Journal: Nature Communications

Article Title: CD248-targeted BBIR-T cell therapy against late-activated fibroblasts in cardiac repair after myocardial infarction

doi: 10.1038/s41467-025-56703-2

Figure Lengend Snippet: a UMAP clustering of 13441 cardiac interstitial cells (left) and fibroblasts (right) isolated from C57BL/6 J wild-type (WT) mice at 7 and 14 days post-sham and post-MI surgery. Each dot represents a single cell. The expression of known marker genes annotated cell type. b UMAP and violin plots depict Postn , Acta2 , and Comp expression in fibroblasts. c Triple IF staining for periostin (green) and αSMA (red) in the peri-infarct zone of the hearts of C57BL/6 J WT mice 0, 7, 14, 21, and 28 days after MI surgery. Nuclei were stained blue. Scale bar = 100 μm. Quantification of the number of periostin + αSMA + F-Myo and periostin + αSMA - F-Act in each high-power field (HPF). n = 5 mice at each time point after MI. d Flow cytometry of fibroblasts isolated from hearts of C57BL/6 J WT and Pdgfra-CRE:tdTomato mice. Total cardiac fibroblasts were lineage traced by pdgfrα. F-Myo and F-Act were marked with antibodies against periostin-AF488 and αSMA-AF647. Quantification of the pdgfrα + fibroblast ratio in live cells, periostin + fibroblasts ratio in pdgfrα + fibroblasts, periostin + αSMA + F-Myo ratio in pdgfrα + fibroblasts, and periostin + αSMA - F-Act ratio in pdgfrα + fibroblasts. n = 5 mice at each time point after MI. e Spatial distribution of the F-Act and F-Myo subpopulation in mouse heart sections from sham and 10 days post-MI conditions, visualized through the integration of scRNA-seq and stereo-seq methodologies. c, d Data are the mean ± SEM and p -values are displayed in the bar charts, one-way ANOVA followed by Tukey’s multiple comparisons tests. Source data are provided as a Source Data file.

Article Snippet: The antibodies used in this study included Periostin (Abcam, ab14041), αSMA-Alexa Fluor 647 (Novus, NBP2-34522), CD248-Alexa Fluor 647 (Santa Cruz, sc-377221), CD3-FITC (BioLegend, 317306 for human and 100204 for mouse), CD4-APC (BioLegend, 344614 for human and 100412 for mouse), CD8-PE (BioLegend, 344706 for human and 100708 for mouse), and goat anti-rabbit IgG (1:1000, Invitrogen, A11008 for Alexa Fluor 488).

Techniques: Isolation, Expressing, Marker, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: CD248-targeted BBIR-T cell therapy against late-activated fibroblasts in cardiac repair after myocardial infarction

doi: 10.1038/s41467-025-56703-2

Figure Lengend Snippet: a Triple IF staining for periostin (green) and CD248 (red) in the peri-infarct zone of the hearts of C57BL/6J WT mice 0, 7, 14, 21, and 28 days after MI surgery. Nuclei were stained blue. Scale bar = 100 μm. Quantification of the periostin + CD248 + cell to DAPI ratio in each HPF. n = 5 mice at each time point after MI. Triple IF staining of periostin, αSMA, COMP (stained green), and CD248 (stained red) in the hearts of MI mouses ( b ) and a patient with ischemic cardiomyopathy ( c ). Nuclei were stained blue. Scale bar =100 μm. IF staining of mouse heart was repeated in heart sections from 5 independent mice, while IF of human heart was repeated 5 times in heart sections from one patient with ischemic cardiomyopathy, yielding similar results. d Flow cytometry of cells isolated from hearts of C57BL/6 J WT mouses and Pdgfra-CRE:tdTomato mice. Total cardiac fibroblasts were lineage traced by pdgfrα. Quantification of the CD248 + cell ratio in live cells, pdgfrα + fibroblast ratio in CD248 + cells, CD248 + cell ratio in pdgfrα + fibroblasts, periostin + CD248 + cell ratio in pdgfrα + fibroblasts, CD248 + cell ratio in periostin + activated fibroblasts, and periostin + activated fibroblasts in CD248 + cells. n = 5 mice at each time point after MI. a, d Data are the mean ± SEM and p -values are displayed in the bar charts, one-way ANOVA followed by Tukey’s multiple comparisons tests. Source data are provided as a Source Data file.

Article Snippet: The antibodies used in this study included Periostin (Abcam, ab14041), αSMA-Alexa Fluor 647 (Novus, NBP2-34522), CD248-Alexa Fluor 647 (Santa Cruz, sc-377221), CD3-FITC (BioLegend, 317306 for human and 100204 for mouse), CD4-APC (BioLegend, 344614 for human and 100412 for mouse), CD8-PE (BioLegend, 344706 for human and 100708 for mouse), and goat anti-rabbit IgG (1:1000, Invitrogen, A11008 for Alexa Fluor 488).

Techniques: Staining, Flow Cytometry, Isolation

Journal: Nature Communications

Article Title: CD248-targeted BBIR-T cell therapy against late-activated fibroblasts in cardiac repair after myocardial infarction

doi: 10.1038/s41467-025-56703-2

Figure Lengend Snippet: a Schematic diagram of the structure of the BBIR-T system. Created in BioRender. Haiting, C. (2025) https://BioRender.com/l46n352 . b Schematic images of dcAv. BBIR-28-137ζ and CD19. CAR-137ζ sequence. c Western blotting shows exogenous CD3ζ expression in HEK-293T cells transduced with lentivirus (LV)-dcAv.BBIR and LV-anti-CD19.CAR. The samples derive from the same experiment and that gels/blots were processed in parallel. Quantifying the relative protein expression of CD3ζ to GAPDH. n = 4 cell culture wells of each group. d Flow cytometry of human peripheral T cells transduced with LV-dcAv.BBIR and LV-anti-CD19.CAR. Quantifying GFP + cells ratio to total T cells and biotin + cells ratio to GFP + cells. n = 5 cell culture wells of each group. e Western blotting by BF shows CD248 expression in NIH-3T3 cells with or without Cd248 overexpression(OE) or knockout(KO). The samples were derived from the same experiment and that gels/blots were processed in parallel. Quantifying the relative protein expression of CD248 to GAPDH. n = 4 cell culture wells of each group. f Triple IF staining of periostin, αSMA, COMP (green), and biotinylated anti-CD248 F(ab’)2 (shorted as BF) (red) in the peri-infarct zone 14 days after MI surgery. Nuclei were stained blue. Scale bar =100 μm. Each experiment was repeated 5 times independently with similar results. c-e Data are the mean ± SEM and p -values are displayed in the bar charts, one-way ANOVA followed by Tukey’s multiple comparisons tests. Source data are provided as a Source Data file.

Article Snippet: The antibodies used in this study included Periostin (Abcam, ab14041), αSMA-Alexa Fluor 647 (Novus, NBP2-34522), CD248-Alexa Fluor 647 (Santa Cruz, sc-377221), CD3-FITC (BioLegend, 317306 for human and 100204 for mouse), CD4-APC (BioLegend, 344614 for human and 100412 for mouse), CD8-PE (BioLegend, 344706 for human and 100708 for mouse), and goat anti-rabbit IgG (1:1000, Invitrogen, A11008 for Alexa Fluor 488).

Techniques: Sequencing, Western Blot, Expressing, Transduction, Cell Culture, Flow Cytometry, Over Expression, Knock-Out, Derivative Assay, Staining

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Mice lacking Dectin-1 are protected from unilateral ureteric obstruction (UUO)-induced progressive renal fibrosis. Wild-type (WT) and Dectin-1 knockout (KO) mice challenged to sham or UUO operation. (A) Experimental design of the treatment procedure. (B) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (C) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n = 6 for each group). Scale bar, 50 μm. (D) Quantification of the positive staining area (%) in (C). (E) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (F) Densitometric quantification of blots in (E). (G) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 6 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Also, antibodies against α-SMA (PE, IC1420P) from R&D Systems (Minnesota, USA), antibodies against p-Smad2/3 (PE, 562586) from BD Biosciences (New Jersey, USA), and antibodies against GAPDH (60004-1-Ig) from Proteintech (Chicago, USA).

Techniques: Knock-Out, Staining, Immunohistochemical staining, Western Blot, Control, Expressing

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Deletion of bone marrow (BM)-derived Dectin-1 inhibits unilateral ureteric obstruction (UUO)-induced renal fibrosis. (A) Experimental design of the treatment procedure. (B) Flow cytometric analysis showing the genetic background of CD45 alleles in the wild-type (WT) and Dectin-1 knockout (KO) mice. (C) Successful BM transplantations (BMTs) were confirmed by flow cytometric analysis of peripheral blood cells. (D-I) After successful BMT, mice from 4 groups (WT BM→WT, Dectin-1 KO BM→WT, WT BM→ Dectin-1 KO, and Dectin-1 KO BM→ Dectin-1 KO) were subjected to sham or UUO operation. (D) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (E) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n=3 for each group). Scale bar, 50 μm. (F) Quantification of the positive staining area (%) in (E). (G) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. α-Tubulin was used as loading control (n = 3 for each group). (H) Densitometric quantification of blots in (G). (I) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 3 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Also, antibodies against α-SMA (PE, IC1420P) from R&D Systems (Minnesota, USA), antibodies against p-Smad2/3 (PE, 562586) from BD Biosciences (New Jersey, USA), and antibodies against GAPDH (60004-1-Ig) from Proteintech (Chicago, USA).

Techniques: Derivative Assay, Knock-Out, Staining, Immunohistochemical staining, Western Blot, Control, Expressing

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Macrophage-specific deletion of Dectin-1 protects against unilateral ureteric obstruction (UUO)-induced renal fibrosis. Dectin-1 f/f and Dectin-1 ΔCd68 mice challenged to sham or UUO operation. (A) Experimental design of the treatment procedure. (B) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (C) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n=6 for each group). Scale bar, 50 μm. (D) Quantification of the positive staining area (%) in (C). (E) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (F) Densitometric quantification of blots in (E). (G) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 6 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Also, antibodies against α-SMA (PE, IC1420P) from R&D Systems (Minnesota, USA), antibodies against p-Smad2/3 (PE, 562586) from BD Biosciences (New Jersey, USA), and antibodies against GAPDH (60004-1-Ig) from Proteintech (Chicago, USA).

Techniques: Staining, Immunohistochemical staining, Western Blot, Control, Expressing

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Dectin-1 deficiency reduces macrophage-to-myofibroblast transition (MMT) through TGF-β/Smad pathway. Wild-type (WT) and Dectin-1 knockout (KO) mice subjected to unilateral ureteric obstruction (UUO) operation. (A) Representative flow cytometric images illustrating the expression of α-SMA + in CD45 + CD11b + F4/80 + live cells (left) and the percentage of α-SMA + cells in macrophages and its mean fluorescence intensity levels (2 parts in the right) in kidneys (n=6 for each group). (B) Relative mRNA expression of Acta2 in sorted macrophages from fibrotic kidneys (n = 5 for each group). (C) Schematic of Dectin-1 induced MMT. (D) Representative western blot analysis of TGF-β1 protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (E) Densitometric quantification of blots in (D). (F) Relative mRNA expression of Tgfb1 in kidney tissues (n = 5 for each group). (G) Relative mRNA expression of Tgfb1 in sorted macrophages from fibrotic kidneys (n = 5 for each group). (H) The concentrations of TGF-β1 detected by ELISA in supernatant from sorted macrophages of fibrotic kidneys (n = 5 for each group). (I) Representative flow cytometric images illustrating the expression of p-Smad2/3 + in CD45 + CD11b + F4/80 + live cells (left) and the percentage of p-Smad2/3 + cells in macrophages and its mean fluorescence intensity levels (2 parts in the right) in kidneys (n=6 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Also, antibodies against α-SMA (PE, IC1420P) from R&D Systems (Minnesota, USA), antibodies against p-Smad2/3 (PE, 562586) from BD Biosciences (New Jersey, USA), and antibodies against GAPDH (60004-1-Ig) from Proteintech (Chicago, USA).

Techniques: Knock-Out, Expressing, Fluorescence, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: Dectin-1 is Pathogenic in Chronic Kidney Disease by Promoting Macrophage Infiltration and Transition to Myofibroblast

doi: 10.7150/ijbs.119129

Figure Lengend Snippet: Inhibition of Dectin-1 attenuates chronic kidney disease (CKD) progression induced by unilateral ureteric obstruction (UUO) and ischemia-reperfusion (IR) in mice. (A-G) Laminarin was administered (300mg/kg/day) following sham or UUO surgery. (A) Experimental design of the treatment procedure. (B) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (C) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n=6 for each group). Scale bar, 50 μm. (D) Quantification of the positive staining area (%) in (C). (E) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. GAPDH was used as loading control (n = 6 for each group). (F) Densitometric quantification of blots in (E). (G) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 6 for each group). (H-N) Laminarin was administered (300mg/kg/day) following sham or IR surgery. (H) Experimental design of the treatment procedure. (I) Representative photomicrographs illustrating H&E and Masson's trichrome staining in kidney tissues. Scale bar, 50 μm. (J) Representative photomicrographs illustrating immunohistochemical staining for fibronectin, collagen1, vimentin, and α-SMA in kidney tissues (n=6 for each group). Scale bar, 50 μm. (K) Quantification of the positive staining area (%) in (J). (L) Representative western blot analysis of fibronectin, collagen1, vimentin, and α-SMA protein in the kidney tissues. α-Tubulin was used as loading control (n = 6 for each group). (M) Densitometric quantification of blots in (L). (N) Relative mRNA expression of Fn1 , Col1a1 , Vim , and Acta2 in kidney tissues (n = 6 for each group). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Also, antibodies against α-SMA (PE, IC1420P) from R&D Systems (Minnesota, USA), antibodies against p-Smad2/3 (PE, 562586) from BD Biosciences (New Jersey, USA), and antibodies against GAPDH (60004-1-Ig) from Proteintech (Chicago, USA).

Techniques: Inhibition, Staining, Immunohistochemical staining, Western Blot, Control, Expressing

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: Molecular Therapy. Methods & Clinical Development

Article Title: Adipose Mesenchymal Cells-Derived EVs Alleviate DOCA-Salt-Induced Hypertension by Promoting Cardio-Renal Protection

doi: 10.1016/j.omtm.2019.11.002

Figure Lengend Snippet: ASC-EV Treatment Promoted Important Attenuation of Kidney Fibrosis in the DOCA-Salt Model (A) Representative photomicrographs of kidney slices stained with Picrosirius red on 9-week-treated rats from the 4 experimental groups (scale bars, 50 μm). (B) α-SMA expression in the kidney of 9-week-treated rats under the different experimental conditions was determined by immunohistochemistry (n = 4 for each treatment) (scale bars, 50 μm). (C) Quantification of Picrosirius red stained area in kidney sections expressed as a percentage of the total area of the kidney, in 9-week-treated rats, measured for all the animals tested in the 4 experimental groups (SHAM, n = 4; UNX, n = 9; DOCA, n = 13; DOCA-EV, n = 10). (D−F) Western blot analysis of α-SMA and Desmin in the cortex of 9-week-treated UNX (n = 6), DOCA, and DOCA-EV (n = 8) rats. GAPDH was used as loading control. Values represent the fold increase with respect to UNX (considered as the reference = 1). (G−I) Analysis of COL1A1 , COL4A1 , and FN mRNA levels in the renal tissue in control SHAM (n = 3) and in all the uninephrectomized animal groups (UNX, n = 6; DOCA and DOCA-EV, n = 7). qRT-PCR data were expressed as RQ and UNX was used as the reference sample. Data represent mean ± SE. *indicates statistical difference to SHAM group, # indicates statistical difference to UNX group, and § indicates statistical difference to DOCA group; p < 0.05.

Article Snippet: The following primary antibodies were used: GAPDH, sc-32233 (1:1,000, Santa Cruz); α-SMA, MAB1420 (1:1,000, R&D Systems, Minneapolis, MN, USA); Desmin, ab32362 (1:1,000, Abcam); B cell lymphoma 2 (Bcl2), 2876 (1:1,000, Cell Signaling Technology, Danvers, MA, USA); and Bcl-2-associated X (Bax), 2772S (1:500, Cell Signaling Technology).

Techniques: Staining, Expressing, Immunohistochemistry, Western Blot, Control, Quantitative RT-PCR

Journal: JACC: Basic to Translational Science

Article Title: Bioprosthetic Valve Deterioration

doi: 10.1016/j.jacbts.2023.01.003

Figure Lengend Snippet: Sparse Collagen Fibers and Fewer Mesenchymal Cells in Bioprosthetic Valves (A) Masson’s trichrome staining and immunohistochemical staining for vimentin and anti-α-smooth muscle actin (SMA) of explanted bioprosthetic valves (eBVs) and normal native and aortic valve stenosis (AS) valves. The left panels are whole views of the valve leaflets. Scale bars, 100 μm. The right panels show colocalizations of vimentin and αSMA in the valves. (B) Quantitative data of the ratios (%) of red/blue areas of Masson’s trichrome staining, vimentin-positive cells, and α-SMA-positive cells in the valves. Statistical analyses were performed using the Kruskal-Wallis test with Dunn’s multiple-comparisons test (all comparisons). ∗∗ P < 0.01 and ∗∗∗ P < 0.001.

Article Snippet: Anti–α-smooth muscle actin (SMA) antibody was obtained from Novus Biologicals.

Techniques: Staining, Immunohistochemical staining

Journal: JACC: Basic to Translational Science

Article Title: Bioprosthetic Valve Deterioration

doi: 10.1016/j.jacbts.2023.01.003

Figure Lengend Snippet: Protein Profiling in Tissue Lysates From the Explanted Native Valves (AS and Normal) and eBVs (A) Two-dimensional gel electrophoresis of tissue lysates from native and bioprosthetic valves. The left panels are the enlarged figures of the areas surrounded by black broken lines in the right panels . (B) Western blot analyses for fibrinogen, plasminogen (PLG), apolipoprotein E (APOE), and anti–serum amyloid P component (APCS) in tissue lysates from the explanted native and bioprosthetic valves. (C) Statistical quantifications of the western blot results are presented. Analysis of variance, followed by Tukey’s multiple-comparisons test (all comparisons), was used for statistical analyses of the fibrinogen, APCS, and APOE levels. The Kruskal-Wallis test (all comparisons) was used for statistical analyses of PLG levels. ∗ P < 0.05 and ∗∗∗ P < 0.001. FGB = fibrinogen β-chain; other abbreviations as in Figure 1 .

Article Snippet: Anti–α-smooth muscle actin (SMA) antibody was obtained from Novus Biologicals.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot

Journal: JACC: Basic to Translational Science

Article Title: Bioprosthetic Valve Deterioration

doi: 10.1016/j.jacbts.2023.01.003

Figure Lengend Snippet: Tissue Localization of Fibrinogen, PLG, Anti-APOE, and Anti-APCS (A) Whole–valve leaflet views of the immunohistochemical images of the preimplanted and explanted bioprosthetic valves (BVs) and normal valve (left panels) . Porcine-derived eBV6, which strongly detected fibrinogen and PLG by western blotting, was stained using antifibrinogen or PLG antibodies. Bovine-derived eBV5, which was strongly detected by APOE and APCS using western blotting, was stained using anti-APOE and APCS antibodies. Preimplanted porcine- and bovine-derived BVs were used according to immunohistochemical staining against each species. The right panels show high magnification of the areas surrounded by lines in the whole view of the left panels . Scale bars, 200 μm. Arrowheads indicate strongly stained areas. (B) Immunofluorescence costaining of fibrinogen/CD68 or PLG/CD68 in the BV. Scale bars, 20 μm. Abbreviations as in Figures 1 , , and .

Article Snippet: Anti–α-smooth muscle actin (SMA) antibody was obtained from Novus Biologicals.

Techniques: Immunohistochemical staining, Derivative Assay, Western Blot, Staining, Immunofluorescence

Journal: JACC: Basic to Translational Science

Article Title: Bioprosthetic Valve Deterioration

doi: 10.1016/j.jacbts.2023.01.003

Figure Lengend Snippet: Histologic Evaluations of Explanted IBTA Valves (A) HE- and Masson’s trichrome–stained whole-leaflet views of the preimplanted Biosheet and explanted in-body tissue architecture (IBTA) valves (implantation term 3 and 12 months) (top panel) . Scale bars, 500 μm. Macroscopic views of the explanted valves (bottom panel) . (B) Masson’s trichrome, HE, and IHC for vimentin and FGB of the areas surrounded by lines in the whole view of right panels in A . Scale bars, 200 μm. (C) Surface structures of Masson’s trichrome–stained IBTA and pericardium-derived bioprosthetic valves for 12 months of implantation. Arrowheads indicate digested collagen fibers. (D) Quantifications of the infiltration foci per leaflet (top panel) and stained areas of immunohistochemistry against FGB (bottom panel) . Ratios were calculated by dividing brown-stained areas by the entire area of the leaflets. Statistical analysis was conducted using analysis of variance, followed by Sidak’s multiple-comparisons test. (E) Whole-leaflet images of the explanted normal, IBTA (3 and 12 months), and pericardium-derived valves (3 and 12 months). All leaflets were immune stained with anti-FGB antibodies. Scale bar, 1 mm. (F) Schematic diagram of molecular mechanism regarding circulating proteins’ and cells’ infiltration. ∗ P < 0.05 and ∗∗∗ P < 0.001. PGB = plasminogen; other abbreviations as in Figures 3 and .

Article Snippet: Anti–α-smooth muscle actin (SMA) antibody was obtained from Novus Biologicals.

Techniques: Staining, Derivative Assay, Immunohistochemistry