Journal: International Journal of Molecular Sciences

Article Title: Serpine1 Regulates the Enhanced Inhibitory Effect of CHIR99021 Combined with Fibroblast Growth Factor 2 on Myocardial Fibrosis After Myocardial Infarction in Mice

doi: 10.3390/ijms27041627

Figure Lengend Snippet: CHIR99021 combined with FGF2 inhibits collagen secretion and activation of cardiac fbroblasts. ( A ) The individual and combined effects of CHIR99021 and FGF2 led to significant changes in the morphology of cardiac fibroblasts. Scale bar: 100 μm. ( B ) Immunofluorescence staining of α-SMA, ColI, and ColIII in CFs treated with CHIR99021 and/or FGF2. Scale bar: 50 μm. ( C ) Quantitative analysis of α-SMA, ColI, and ColIII expression in CFs following treatment. n = 3. * p < 0.05, ** p < 0.01. ( D ) Western blotting analysis of α-SMA expression in CFs under different treatment conditions. n = 3. ** p < 0.01. ( E ) Quantification of hydroxyproline content in CFs treated with CHIR99021 and/or FGF2. n = 3. * p < 0.05.

Article Snippet: CFs were seeded into 96-well plates at 3000 cells/well and treated with CHIR99021 (10 μM), FGF2 (20 ng/mL), or both for 24 h. Immunofluorescence staining was performed for α-SMA, ColI, ColIII, Serpine1 and Cryab using the following antibodies: α-SMA (ET1607-53, HuaBio, 1:1000, Hangzhou, China), ColI (HA722517, HuaBio, 1:500, Hangzhou, China), and ColIII (HA720050, HuaBio, 1:500, Hangzhou, China), Serpine1 ( HA500124 , HuaBio, 1:100, Hangzhou, China) and Cryab (CY6897, Abways, 1:100, Shanghai, China).

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Serpine1 Regulates the Enhanced Inhibitory Effect of CHIR99021 Combined with Fibroblast Growth Factor 2 on Myocardial Fibrosis After Myocardial Infarction in Mice

doi: 10.3390/ijms27041627

Figure Lengend Snippet: CHIR99021 combined with FGF2 inhibits the expression of genes related to fibrosis, n = 3. ( A ) Relative mRNA expression levels of fibrosis-related genes ( Acta2 , Col3a1 , Col1a1 and Serpine1 ) in CFs under different treatment conditions, as determined by qPCR. Gapdh was used as the internal control. * p < 0.05, ** p < 0.01. ( B ) Immunofluorescence staining showing the expression of α-SMA, ColIII, and Serpine1 in CFs. Scale bar: 100 μm. ( C ) Quantitative determination of hydroxyproline content in the culture supernatant of CFs following various treatments. * p < 0.05. ( D ) Western blotting analysis showing the protein expression levels of α-SMA, Col III, and Serpine1 in CFs treated with CHIR99021 and FGF2. ( E ) Quantitative analysis of the Western blot results. Scale bar: 100 μm. * p < 0.05, ** p < 0.01.

Article Snippet: CFs were seeded into 96-well plates at 3000 cells/well and treated with CHIR99021 (10 μM), FGF2 (20 ng/mL), or both for 24 h. Immunofluorescence staining was performed for α-SMA, ColI, ColIII, Serpine1 and Cryab using the following antibodies: α-SMA (ET1607-53, HuaBio, 1:1000, Hangzhou, China), ColI (HA722517, HuaBio, 1:500, Hangzhou, China), and ColIII (HA720050, HuaBio, 1:500, Hangzhou, China), Serpine1 ( HA500124 , HuaBio, 1:100, Hangzhou, China) and Cryab (CY6897, Abways, 1:100, Shanghai, China).

Techniques: Expressing, Control, Immunofluorescence, Staining, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Serpine1 Regulates the Enhanced Inhibitory Effect of CHIR99021 Combined with Fibroblast Growth Factor 2 on Myocardial Fibrosis After Myocardial Infarction in Mice

doi: 10.3390/ijms27041627

Figure Lengend Snippet: The combination of CHIR99021 and FGF2 inhibits fibrotic remodeling after myocardial infarction. ( A ) Schematic representation of the myocardial infarction model and drug administration protocol in mice. ( B ) Masson’s trichrome staining of cardiac tissue from each group. Scale bar: 1000 μm. ( C ) is an enlarged part of ( B ). Scale bar: 50 μm. ( D ) Immunofluorescence staining of α-actinin and ColIII in the myocardial infarction area. Scale bar: 20 μm. ( E ) Immunofluorescence staining of Serpine1 and ColIII in the myocardial infarction area. Scale bar: 50 μm.

Article Snippet: CFs were seeded into 96-well plates at 3000 cells/well and treated with CHIR99021 (10 μM), FGF2 (20 ng/mL), or both for 24 h. Immunofluorescence staining was performed for α-SMA, ColI, ColIII, Serpine1 and Cryab using the following antibodies: α-SMA (ET1607-53, HuaBio, 1:1000, Hangzhou, China), ColI (HA722517, HuaBio, 1:500, Hangzhou, China), and ColIII (HA720050, HuaBio, 1:500, Hangzhou, China), Serpine1 ( HA500124 , HuaBio, 1:100, Hangzhou, China) and Cryab (CY6897, Abways, 1:100, Shanghai, China).

Techniques: Staining, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Serpine1 Regulates the Enhanced Inhibitory Effect of CHIR99021 Combined with Fibroblast Growth Factor 2 on Myocardial Fibrosis After Myocardial Infarction in Mice

doi: 10.3390/ijms27041627

Figure Lengend Snippet: Role of Serpine1 in Cardiac Fibroblast Activation and Collagen Expression. ( A ) Serpine1 knockdown and overexpression on the morphological changes in cardiac fibroblasts. Scale bar: 100 μm. ( B ) Western blotting analysis of ColIII, α-SMA, and Col I protein expression in CFs following Serpine1 knockdown. n = 3. ** p < 0.01, *** p < 0.001. ( C ) Immunofluorescence analysis of Serpine1, α-SMA, ColIII and Cryab expression in CFs after Serpine1 knockdown and overexpression. Scale bar: 50 μm.

Article Snippet: CFs were seeded into 96-well plates at 3000 cells/well and treated with CHIR99021 (10 μM), FGF2 (20 ng/mL), or both for 24 h. Immunofluorescence staining was performed for α-SMA, ColI, ColIII, Serpine1 and Cryab using the following antibodies: α-SMA (ET1607-53, HuaBio, 1:1000, Hangzhou, China), ColI (HA722517, HuaBio, 1:500, Hangzhou, China), and ColIII (HA720050, HuaBio, 1:500, Hangzhou, China), Serpine1 ( HA500124 , HuaBio, 1:100, Hangzhou, China) and Cryab (CY6897, Abways, 1:100, Shanghai, China).

Techniques: Activation Assay, Expressing, Knockdown, Over Expression, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Serpine1 Regulates the Enhanced Inhibitory Effect of CHIR99021 Combined with Fibroblast Growth Factor 2 on Myocardial Fibrosis After Myocardial Infarction in Mice

doi: 10.3390/ijms27041627

Figure Lengend Snippet: CHIR99021 and FGF2 regulate Serpine1 through the TGF-β and FAK signaling pathway. ( A ) Western blot assay showing the changes in protein expression of fibroblast activation markers (ColIII; and α-SMA), Smad2/3 and FAK through the overexpression of Serpine1 in cardiac fibroblasts following the indicated treatments. ( B ) Statistical analysis of relative protein expression levels n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: CFs were seeded into 96-well plates at 3000 cells/well and treated with CHIR99021 (10 μM), FGF2 (20 ng/mL), or both for 24 h. Immunofluorescence staining was performed for α-SMA, ColI, ColIII, Serpine1 and Cryab using the following antibodies: α-SMA (ET1607-53, HuaBio, 1:1000, Hangzhou, China), ColI (HA722517, HuaBio, 1:500, Hangzhou, China), and ColIII (HA720050, HuaBio, 1:500, Hangzhou, China), Serpine1 ( HA500124 , HuaBio, 1:100, Hangzhou, China) and Cryab (CY6897, Abways, 1:100, Shanghai, China).

Techniques: Western Blot, Expressing, Activation Assay, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

doi: 10.3390/ijms24020932

Figure Lengend Snippet: L1 binding partners assayed in this study.

Article Snippet: The siRNAs for murine WDR5 (sc-61799), SFPQ (PSF; sc-38305), NonO (p54/nrb; sc-38164), PSPC1 (sc-152566), PPARγ (PPARgamma; sc-29456), HistHe (Histone cluster 1 H1E; sc-37975), NDUFV2 (sc-149892), Hsc70 (HSC 70; sc-35593), and SYT1 (Synaptotagmin I; sc-41311) were from Santa Cruz Biotechnology.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

doi: 10.3390/ijms24020932

Figure Lengend Snippet: L1-55 interacts with MeCP2, HP1, and HistH1 but not with other verified or putative L1 binding partners in cultured cortical neurons. Neurons were treated with the vehicle dimethyl sulfoxide (DMSO) (+DMSO) or with the γ-secretase inhibitor DAPT and were then subjected to proximity ligation with L1 antibodies and antibodies against NDUFV2, SFPQ, NonO, PSPC1, WDR5, TOP1, hnRNP A isoforms, HistH1, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, AR, VDR, MeCP2, or HP1γ. Nuclei are stained with DAPI (4′,6-diamidino-2-phenylindole). ( a , b ) Representative images of DMSO- and DAPT-treated neurons stained with mouse L1 antibody C-2 and a rabbit antibody against hnRNP A isoforms ( a ) or HistH1 ( b ) are shown. Scale bar: 10 µm. ( c ) The mean values + SD are from two independent experiments and show average numbers of red dots per cell after DAPT-treatment relative to control (values of vehicle control set to 100%) (**** p < 0.001; one-way ANOVA with Dunn’s multiple comparison test). Values obtained for proteins known to bind to L1-55 served as positive controls and are marked in dark gray. ( d ) Non-nuclear fractions from wild-type (WT) and L1-deficient (KO) mice were used for immunoprecipitation with mouse HistH1 or NDUFV2 antibodies immobilized to Protein G. Fractions (input) and immunoprecipitates (IP) were subjected to Western blot analysis with L1 antibody C-2. Arrows indicate L1-55 and L1-70, and the arrowhead indicates an unknown L1 band of approximately 75 kDa.

Article Snippet: The siRNAs for murine WDR5 (sc-61799), SFPQ (PSF; sc-38305), NonO (p54/nrb; sc-38164), PSPC1 (sc-152566), PPARγ (PPARgamma; sc-29456), HistHe (Histone cluster 1 H1E; sc-37975), NDUFV2 (sc-149892), Hsc70 (HSC 70; sc-35593), and SYT1 (Synaptotagmin I; sc-41311) were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Cell Culture, Ligation, Staining, Control, Comparison, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

doi: 10.3390/ijms24020932

Figure Lengend Snippet: In cultured cortical neurons, L1 interacts with several binding partners via its KDET motif. Cultured cortical neurons were treated with vehicle, tat-KDET peptide, or tat-QNQS control peptide, followed by treatment without ( a ) and with ( b ) L1 antibody 557 and proximity ligation with a L1 antibody and an antibody against MeCP2, NDUFV2, SFPQ, NonO, PSPC1, WDR5, TOP1, HistH1, Nup93, Hsc70, SYT1, ERα, or PPARγ. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatment with vehicle set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni´s multiple comparison test).

Article Snippet: The siRNAs for murine WDR5 (sc-61799), SFPQ (PSF; sc-38305), NonO (p54/nrb; sc-38164), PSPC1 (sc-152566), PPARγ (PPARgamma; sc-29456), HistHe (Histone cluster 1 H1E; sc-37975), NDUFV2 (sc-149892), Hsc70 (HSC 70; sc-35593), and SYT1 (Synaptotagmin I; sc-41311) were from Santa Cruz Biotechnology.

Techniques: Cell Culture, Binding Assay, Control, Ligation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions

doi: 10.3390/ijms24020932

Figure Lengend Snippet: Reduction of SFPQ, NonO, WDR5, NDUFV2, SYT1, Hsc70, and HistH1e expression by siRNAs inhibits L1-dependent neurite outgrowth. Cortical neurons were not treated (no), treated without (mock), or treated with siRNAs specific for SFPQ, NonO, PSPC1, WDR5, NDUFV2, SYT1, Hsc70, and HistH1e. Neurons were then treated without or with antibody 557. ( a ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.0001 relative to L1 antibody-stimulated mock-transfected neurons, #### p < 0.0001 relative to non-stimulated mock-transfected neurons; one-way ANOVA with Dunn´s multiple comparison test). ( b ) Western blot analysis of lysates from mock-transfected neurons or neurons transfected with siRNAs using the corresponding antibodies. Ponceau S staining of a prominent 35-kDa band served as loading control. ( c ) Immunostaining of mock-transfected neurons or neurons transfected with NDUFV2 siRNA using a NDUFV2 antibody. Scale bar: 10 µm.

Article Snippet: The siRNAs for murine WDR5 (sc-61799), SFPQ (PSF; sc-38305), NonO (p54/nrb; sc-38164), PSPC1 (sc-152566), PPARγ (PPARgamma; sc-29456), HistHe (Histone cluster 1 H1E; sc-37975), NDUFV2 (sc-149892), Hsc70 (HSC 70; sc-35593), and SYT1 (Synaptotagmin I; sc-41311) were from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Comparison, Western Blot, Staining, Control, Immunostaining