p38 mapk antagonist sb203580 (Selleck Chemicals)

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Selleck Chemicals p38 mapk antagonist sb203580

Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by <t>p38</t> <t>MAPK</t> in fibroblasts from experimental models of pulmonary hypertension (PH). A : cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. *** P < 0.001. B : this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of <t>p38</t> <t>MAPK</t> with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C : PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± <t>SB203580</t> (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. ** P < 0.005. D : PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. * P < 0.05; ** P < 0.005. E : PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. *** P < 0.005. F : PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. *** P < 0.001.

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1) Product Images from "The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension"

Article Title: The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

doi: 10.1152/ajplung.00038.2015

Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A : cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. *** P < 0.001. B : this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C : PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. ** P < 0.005. D : PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. * P < 0.05; ** P < 0.005. E : PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. *** P < 0.005. F : PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. *** P < 0.001.

Figure Legend Snippet: Proproliferative fibroblast phenotype and paracrine smooth muscle proliferation mediated by p38 MAPK in fibroblasts from experimental models of pulmonary hypertension (PH). A : cells were isolated from the 2nd order division of the pulmonary artery and grown in normoxic culture. The effect of serum stimulation was observed on these cells. At baseline with no serum stimulation, the chronic hypoxic (CH) and monocrotaline (MCT) pulmonary artery fibroblast (PAF) had an increased proliferative rate compared with the PAF derived from normoxic wild-type rats. This was also seen with low dose 1% serum stimulation. The effect was lost at 5% serum stimulation, which may reflect cell contact inhibition. The thymidine incorporation assay was used to assess the proliferation of the cells, and the results presented are representative of 3 experiments with triplicate values in each experiment. Values are means ± SE. *** P < 0.001. B : this immunoblot shows that under normoxic conditions the PAFs derived from both CH and MCT animals show a constitutive activation of p38 MAPK with increased levels of p-p38 detected. Cells were from passage 3 and were quiescesed in serum free medium for 24 h before harvest. Immunoblot for phospho-p38 and total p38 MAPK was performed and representative of 3 experiments. C : PAFs derived from MCT animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to smooth muscle cells for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of pulmonary artery smooth muscle cells (PASMCs) while this effect was lost in the conditioned media from the PAFs, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. ** P < 0.005. D : PAFs derived from CH animals were isolated and then quiescesed for 24 h in serum free media. After 24 h, cells were stimulated with either 1% serum or some remained in serum free media ± SB203580 (p38 inhib). The conditioned media were aspirated from the wells after 24 h and passed through a cell sieve to give conditioned media from serum free cells ± SB203580 (SF cond med, SF cond med + p38 inhib) and 1% stimulated cells ± SB203580 (1% cond med, 1% cond med + p38 inhib). The conditioned media were added to normal PASMCs for 48 h before a proliferation assay was performed. Both SF cond med and 1% cond med resulted in increased proliferation of PASMCs while this effect was lost in the conditioned media from the PAF, which had been coincubated with SB203580. There was no effect of SB203580 directly on the PASMCs. Values are means ± SE. Data shown from 3 experiments. * P < 0.05; ** P < 0.005. E : PAF from normal animals were exposed to 48 h normoxia or hypoxia ± SB203580. The supernatant was collected and analyzed using cytokine array. The above are representative of triplicate samples from 3 different animals. Exposure times for each blot was the same. The results for IL-6 are shown and are presented as a relative density plot vs. the positive control blots. *** P < 0.005. F : PASMCs are quiescesed and then incubated with conditioned media derived from normoxic or hypoxic PAF. The hypoxic conditioned media induced PASMC proliferation. When both the conditioned media and anti-IL-6 were added to the PASMCs, there was an inhibition in the proliferative stimulus. Results are means ± SE from 3 replicates from 3 different animals. *** P < 0.001.

Techniques Used: Isolation, Derivative Assay, Inhibition, Thymidine Incorporation Assay, Western Blot, Activation Assay, Proliferation Assay, Positive Control, Incubation

Increased expression of p38 MAPK α-isoform in animal models of PH. A : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using the BCA method. Equal concentrations were then loaded on a gel and blotted for p38 MAPKα and β-actin for loading control. There are 3 wells for each condition. Immunoblot shown is best representative of 3 experiments using 3 different animals with each condition. B : densitometry of immunoblot in A . Values are mean arbitrary values from 3 immunoblots expressed relative to the value for β-actin. * P < 0.05 by ANOVA. C : lung sections (5 mm) were prepared from normal, CH, and MCT animals. Sections were stained for p38 MAPKα using 1:400 dilution. Magnification: ×20. Bar represents = 150 mm. Arrows identify blood vessels. D : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then used in a p38 MAPK activity assay using immunoprecipitation and the phosphorylation of activating transcription factor-2 (ATF-2) as a read out. Immunoblot shown is representative of 3 experiments using 3 different animals. E and F : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phosphorylated p38 MAPK and β-actin for loading control. Immunoblot shown is representative of 3 experiments using 3 different animals with each condition. Densitometry is shown for remaining blots. * P < 0.05.

Figure Legend Snippet: Increased expression of p38 MAPK α-isoform in animal models of PH. A : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using the BCA method. Equal concentrations were then loaded on a gel and blotted for p38 MAPKα and β-actin for loading control. There are 3 wells for each condition. Immunoblot shown is best representative of 3 experiments using 3 different animals with each condition. B : densitometry of immunoblot in A . Values are mean arbitrary values from 3 immunoblots expressed relative to the value for β-actin. * P < 0.05 by ANOVA. C : lung sections (5 mm) were prepared from normal, CH, and MCT animals. Sections were stained for p38 MAPKα using 1:400 dilution. Magnification: ×20. Bar represents = 150 mm. Arrows identify blood vessels. D : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then used in a p38 MAPK activity assay using immunoprecipitation and the phosphorylation of activating transcription factor-2 (ATF-2) as a read out. Immunoblot shown is representative of 3 experiments using 3 different animals. E and F : lungs from normal, CH, and MCT animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phosphorylated p38 MAPK and β-actin for loading control. Immunoblot shown is representative of 3 experiments using 3 different animals with each condition. Densitometry is shown for remaining blots. * P < 0.05.

Techniques Used: Expressing, Protein Concentration, Control, Western Blot, Staining, Activity Assay, Immunoprecipitation, Phospho-proteomics

PH is prevented by administration of SB203580, a p38 MAPKα inhibitor. A and B : animals were exposed to a hypobaric hypoxic environment for 2 wk. Some animals received daily injections of SB203580. Hemodynamics ( A ) and hematocrit ( B ) were measured after 2 wk. RVSP, right ventricular (RV) systolic pressure. Data represent mean values ± SE. Total animals n = 5–6 per group. C : hearts were isolated from the animals and the RV was dissected out from the left ventricle (LV) and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. The total RV weight was also plotted. Values are means ± SD; n = 5 per group. ** P < 0.05. D : noninvasive systemic blood pressure taken by tail-cuff measurement. E and F : rats were exposed to hypoxia ± SB203580. The lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five random fields per slide were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized and are presented as numbers per total number of vessels present in each field. Groups analyzed by ANOVA for overall change with posttest analysis; n = 5 animals. *** P < 0.001, for A – C and E .

Figure Legend Snippet: PH is prevented by administration of SB203580, a p38 MAPKα inhibitor. A and B : animals were exposed to a hypobaric hypoxic environment for 2 wk. Some animals received daily injections of SB203580. Hemodynamics ( A ) and hematocrit ( B ) were measured after 2 wk. RVSP, right ventricular (RV) systolic pressure. Data represent mean values ± SE. Total animals n = 5–6 per group. C : hearts were isolated from the animals and the RV was dissected out from the left ventricle (LV) and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. The total RV weight was also plotted. Values are means ± SD; n = 5 per group. ** P < 0.05. D : noninvasive systemic blood pressure taken by tail-cuff measurement. E and F : rats were exposed to hypoxia ± SB203580. The lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five random fields per slide were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized and are presented as numbers per total number of vessels present in each field. Groups analyzed by ANOVA for overall change with posttest analysis; n = 5 animals. *** P < 0.001, for A – C and E .

Techniques Used: Isolation, Staining

PH in 2 in vivo animal models is reversed by the administration of SB203580, a p38 MAPKα inhibitor. A : animals were exposed to a hypobaric hypoxic environment for 2 wk and then p38 MAPK inhibition was commenced. Hemodynamics and RVSP were measured after 4 wk. Data represent mean values ± SE. Total animals n = 5–6 per group. ** P < 0.01; *** P < 0.001, for normal relative to all other conditions. B : hearts were isolated form the animals and the RV dissected out from the LV and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. Values are means ± SE; n = 6–7. ** P < 0.01; *** P < 0.005. C : hematocrit ratio. D : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. ** P < 0.01; *** P < 0.001. E : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 7 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. ** P < 0.01; *** P < 0.001. F and G : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 6–7 per group. * P < 0.05; *** P < 0.001, for F . * P < 0.05; ** P < 0.01, for G . H : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. * P < 0.05; *** P < 0.001. I : lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 MCT control.

Figure Legend Snippet: PH in 2 in vivo animal models is reversed by the administration of SB203580, a p38 MAPKα inhibitor. A : animals were exposed to a hypobaric hypoxic environment for 2 wk and then p38 MAPK inhibition was commenced. Hemodynamics and RVSP were measured after 4 wk. Data represent mean values ± SE. Total animals n = 5–6 per group. ** P < 0.01; *** P < 0.001, for normal relative to all other conditions. B : hearts were isolated form the animals and the RV dissected out from the LV and septum. The ventricles were dry blotted and then weighed, and the ratio was calculated. Values are means ± SE; n = 6–7. ** P < 0.01; *** P < 0.005. C : hematocrit ratio. D : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. ** P < 0.01; *** P < 0.001. E : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 7 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. ** P < 0.01; *** P < 0.001. F and G : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 6–7 per group. * P < 0.05; *** P < 0.001, for F . * P < 0.05; ** P < 0.01, for G . H : the lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. * P < 0.05; *** P < 0.001. I : lungs were removed after experiment and sections (5 mm) cut. These were stained for α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups were analyzed by ANOVA for overall change with posttest analysis; n = 6 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 MCT control.

Techniques Used: In Vivo, Inhibition, Isolation, Staining, Control, Injection

PH in a reversal strategy in 2 in vivo animal models by administration of PH-797804, a more selective p38 MAPKα inhibitor. A and B : animals were exposed to CH and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RV hypertrophy (RVH) were measured after 4 wk. Data represent mean values ± SE. * P < 0.05; ** P < 0.01 for A . ** P < 0.01; *** P < 0.001, for B . C : the lungs were removed after experiment and sections (5 mm) were cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05 for complete muscularized group in drug-treated vs. day 14 hypoxic control. D : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. E and F : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 14–15 per group. *** P < 0.001 for E . * P < 0.05; ** P < 0.01 for F . G : the lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05, for complete muscularized group in drug-treated vs. day 14 hypoxic control. H : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for drug-treated vs. day 14 MCT control.

Figure Legend Snippet: PH in a reversal strategy in 2 in vivo animal models by administration of PH-797804, a more selective p38 MAPKα inhibitor. A and B : animals were exposed to CH and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RV hypertrophy (RVH) were measured after 4 wk. Data represent mean values ± SE. * P < 0.05; ** P < 0.01 for A . ** P < 0.01; *** P < 0.001, for B . C : the lungs were removed after experiment and sections (5 mm) were cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially, or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05 for complete muscularized group in drug-treated vs. day 14 hypoxic control. D : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized and the percentage of muscularized vessels calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for hypoxic drug-treated vs. day 14 hypoxic control. E and F : animals were injected with MCT and after 2 wk p38 MAPK inhibition was commenced with daily injections. Hemodynamics and RVH were measured after 4 wk. Data represent mean values ± SE. Total animals n = 14–15 per group. *** P < 0.001 for E . * P < 0.05; ** P < 0.01 for F . G : the lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin and the vessels <80 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as completely, partially or nonmuscularized. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. ** P < 0.01; ■ P < 0.05, for complete muscularized group in drug-treated vs. day 14 hypoxic control. H : lungs were removed after experiment and sections (5 mm) cut. These were stained with α-smooth muscle actin, and the vessels <100 mm were analyzed for degree of muscularization. Five to 10 random fields (×40) were analyzed with 3 slides per animal. The vessels were categorized as muscularized or nonmuscularized, and the percentage of muscularized vessels was calculated. Groups analyzed by ANOVA for overall change with posttest analysis; n = 10 animals. **** P < 0.0001; ■ P < 0.05 for drug-treated vs. day 14 MCT control.

Techniques Used: In Vivo, Inhibition, Staining, Control, Injection

p38 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B : supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. * P < 0.05 ** P < 0.005. C : PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D : PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. ** P < 0.01; *** P < 0.01, for C and D . E : PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. * P < 0.05; ** P < 0.005; *** P < 0.0001. F : PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. **** P < 0.0001. G : PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. ** P < 0.01. H and I : PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. *** P < 0.005; **** P < 0.001, for I .

Figure Legend Snippet: p38 MAPKα inhibition reduces interleukin (IL)-6 generation and signaling as a potential mechanism for beneficial effects in PH. A and B : supernatant from normoxic and hypoxic PAF ± SB203580 was analyzed using a quantitative ELISA to determine actual amounts released. Cells were growth arrested in serum free media for 24 h before hypoxic exposure. The values are mean ± SE values from triplicate wells for each sample and experiment repeated 3 times using cells from 3 animals. * P < 0.05 ** P < 0.005. C : PAF were exposed to hypoxia and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using quantitative (q)RT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. D : PAF were exposed to hypoxia in the presence of SB203580 and then RNA isolated after 48 h. The RNA was reverse transcribed into cDNA and then analyzed using qRT-PCR. The mRNA increased and peaked at 12 h of hypoxia. Values represent ratio of increase of IL-6 gene mRNA relative to the housekeeping gene of β-actin and calculated using comparative Ct method. Values are representative of triplicate samples from 3 experiments using 3 different animals. ** P < 0.01; *** P < 0.01, for C and D . E : PAF were isolated and incubated with IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-6) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are mean values ± SE and are representative of duplicate experiments performed on cells from 3 different animals. * P < 0.05; ** P < 0.005; *** P < 0.0001. F : PASMCs were exposed to IL-6 (100 ng/ml) ± soluble IL-6 receptor (sIL-r) and then using DNA synthesis as a marker of cell proliferation, the response was observed. Data are means ± and are representative of duplicate experiments performed on cells from 3 different animals. **** P < 0.0001. G : PASMCs were growth arrested for 24 h and then incubated with serum free media, IL-6 or IL-6 and anti-IL-6 antibody. Thymidine assay was used to quantify DNA synthesis, a measure of cell proliferation. Results are plotted as counts per million. Values are means ± SE and represent mean of 3 experiments on cells from same animal. A total of 3 different animals were used. ** P < 0.01. H and I : PASMCs were stimulated with 100 ng/ml IL-6 (+) or without (−) and the protein harvested at baseline, 15 min, 30 min, 1 h, and 4 h. The cell lysates were immunoblotted for phosphorylated STAT3 and total STAT3. Experiment was repeated 3 times; blots above are representative of those experiments. H shows densitometry from repeat blots. *** P < 0.005; **** P < 0.001, for I .

Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Incubation, DNA Synthesis, Marker

p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A : lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B : lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05 ** P < 0.01. C and D : lungs from CH 28-day controls and SB203580 -treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. ** P < 0.001. E : lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05; ns is not significant by ANOVA. F : PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. *** P < 0.005. G : PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. * P < 0.01; *** P < 0.005. H : fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. ** P < 0.01, *** P < 0.001.

Figure Legend Snippet: p38 MAPK inhibition in vivo leads to reduced IL-6 in experimental models of PH. A : lungs were isolated from CH animals in the prevention study with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. B : lungs were isolated from normal and CH animals after treatment in reversal strategy with SB203580 and homogenized. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Drug-treated and normal control animals are at 28 days. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05 ** P < 0.01. C and D : lungs from CH 28-day controls and SB203580 -treated hypoxic animals were harvested and homogenized with a cocktail of phosphatase and kinase inhibitors. The protein concentration was quantified using BCA method. Equal concentrations were then loaded on a gel and blotted for phospho-STAT3 and total STAT3. Immunoblot shown is best representative of 3 experiments using lungs from 3 different animals with each condition. Densitometry is shown of other blots. ** P < 0.001. E : lungs were isolated from MCT and normal control animals and homogenized. Inhibitor used was SB203580. The protein concentration was normalized by protein concentration as per BCA method. ELISA was used to analyze for IL-6 levels in the lung tissue. Data shown are means ± SE from triplicate samples from 4/5 animals in each group. * P < 0.05; ns is not significant by ANOVA. F : PH-797804 reduces serum IL-6 in reversal of CH-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 11. *** P < 0.005. G : PH-797804 reduces serum IL-6 in reversal of MCT-induced PH. Serum was collected from animals at the time of cardiac puncture and stored at −80°C until analysis could be performed. ELISA for IL-6 was performed on serum samples. Values shown are means ± SE. Samples were analyzed in duplicate and total animal number n = 12. * P < 0.01; *** P < 0.005. H : fibroblasts undergo phenotypic switch back to normal after p38 MAPK inhibition. PAF were cultured from pulmonary arteries derived from normal, experimental models of PH (CH and MCT) and from animals after treatment with p38 MAPK inhibition PH-787904 for 2 wk. Cells were challenged with or without serum to assess proliferation; n = 3–4 per group. Experiment repeated 3 times. ** P < 0.01, *** P < 0.001.

Techniques Used: Inhibition, In Vivo, Isolation, Protein Concentration, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Cell Culture, Derivative Assay

Phospho-p38 MAPK and p38 MAPKα expression in explanted lungs from patients with idiopathic pulmonary arterial hypertension (IPAH). A : sections of 5 mm were taken. Then, normal control lung and IPAH lung are stained for phospho-p38 MAPK at dilution of 1:300. The isotype on IPAH lung is also shown. This dilution was optimally assessed for. Objective: ×20. Bar = 150 mm. B : high-power microscopy shows that there is strong staining for phospho-p38 MAPK in the intima, media, and the adventitia (arrows). Objective lens: ×20 and ×40. Bar = 50 mm. C : sections of control lung ( A ) and IPAH lung ( B ) were stained for p38 MAPKα at dilution of 1:300. Objective lens × 20. Bar = 150 mm. D : staining for p38 MAPKa showed increased cytosolic staining in the IPAH lung ( right ) compared with control lung ( left ). E : high-power view (×40) of staining with isotype and p38 MAPKα in a vessel in IPAH lung. This shows staining throughout the vessel layers but especially in adventitia and fibroblast cells (arrow). Bar = 50 mm. F : low-power view of a plexiform lesion. Staining for p38 MAPKα using 1:300 dilution. Objective lens × 20. Bar = 150 mm. G : histological scoring shows increased p38 MAPKα staining throughout the vascular wall. With the use of a well-validated histological scoring system (Allred), the vascular wall cells were scored for intensity of staining. The intensity multiplied by the number of vessels with that intensity determines the values. Values shown are from 5 random high-power fields from 2 slides. **** P < 0.0001 by ANOVA. ** P < 0.001 for individual IPAH vs. control columns.

Figure Legend Snippet: Phospho-p38 MAPK and p38 MAPKα expression in explanted lungs from patients with idiopathic pulmonary arterial hypertension (IPAH). A : sections of 5 mm were taken. Then, normal control lung and IPAH lung are stained for phospho-p38 MAPK at dilution of 1:300. The isotype on IPAH lung is also shown. This dilution was optimally assessed for. Objective: ×20. Bar = 150 mm. B : high-power microscopy shows that there is strong staining for phospho-p38 MAPK in the intima, media, and the adventitia (arrows). Objective lens: ×20 and ×40. Bar = 50 mm. C : sections of control lung ( A ) and IPAH lung ( B ) were stained for p38 MAPKα at dilution of 1:300. Objective lens × 20. Bar = 150 mm. D : staining for p38 MAPKa showed increased cytosolic staining in the IPAH lung ( right ) compared with control lung ( left ). E : high-power view (×40) of staining with isotype and p38 MAPKα in a vessel in IPAH lung. This shows staining throughout the vessel layers but especially in adventitia and fibroblast cells (arrow). Bar = 50 mm. F : low-power view of a plexiform lesion. Staining for p38 MAPKα using 1:300 dilution. Objective lens × 20. Bar = 150 mm. G : histological scoring shows increased p38 MAPKα staining throughout the vascular wall. With the use of a well-validated histological scoring system (Allred), the vascular wall cells were scored for intensity of staining. The intensity multiplied by the number of vessels with that intensity determines the values. Values shown are from 5 random high-power fields from 2 slides. **** P < 0.0001 by ANOVA. ** P < 0.001 for individual IPAH vs. control columns.

Techniques Used: Expressing, Control, Staining, Microscopy

Planar Chromatography:

Article Title: Downregulation of mitofusin2 disrupts vascular endothelial barrier function by modulating connexin43 redistribution
Article Snippet: Antibodies against the following proteins were used: mfn2 (1:1000; Abcam, Cambridge, UK); N-Cadherin (1:1000; Cell Signaling Technology, Danvers, MA, USA); ZO-1 (1:1000; Life Technologies, Waltham, MA, USA); Claudin-5 (1:1000; Thermo Fisher Scientific, Waltham, MA, USA); VE-Cadherin (1:500; Invitrogen, Waltham, MA, USA); CK1 (1:1000; Cell Signaling Technology, Danvers, MA, USA); phospho-(Ser)PKC (1:1000; Cell Signaling Technology, Danvers, MA, USA); ubiquitin (1:500; Thermo Fisher Scientific, Waltham, MA, USA); Cx43 (1:1000; Sigma-Aldrich, St. Louis, MO, USA); phospho-Cx43 at Ser262 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA); phospho-Cx43 at Ser282 (1:1000; Thermo Fisher Scientific, Waltham, MA, USA); phospho-Cx43 at Ser368 (1:500; Thermo Fisher Scientific, Waltham, MA, USA); ERK (1:1000; Pierce, Rockford, IL, USA); phospho-ERK (1:500; Thermo Fisher Scientific, Waltham, MA, USA); p38 (1:1500; SigmaAldrich, St. Louis, MO, USA); WGA-conjugated Alexa Fluor 488 (1:200; Thermo Fisher Scientific, Waltham, MA, USA); EEA1 (1:1000; Cell Signaling Technology, Danvers, MA, USA); and β-actin (1:5000; Sigma-Aldrich, St. Louis, MO, USA). .. Other compounds included cycloheximide (Selleck Chemicals, Houston, TX, USA); LPS (Escherichia coli 0111: B4; Sigma-Aldrich, St. Louis, MO, USA); carboxyl terminus 1 peptide (a-CT1; American Peptide Company, Sunnyvale, CA, USA); NPC-15437 (PKC inhibitor; Abcam, Cambridge, UK); D4474 (CK1 inhibitor; Abcam, Cambridge, UK); SB203580 (p38 inhibitor; Selleck Chemicals, Houston, TX, USA); U-73122 (PLC/ DAG inhibitor; MedChemExpress, Monmouth Junction, NJ, USA); brefeldin A (MedChemExpress, Monmouth Junction, NJ, USA); GeneArt CRISPR Nuclease Vector Kit (Invitrogen, Carlsbad, California, USA) and Mito Tracker Deep Red (1:50000; Thermo Fisher Scientific, Waltham, MA, USA). .. Mutant plasmids of Cx43 at site s368 (S368A, A368E) were obtained from ADDGENE.

CRISPR:

Plasmid Preparation:

Incubation:

Article Title: Sphingosine-1-phosphate receptor 1 enhances olfactory receptor 51E1-mediated inhibition of proliferation via Src/JNK signaling in prostate cancer cells
Article Snippet: Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Inc.). .. For pathway inhibitor experiments, cells were pre-treated for 1 h with the indicated pathway inhibitors (SQ-22536, 100 μM (cat. no. 568500; MilliporeSigma; PubChem ID 5270); SU6656, 10 μM (cat. no. 572635; MilliporeSigma; PubChem ID 5312137); dasatinib, 10 nM (cat. no. CDS023389; MilliporeSigma; PubChem ID 3062316); SP600125, 10 μM (cat. no. HY-12041; MilliporeSigma; PubChem ID NSC75890); SB203580, 10 μM (cat. no. S1076; Selleck Chemicals; PubChem ID 176155); U0126, 1 μM (cat. no. HY-12031A; MilliporeSigma; PubChem ID 3006531); AG490, 10 μM (cat. no. HY-12000; MilliporeSigma; PubChem ID 5328779), followed by incubation with or without NA for 48 h. Subsequently, 10 μl of CCK-8 reagent was added to each well and incubated for 4 h at 37°C. .. For pathway inhibitor experiments, cells were pre-treated for 1 h with the indicated pathway inhibitors (SQ-22536, 100 μM (cat. no. 568500; MilliporeSigma; PubChem ID 5270); SU6656, 10 μM (cat. no. 572635; MilliporeSigma; PubChem ID 5312137); dasatinib, 10 nM (cat. no. CDS023389; MilliporeSigma; PubChem ID 3062316); SP600125, 10 μM (cat. no. HY-12041; MilliporeSigma; PubChem ID NSC75890); SB203580, 10 μM (cat. no. S1076; Selleck Chemicals; PubChem ID 176155); U0126, 1 μM (cat. no. HY-12031A; MilliporeSigma; PubChem ID 3006531); AG490, 10 μM (cat. no. HY-12000; MilliporeSigma; PubChem ID 5328779), followed by incubation with or without NA for 48 h. Subsequently, 10 μl of CCK-8 reagent was added to each well and incubated for 4 h at 37°C.

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p38 mapk antagonist sb203580 (Selleck Chemicals)

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1) Product Images from "The reversal of pulmonary vascular remodeling through inhibition of p38 MAPK-alpha: a potential novel anti-inflammatory strategy in pulmonary hypertension"

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