bound 6  (Landauer Inc)

Journal: Physiological Reports

Article Title: ERK3 is involved in regulating cardiac fibroblast function

doi: 10.14814/phy2.16108

Figure Lengend Snippet: Effect of Small Interfering (si)RNA‐Mediated ERK3 Knockdown on the abundance of transcripts for fetal proteins in ventricular fibroblasts and myofibroblasts. Ventricular fibroblasts from ERK3 +/+ mice were isolated and cultured on “compliant” (c) collagen‐coated, hydrogel‐bound (elastic modulus 8‐kPa) polystyrene plates or “non‐compliant” (elastic modulus 10 7 ‐kPa) uncoated standard cell culture dishes (nc), resulting in quiescent fibroblasts or myofibroblasts, respectively. Quiescent fibroblasts or myofibroblasts were transiently transfected with either siRNA for ERK3 (si) or a scrambled RNA sequence (sc) as described under Methods. In both cases, after 12 h of serum deprivation, cells were incubated in serum‐free M199 in the presence or absence of 1 μM angiotensin II (Ang II) or transforming growth factor‐β1 (TGF‐β) for 24 h. (a) Immunoblot assay showing siRNA reduced the abundance of ERK3 immunoreactivity in myofibroblast lysates from two independent experiments using separate cell preparations (a, b). The numbers at the right indicate the position of molecular mass markers (in kDa). (b) An immunoblot assay showing the abundance of smooth muscle α‐actin (αSMA) immunoreactivity in lysates of fibroblasts that were grown on compliant (c) and non‐compliant (nc) substrates. Results from two independent cell preparations (a, b) are shown. The numbers at the right indicate the position of molecular mass markers (in kDa). The relative abundance of the mRNA (c, f) A‐type natriuretic peptide ( Nppa ), (d, g) B‐type natriuretic peptide ( Nppb ), and (e, h) β‐myosin heavy chain ( Myh7 ) in quiescent fibroblasts (c–e) and myofibroblasts (f–h) was evaluated by qPCR. The data shown was normalized to Gapdh mRNA. Data shown are mean ± SEM for n = 3 cell preparations. Statistical analysis was by 2‐way ANOVA followed by Tukey's post hoc tests was performed. *** p < 0.001.

Article Snippet: All standard culture plates were from Sarstedt Inc. Collagen‐coated hydrogel‐bound (8‐kPa) polystyrene 35 mm (#PS35‐COL‐8) and 6‐well (#SW6‐COL‐8) plates were from Matrigen.

Techniques: Isolation, Cell Culture, Transfection, Sequencing, Incubation, Western Blot

Journal: Physiological Reports

Article Title: ERK3 is involved in regulating cardiac fibroblast function

doi: 10.14814/phy2.16108

Figure Lengend Snippet: Small interfering (si)RNA‐mediated knockdown of ERK3 in fibroblasts reduced the abundance of Col1a1 mRNA but not type 1 collagen immunoreactivity. Ventricular fibroblasts from ERK3 +/+ mice were isolated and cultured on (a, c, d) “compliant” (elastic modulus 8‐kPa) or (b, e, f) “non‐compliant” (elastic modulus 10 7 ‐kPa) culture dishes, resulting in quiescent fibroblasts or myofibroblasts, respectively. Quiescent fibroblasts or myofibroblasts were transiently transfected with either siRNA for ERK3 (siRNA) or a scrambled RNA sequence (scRNA) as described under Methods. In both cases, after 12 h of serum deprivation, cells were incubated in serum‐free M199 in the absence (control) or presence of 1 μM angiotensin II (Ang II) or transforming growth factor‐β1 (TGF‐β) for 24 h. (a, b) Col1a1 mRNA was quantified by qPCR and normalized Gapdh mRNA. (c‐f) Type 1 collagen immunoreactivity was measured in cell lysates (c, e; 20 μg) or conditioned media (d, f) by immunoblot assay. Results were normalized to cell number and then control fibroblasts transfected with scRNA. Representative immunoblots are shown above the histograms. GAPDH immunoreactivity was employed as a loading control when determining intracellular collagen immunoreactivity. Numbers on the right of the immunoblot images indicate the position of the molecular mass markers (in kDa). Values in the histograms show the mean ± SEM of three independent experiments. Each experiment was performed with fibroblasts isolated from a separate mouse. Statistical analysis was by 2‐way ANOVA followed by Tukey's post hoc tests was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: All standard culture plates were from Sarstedt Inc. Collagen‐coated hydrogel‐bound (8‐kPa) polystyrene 35 mm (#PS35‐COL‐8) and 6‐well (#SW6‐COL‐8) plates were from Matrigen.

Techniques: Isolation, Cell Culture, Transfection, Sequencing, Incubation, Western Blot

Journal: Physiological Reports

Article Title: ERK3 is involved in regulating cardiac fibroblast function

doi: 10.14814/phy2.16108

Figure Lengend Snippet: MMP‐2 and MMP‐9 activity in ERK3‐deficient quiescent ventricular fibroblasts. Ventricular fibroblasts isolated from ERK3 +/+ mice, maintained on “compliant” (elastic modulus 8‐kPa) plates, resulting in quiescent fibroblasts, were transiently transfected with either siRNA for ERK3 (siRNA) or a scrambled RNA sequence (scRNA). After 12 h of serum deprivation, cells were incubated in serum‐free M199 in the absence (control) or presence of 1 μM angiotensin II (Ang II) or transforming growth factor‐β1 (TGF‐β) for 24 h, and both the cell lysate (20 μg) and conditioned media used for the measurement of MMP‐2 and MMP9 activity by gelatin zymography. (a) Representative zymogram showing bands of cell‐associated pro‐MMP‐2, MMP‐2, and MMP9‐NGAL (MMP9/N) activity. Numbers on the right of the image indicate the position of molecular weight markers (in kDa). Quantitative analysis of MMP‐2 activity (b) and MMP‐9‐NGAL activity (c) normalized to that of fibroblasts transfected with scRNA and maintained in serum‐free media. (d) Representative zymogram showing bands of secreted pro‐MMP‐2, MMP‐2, and MMP9 activity. Numbers on the right of the image indicate the position of molecular weight markers (in kDa). Quantitative analysis of MMP‐2 activity (e) and MMP‐9 activity (f) normalized to that of fibroblasts transfected with scRNA and maintained in serum‐free media. The data shown are mean ± SEM of assessments performed in fibroblasts isolated from three different mice. Statistical analysis was by 2‐way ANOVA followed Tukey's post hoc test for multiple comparisons. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: All standard culture plates were from Sarstedt Inc. Collagen‐coated hydrogel‐bound (8‐kPa) polystyrene 35 mm (#PS35‐COL‐8) and 6‐well (#SW6‐COL‐8) plates were from Matrigen.

Techniques: Activity Assay, Isolation, Transfection, Sequencing, Incubation, Zymography, Molecular Weight

Journal: bioRxiv

Article Title: ERK3 Is Involved in Regulating Cardiac Fibroblast Function

doi: 10.1101/2023.12.05.570171

Figure Lengend Snippet: Ventricular fibroblasts from ERK3 +/+ mice were isolated and cultured on ‘compliant’ (c) collagen-coated, hydrogel-bound (elastic modulus 8-kPa) polystyrene plates or ‘non-compliant’ (elastic modulus 10 7 -kPa) uncoated standard cell culture dishes (nc), resulting in quiescent fibroblasts or myofibroblasts, respectively. Quiescent fibroblasts or myofibroblasts were transiently transfected with either siRNA for ERK3 (si) or a scrambled RNA sequence (sc) as described under Methods. In both cases, after 12 h of serum deprivation, cells were incubated in serum-free M199 in the presence or absence of 1 μM angiotensin II (Ang II) or transforming growth factor-β1 (TGF-β) for 24 h. ( A ) Immunoblot assay showing siRNA reduced the abundance of ERK3 immunoreactivity in myofibroblast lysates from two independent experiments using separate cell preparations (a, b). The numbers at the right indicate the position of molecular mass markers (in kDa). ( B ) An immunoblot assay showing the abundance of smooth muscle α-actin (αSMA) immunoreactivity in lysates of fibroblasts that were grown on compliant (c) and non-compliant (nc) substrates. Results from two independent cell preparations (a, b) are shown. The numbers at the right indicate the position of molecular mass markers (in kDa). The relative abundance of the mRNA ( C, F ) A-type natriuretic peptide ( Nppa ), ( D, G ) B- type natriuretic peptide ( Nppb ), and ( E, H ) β-myosin heavy chain ( Myh7 ) in quiescent fibroblasts ( C - E ) and myofibroblasts ( F - H ) was evaluated by qPCR and. The data shown was normalized to Gapdh mRNA. Data shown are mean ± SEM for n = 3 cell preparations. Statistical analysis was by 2-way ANOVA followed by Tukey’s post hoc tests was performed. ***P < 0.001.

Article Snippet: All standard culture plates were from Sarstedt, Inc. Collagen-coated hydrogel-bound (8-kPa) polystyrene 35 mm (#PS35-COL-8) and 6-well (#SW6-COL-8) plates were from Matrigen.

Techniques: Isolation, Cell Culture, Transfection, Sequencing, Incubation, Western Blot

Journal: bioRxiv

Article Title: ERK3 Is Involved in Regulating Cardiac Fibroblast Function

doi: 10.1101/2023.12.05.570171

Figure Lengend Snippet: Ventricular fibroblasts from ERK3 +/+ mice were isolated and cultured on ( A, C, D ) ‘compliant’ collagen-coated, hydrogel-bound (elastic modulus 8-kPa) plates or ( B, E, F ) ‘non-compliant’ (elastic modulus 10 7 -kPa) uncoated standard cell culture dishes, resulting in quiescent fibroblasts or myofibroblasts, respectively. Quiescent fibroblasts or myofibroblasts were transiently transfected with either siRNA for ERK3 (siRNA) or a scrambled RNA sequence (scRNA) as described under Methods. In both cases, after 12 h of serum deprivation, cells were incubated in serum-free M199 in the absence (control) or presence of 1 μM angiotensin II (Ang II) or transforming growth factor-β1 (TGF-β) for 24 h. (A, B) Col1a1 mRNA was quantified by qPCR and normalized Gapdh mRNA. ( C-F ) Type 1 collagen immunoreactivity was measured in cell lysates ( C, E ; 20μg) or conditioned media ( D, F ) by immunoblot assay. Results were normalized to cell number and then control fibroblasts transfected with scRNA. Representative immunoblots are shown above the histograms. GAPDH immunoreactivity was employed as a loading control when determining intracellular collagen immunoreactivity. Numbers on the right of the immunoblot images indicate the position of the molecular mass markers (in kDa). Values in the histograms show the mean ± SEM of three independent experiments. Each experiment was performed with fibroblasts isolated from a separate mouse. Statistical analysis was by 2-way ANOVA followed by Tukey’s post hoc tests was performed. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: All standard culture plates were from Sarstedt, Inc. Collagen-coated hydrogel-bound (8-kPa) polystyrene 35 mm (#PS35-COL-8) and 6-well (#SW6-COL-8) plates were from Matrigen.

Techniques: Isolation, Cell Culture, Transfection, Sequencing, Incubation, Western Blot

Journal: bioRxiv

Article Title: ERK3 Is Involved in Regulating Cardiac Fibroblast Function

doi: 10.1101/2023.12.05.570171

Figure Lengend Snippet: Ventricular fibroblasts isolated from ERK3 +/+ mice, maintained on ‘compliant’ collagen-coated, hydrogel-bound (elastic modulus 8-kPa) plates, resulting in quiescent fibroblasts, were transiently transfected with either siRNA for ERK3 (siRNA) or a scrambled RNA sequence (scRNA). After 12 h of serum deprivation, cells were incubated in serum-free M199 in the absence (control) or presence of 1 μM angiotensin II (Ang II) or transforming growth factor-β1 (TGF-β) for 24 h, and both the cell lysate (20 μg) and conditioned media used for the measurement of MMP-2 and MMP9 activity by gelatin zymography. ( A ) Representative zymogram showing bands of cell-associated pro-MMP-2, MMP-2, and MMP9-NGAL (MMP9/N) activity. Numbers on the right of the image indicate the position of molecular weight markers (in kDa). Quantitative analysis of MMP-2 activity ( B ) and MMP-9-NGAL activity ( C ) normalized to that of fibroblasts transfected with scRNA and maintained in serum-free media. ( D ) Representative zymogram showing bands of secreted pro-MMP-2, MMP-2, and MMP9 activity. Numbers on the right of the image indicate the position of molecular weight markers (in kDa). Quantitative analysis of MMP-2 activity ( E ) and MMP-9 activity ( F ) normalized to that of fibroblasts transfected with scRNA and maintained in serum-free media. The data shown are mean ± SEM of assessments performed in fibroblasts isolated from 3 different mice. Statistical analysis was by 2-way ANOVA followed Tukey’s post hoc test for multiple comparisons. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: All standard culture plates were from Sarstedt, Inc. Collagen-coated hydrogel-bound (8-kPa) polystyrene 35 mm (#PS35-COL-8) and 6-well (#SW6-COL-8) plates were from Matrigen.

Techniques: Isolation, Transfection, Sequencing, Incubation, Activity Assay, Zymography, Molecular Weight

Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: Knockdown of Trex1 increases the levels of (6-4)PP- and CPD-containing sedDNAs. ( A ) HeLa cells were treated as in Figure except that sedDNAs were immunoprecipitated with an antibody against (6-4)PPs. ( B ) Cells were processed as in (A) except that an anti-CPD antibody was used for immunoprecipitation. The graphs show the relative level of sedDNAs from at least three independent experiments.

Article Snippet: Briefly, the samples were immunoprecipitated with Dynabeads bound to anti-(6-4)PP or anti-CPD antibody (Cosmo Bio), washed with Wash Buffer I (20 mM Tris–HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 0.1% SDS), Wash Buffer II (20 mM Tris–HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), Wash Buffer III (10 mM Tris–HCl pH 8.0, 1 mM EDTA, 150 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate), Wash Buffer IV (100 mM Tris–HCl pH 8.0, 1 mM EDTA, 500 mM LiCl, 1% Nonidet-P40, 1% sodium deoxycholate), and twice with Tris-EDTA buffer.

Techniques: Knockdown, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: Overexpression of Trex1 decreases sedDNA abundance in UV-irradiated cells. ( A ) HeLa cells were transfected with expression vectors expressing the indicated fusion proteins, exposed to UV, and then harvested for analysis of sedDNAs. Quantitation of relative sedDNAs from five independent experiments. ( B , C ) Cells were processed as in (A) except that sedDNAs were purified and then immunoprecipitated with anti-(6-4)PP or anti-CPD antibodies.

Article Snippet: Briefly, the samples were immunoprecipitated with Dynabeads bound to anti-(6-4)PP or anti-CPD antibody (Cosmo Bio), washed with Wash Buffer I (20 mM Tris–HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 0.1% SDS), Wash Buffer II (20 mM Tris–HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), Wash Buffer III (10 mM Tris–HCl pH 8.0, 1 mM EDTA, 150 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate), Wash Buffer IV (100 mM Tris–HCl pH 8.0, 1 mM EDTA, 500 mM LiCl, 1% Nonidet-P40, 1% sodium deoxycholate), and twice with Tris-EDTA buffer.

Techniques: Over Expression, Irradiation, Transfection, Expressing, Quantitation Assay, Purification, Immunoprecipitation

Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Products Cat#SMI-21R-500; RRID: AB_509979 Mouse anti-NG2, clone 9.2.27 Millipore Cat# MAB2029; RRID: AB_94509 Rabbit anti-human PDGFRa, clone D13C6 Cell Signal Cat#5241S; RRID: AB_10692773 Rabbit anti-Transferrin antibody (discontinued) Abcam Cat#ab9538; RRID: AB_307325 Mouse anti-human Transferrin, clone HT1/13.6.3 MP Biomedicals Cat#AM025; RRID: AB_10059965 Rabbit anti-CNP Abcam Cat#18527; RRID: AB_470266 Mouse anti-human cytoplasmic antigen, STEM121 Takara Bio Cat# AB-121-U-050; RRID:AB_2632385 Goat anti-mouse IgG (H+L) Alexa Fluor 647 ThermoFisher Cat#A-21235; RRID: AB_2535804 Goat anti-mouse IgG1 Alexa Fluor 488 ThermoFisher Cat#A-21121; RRID: AB_2535764 Goat anti-mouse IgG (H+L) Alexa Fluor 488 ThermoFisher Cat#A-11029; RRID: AB_2534088 Goat anti-mouse IgG (H+L) Alexa Fluor 568 ThermoFisher Cat#A-11031; RRID: AB_144696 Goat anti-mouse IgG1 Alexa Fluor 568 ThermoFisher Cat#A-21124; RRID: AB_2535766 Goat anti-Rabbit IgG (H+L) Alexa Fluor 647 ThermoFisher Cat#A-21245; RRID: AB_2535813 Goat anti-Rabbit IgG (H+L) Alexa Fluor 568 ThermoFisher Cat#A-11036; RRID: AB_2534094 Goat anti-Rabbit IgG (H+L) Alexa Fluor 488 ThermoFisher Cat#A-11034; RRID: AB_2576217 Goat anti-Rat IgG (H+L) Alexa Fluor 647 ThermoFisher Cat#A-21247; RRID: AB_141778 Goat anti-Rat IgG (H+L) Alexa Fluor 568 ThermoFisher Cat#A-11077; RRID: AB_2534121 Goat anti-Rat IgG (H+L) Alexa Fluor 488 ThermoFisher Cat#A-11006; RRID: AB_2534074 Antibodies used in vitro Mouse anti-PSA-NCAM, clone 2-2B Millipore Cat#MAB5324; RRID: AB_95211 Mouse anti-PSA-NCAM supernatant, clone 5A5 DSHB Cat#5A5; RRID: AB_528392 Mouse anti-A2B5 supernatant, clone 105 ATCC Cat#CRL-1520; RRID: CVCL_7946 Mouse anti-CD140a, clone AR1 BD Biosci.

Techniques: Marker, In Vitro, Recombinant, Software, Microscopy, Lysis

Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-human Transferrin, clone HT1/13.6.3 , MP Biomedicals , Cat#AM025; RRID: AB_10059965.

Techniques: Marker, In Vitro, Recombinant, Software, Microscopy, Lysis