiation medium (D). Their relative amount was evaluated by RT-qPCR using specific primers (see Materials and Methods). The graphs depict relative mRNA amount (normalized to Hmbs ) for Lap2a and Lap2b. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). For each analysis, samples of LAP2a +/+ in proliferation (green circle) were used to arbitrarily define a reference value equal to 1.00. Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 3 or 4 independent differentiation experiments for the LAP2a -/- clone). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 (Mann Whitney test). ( B ) Whole cell protein extracts were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (Prolif., P) or incubated for 7 days in differentiation medium (Diff., D) and analysed by western blot. The latter were revealed with the rabbit anti TMPO Ab to detect LAP2a (alpha), LAP2b (beta) and a short LAP2b-like isoform (bsh). ( C-E ) The graphs depict the relative protein amount (i.e. mean ECL signal intensity (a.u) ± s.e.m normalized to Red ponceau) for LAP2a, LAP2 b, LAP2a:LAP2b, and LAP2bsh as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). For each analysis, samples of LAP2a +/+ in proliferation were used to arbitrarily define a reference value equal to 1.00 (green circle). The graphs present the individual values and means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 3 to 5 independent differentiation experiments for the LAP2a -/- clone). * p<0.05; ** p<0.01; *** p<0.001 (Mann Whitney test).

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: Expressing, In Vitro, CRISPR, Clone Assay, Incubation, Quantitative RT-PCR, MANN-WHITNEY, Western Blot

Journal: medRxiv

Article Title: GDNF levels regulate lumbar motor neuron physiology and determine life expectancy in limb-onset ALS

doi: 10.64898/2025.12.05.25341683

Figure Lengend Snippet: (A) Analysis of RET-positive neurons in the lumbar spinal cord (n=3 animals per group, 6 sections per animal) reveals no difference between the genotypes (B-C) Analyses of NMJs at 35 weeks of age showed significantly more occupied (p=0.0033) NMJs in Gdnf wt/hyper mice and less denervated (p=0.0036) NMJs compared to the Gdnf wt/wt animals (n=5 neurofilament (2H3) and synaptic vesicles (SV2) were stained to visualize the axons and pre-synaptic nerve terminals, α-bungarotoxin (α-BTX) was used to visualize the endplate (stains acetylcholine receptors in skeletal muscle fibers). ( Gdnf wt/wt n=5 and n=6 Gdnf wt/hyper ). (D) Representative images of an NMJ in a 35-week-old Gdnf wt/wt and Gdnf wt/hyper mouse in SOD1 G93A background (scale bar = 10µm). Note the lack of neuronal staining in Gdnf wt/wt in SOD1 G93A background, indicating degeneration of MN axons.

Article Snippet: Muscle sections were blocked with PBS-Triton X-100 (0.5%) and incubated for 24h with anti-SV2 and anti-2H3 primary antibodies (1:100, DSHB, AB_2315387, and AB_531793), recognizing neurofilament (2H3) and synaptic vesicles (SV2) to visualize the axons and pre-synaptic nerve terminals.

Techniques: Staining