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4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and <t>β</t> -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p
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1) Product Images from "Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1"

Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2018/2747018

4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and β -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p
Figure Legend Snippet: 4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and β -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p

Techniques Used: Expressing, MTT Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Quantitative RT-PCR

4-PG prevents LPS-induced acute lung injury and upregulates HO-1 expression. (a) The scheme depicts the experimental protocol used to assess the protective effect of pterostilbene 4′-glucoside (4-PG) and pterostilbene (PTER) on LPS-induced ALI. 10-week-old mice were injected with 4-PG (10 mg/kg, i.p.) and PTER (10 mg/kg, i.p.) for 4 days prior to intranasal administration of LPS (2.5 mg/kg) for 24 h. (b) Chemical structures of 4-PG and PTER. (c) Lung sections were stained with hematoxylin and eosin (H E) for morphological evaluation, and the representative lung sections of each group are shown. Scale bar = 100 μ m. (left). Quantitative analysis of histologic lung section by lung injury score for six experimental groups. The score generates the average of two independent investigators (right). (d, e) The mRNA expression of proinflammatory cytokines and chemokines (TNF- α , IL-6, IL-1 β , CXCL1, and CXCL2) in lung tissues was detected by RT-PCR. Furthermore, mRNA and protein levels of HO-1 were assessed by RT-PCR (f, left: HO-1mRNA levels, right: quantification of the relative band density) and Western blotting (g, left: HO-1 protein levels, right: quantification of the relative band density) from lung tissues, respectively. 18S and β -actin were used as internal controls. Data were expressed as mean ± SD ( n = 5 per group); ∗∗ p
Figure Legend Snippet: 4-PG prevents LPS-induced acute lung injury and upregulates HO-1 expression. (a) The scheme depicts the experimental protocol used to assess the protective effect of pterostilbene 4′-glucoside (4-PG) and pterostilbene (PTER) on LPS-induced ALI. 10-week-old mice were injected with 4-PG (10 mg/kg, i.p.) and PTER (10 mg/kg, i.p.) for 4 days prior to intranasal administration of LPS (2.5 mg/kg) for 24 h. (b) Chemical structures of 4-PG and PTER. (c) Lung sections were stained with hematoxylin and eosin (H E) for morphological evaluation, and the representative lung sections of each group are shown. Scale bar = 100 μ m. (left). Quantitative analysis of histologic lung section by lung injury score for six experimental groups. The score generates the average of two independent investigators (right). (d, e) The mRNA expression of proinflammatory cytokines and chemokines (TNF- α , IL-6, IL-1 β , CXCL1, and CXCL2) in lung tissues was detected by RT-PCR. Furthermore, mRNA and protein levels of HO-1 were assessed by RT-PCR (f, left: HO-1mRNA levels, right: quantification of the relative band density) and Western blotting (g, left: HO-1 protein levels, right: quantification of the relative band density) from lung tissues, respectively. 18S and β -actin were used as internal controls. Data were expressed as mean ± SD ( n = 5 per group); ∗∗ p

Techniques Used: Expressing, Mouse Assay, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

2) Product Images from "Dietary Restriction Promotes Vessel Maturation in a Mouse Astrocytoma"

Article Title: Dietary Restriction Promotes Vessel Maturation in a Mouse Astrocytoma

Journal: Journal of Oncology

doi: 10.1155/2012/264039

Dietary restriction increases α -SMA and reduces Factor VIII expression in the CT-2A astrocytoma. The histograms show the average relative expression of the indicated protein normalized to β -actin based on Western blot analysis. Equal amounts of protein were loaded into each lane of the Western blot (25 μ g). Other conditions were as described in Section 2 . Values are expressed as normalized means of three to four independent tissue samples per group ± SEM. The value is significantly different in the tumors of DR-fed mice than in the tumors of AL-fed mice: * P
Figure Legend Snippet: Dietary restriction increases α -SMA and reduces Factor VIII expression in the CT-2A astrocytoma. The histograms show the average relative expression of the indicated protein normalized to β -actin based on Western blot analysis. Equal amounts of protein were loaded into each lane of the Western blot (25 μ g). Other conditions were as described in Section 2 . Values are expressed as normalized means of three to four independent tissue samples per group ± SEM. The value is significantly different in the tumors of DR-fed mice than in the tumors of AL-fed mice: * P

Techniques Used: Expressing, Western Blot, Mouse Assay

Dietary restriction reduces PDGF-R β and VEGF-R2 association in the CT-2A astrocytoma. (a) Confocal analysis of CT-2A tumor tissue double stained for VEGF-R2 (green) and PDGF-R β (red). Results show that the colocolization (yellow) of VEGF-R2 and PDGF-R β is less in the tumors of DR-fed mice than in the tumors of AL-fed mice. White arrow indicates colocolization. All other conditions are as described in Section 2 . (b) The histogram shows that the average relative expression of PDGF-R β normalized to β -actin based on Western blot analysis is similar in the tumors of DR-fed and AL-fed mice. Equal amounts of protein were loaded into each lane (25 μ g), and the other conditions were as described in Section 2 . Values are expressed as normalized means of three to four independent tissue samples per group ± SEM. There is no significant difference between values of DR-fed and AL-fed mice, Student's t- test. Two representative samples are shown for each tissue type.
Figure Legend Snippet: Dietary restriction reduces PDGF-R β and VEGF-R2 association in the CT-2A astrocytoma. (a) Confocal analysis of CT-2A tumor tissue double stained for VEGF-R2 (green) and PDGF-R β (red). Results show that the colocolization (yellow) of VEGF-R2 and PDGF-R β is less in the tumors of DR-fed mice than in the tumors of AL-fed mice. White arrow indicates colocolization. All other conditions are as described in Section 2 . (b) The histogram shows that the average relative expression of PDGF-R β normalized to β -actin based on Western blot analysis is similar in the tumors of DR-fed and AL-fed mice. Equal amounts of protein were loaded into each lane (25 μ g), and the other conditions were as described in Section 2 . Values are expressed as normalized means of three to four independent tissue samples per group ± SEM. There is no significant difference between values of DR-fed and AL-fed mice, Student's t- test. Two representative samples are shown for each tissue type.

Techniques Used: Staining, Mouse Assay, Expressing, Western Blot

3) Product Images from "Protective effects of biochanin A on articular cartilage: in vitro and in vivo studies"

Article Title: Protective effects of biochanin A on articular cartilage: in vitro and in vivo studies

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/1472-6882-14-444

Effects of biochanin A on protein expression of MMP-1,-3, −13, and TIMP-1 in interleukin-1β (IL-1β)–induced chondrocytes. Chondrocytes were incubated for 24 hours with IL-1β or a combination of biochanin A and IL-1β. Protein levels of MMP-1, −3, −13, and TIMP-1 were determined by Western blotting. β-actin was used as a loading control in the Western blotting.
Figure Legend Snippet: Effects of biochanin A on protein expression of MMP-1,-3, −13, and TIMP-1 in interleukin-1β (IL-1β)–induced chondrocytes. Chondrocytes were incubated for 24 hours with IL-1β or a combination of biochanin A and IL-1β. Protein levels of MMP-1, −3, −13, and TIMP-1 were determined by Western blotting. β-actin was used as a loading control in the Western blotting.

Techniques Used: Expressing, Incubation, Western Blot

4) Product Images from "Rhein Induces Apoptosis in Human Breast Cancer Cells"

Article Title: Rhein Induces Apoptosis in Human Breast Cancer Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/952504

Western blotting analysis of caspase 9 in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anticaspase-9 and anti- β actin antibodies.
Figure Legend Snippet: Western blotting analysis of caspase 9 in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anticaspase-9 and anti- β actin antibodies.

Techniques Used: Western Blot, Incubation

Western blotting analysis of p21 expression in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anti-p21 and anti- β actin antibodies.
Figure Legend Snippet: Western blotting analysis of p21 expression in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anti-p21 and anti- β actin antibodies.

Techniques Used: Western Blot, Expressing, Incubation

Expression of HER2/ neu in human breast cancer cells. Vector control MCF-7 cells (MCF-7/VEC) and HER2-overexpressing MCF-7 cells (MCF-7/HER2) were harvested, and cell lysates were analyzed by Western blotting with anti-HER2 and anti- β actin antibodies.
Figure Legend Snippet: Expression of HER2/ neu in human breast cancer cells. Vector control MCF-7 cells (MCF-7/VEC) and HER2-overexpressing MCF-7 cells (MCF-7/HER2) were harvested, and cell lysates were analyzed by Western blotting with anti-HER2 and anti- β actin antibodies.

Techniques Used: Expressing, Plasmid Preparation, Western Blot

Western blotting analysis of ASK1 expression in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anti-ASK1 and anti- β actin antibodies.
Figure Legend Snippet: Western blotting analysis of ASK1 expression in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anti-ASK1 and anti- β actin antibodies.

Techniques Used: Western Blot, Expressing, Incubation

5) Product Images from "Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival"

Article Title: Etoposide-induced protein 2.4 functions as a regulator of the calcium ATPase and protects pancreatic β-cell survival

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.002399

Loss of Ei24 impairs glucose homeostasis. A , expression of Ei24 in islets from ob/ob mice, GK rats at 4 months of age, and C57BL/6 mice fed an HFD for 2 weeks and 8 weeks. β-Actin served as the loading control. CD , chow diet. B , total RNA was prepared from islets of WT and KO mice at 8 weeks of age. The transcription levels of Ei24 mRNA are normalized to β-actin mRNA. The results are representative of three individual experiments. C , Western blotting of Ei24 protein from isolated islets (300 islets/group) from WT and KO mice at 12 weeks of age. β-Actin served as the loading control. D , weight curves for WT and KO mice. The mean ± S.D. ( error bars ) of 15 mice is shown. E , concentration of the basic glucose curve for WT and KO mice. The mean ± S.D. of 10 mice is shown. F and G , glucose tolerance test ( GTT ) results of 10-week-old male ( F ) and female mice ( G ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). H and I , ITT results of 10-week-old male ( H ) and female mice ( I ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). J , in vivo GSIS detection in WT and KO mice. The results are representative of five replicates for each group. K , in vitro GSIS from isolated islets (70/group) of WT and KO mice by a fast digital perfusion system. The results are representative of three individual experiments. Data are expressed as the mean ± S.D. *, p
Figure Legend Snippet: Loss of Ei24 impairs glucose homeostasis. A , expression of Ei24 in islets from ob/ob mice, GK rats at 4 months of age, and C57BL/6 mice fed an HFD for 2 weeks and 8 weeks. β-Actin served as the loading control. CD , chow diet. B , total RNA was prepared from islets of WT and KO mice at 8 weeks of age. The transcription levels of Ei24 mRNA are normalized to β-actin mRNA. The results are representative of three individual experiments. C , Western blotting of Ei24 protein from isolated islets (300 islets/group) from WT and KO mice at 12 weeks of age. β-Actin served as the loading control. D , weight curves for WT and KO mice. The mean ± S.D. ( error bars ) of 15 mice is shown. E , concentration of the basic glucose curve for WT and KO mice. The mean ± S.D. of 10 mice is shown. F and G , glucose tolerance test ( GTT ) results of 10-week-old male ( F ) and female mice ( G ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). H and I , ITT results of 10-week-old male ( H ) and female mice ( I ). Solid lines , WT mice ( n = 8); dashed lines , KO mice ( n = 8). J , in vivo GSIS detection in WT and KO mice. The results are representative of five replicates for each group. K , in vitro GSIS from isolated islets (70/group) of WT and KO mice by a fast digital perfusion system. The results are representative of three individual experiments. Data are expressed as the mean ± S.D. *, p

Techniques Used: Expressing, Mouse Assay, Western Blot, Isolation, Concentration Assay, In Vivo, In Vitro

Loss of Ei24 impairs AMPK activation and induces apoptotic cell death. A , ATP content of islets (40 islets/group), which were cultured in RPMI medium 1640 with 10% FBS from WT and KO mice at 8 weeks of age. Data were normalized to protein content. Data were obtained from three independent experiments. B , Western blotting for p-AMPK, t-AMPK, p-ACC, and t-ACC in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. C , Western blotting for p-CAMKK2 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. D , Western blotting for p-AMPK, t-AMPK, c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) with or without AICAR treatment from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. Data are expressed as the mean ± S.D. ( error bars ): WT versus KO (*, p
Figure Legend Snippet: Loss of Ei24 impairs AMPK activation and induces apoptotic cell death. A , ATP content of islets (40 islets/group), which were cultured in RPMI medium 1640 with 10% FBS from WT and KO mice at 8 weeks of age. Data were normalized to protein content. Data were obtained from three independent experiments. B , Western blotting for p-AMPK, t-AMPK, p-ACC, and t-ACC in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. C , Western blotting for p-CAMKK2 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. D , Western blotting for p-AMPK, t-AMPK, c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) with or without AICAR treatment from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. Data are expressed as the mean ± S.D. ( error bars ): WT versus KO (*, p

Techniques Used: Activation Assay, Cell Culture, Mouse Assay, Western Blot, Isolation

Loss of Ei24 causes apoptosis of pancreatic β cells. A , morphologies of islets. The islets from KO mice became uncompact and transparent compared with the WT islets. B , representative images of H E staining of islets from WT and KO mice at the age of 12 weeks. The degenerative β cells are indicated by black arrowheads. C , representative sections of islets stained for insulin ( red ) and nuclei ( blue ) from WT and KO mice at 12 weeks of age. Scale bars , 10 μm. D , density of β cells determined by counting the number of β cells in islets ( n = 40/group) from WT and KO mice. E , EM micrographs of WT and KO pancreatic β cells. The bottom panel in E shows enlargement of the boxed area in the top panel . The arrows indicate the dense core vesicles. F , the number of dense core vesicles ( DCV ) in pancreatic β cells was counted using Imaris software (cell numbers, n = 48/group). G , Western blotting for proinsulin in the isolated islets (30 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. H , Western blotting for insulin in the isolated islets (60 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. I , Western blotting for c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from five independent experiments. Data are expressed as the mean ± S.D. ( error bars ). *, p
Figure Legend Snippet: Loss of Ei24 causes apoptosis of pancreatic β cells. A , morphologies of islets. The islets from KO mice became uncompact and transparent compared with the WT islets. B , representative images of H E staining of islets from WT and KO mice at the age of 12 weeks. The degenerative β cells are indicated by black arrowheads. C , representative sections of islets stained for insulin ( red ) and nuclei ( blue ) from WT and KO mice at 12 weeks of age. Scale bars , 10 μm. D , density of β cells determined by counting the number of β cells in islets ( n = 40/group) from WT and KO mice. E , EM micrographs of WT and KO pancreatic β cells. The bottom panel in E shows enlargement of the boxed area in the top panel . The arrows indicate the dense core vesicles. F , the number of dense core vesicles ( DCV ) in pancreatic β cells was counted using Imaris software (cell numbers, n = 48/group). G , Western blotting for proinsulin in the isolated islets (30 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. H , Western blotting for insulin in the isolated islets (60 islets/group) from WT and KO mice at 8 weeks of age. β-Actin served as the loading control. Data were obtained from three independent experiments. I , Western blotting for c-PARP, t-PARP, c-caspase-3, and t-caspase-3 in the isolated islets (150 islets/group) from WT and KO mice at 8–10 weeks of age. β-Actin served as the loading control. Data were obtained from five independent experiments. Data are expressed as the mean ± S.D. ( error bars ). *, p

Techniques Used: Mouse Assay, Staining, Software, Western Blot, Isolation

6) Product Images from "Lipoxin A4 ameliorates alveolar fluid clearance disturbance in lipopolysaccharide-induced lung injury via aquaporin 5 and MAPK signaling pathway"

Article Title: Lipoxin A4 ameliorates alveolar fluid clearance disturbance in lipopolysaccharide-induced lung injury via aquaporin 5 and MAPK signaling pathway

Journal: Journal of Thoracic Disease

doi: 10.21037/jtd.2019.08.86

Effect of LXA4 on the expression of aquaporin 5(AQP5) in LPS-induced acute lung injury. (A,B,C,D) The immunohistochemistry staining was performed to detect AQP5 protein expression (brown) in lung tissue section from each group. The representative images of each group were shown. (E) The protein expression of AQP5 in lung tissue from each group measured by western blot analysis. (F) The densitometry values were normalized to β-actin respectively for AQP5 protein. All data represent the means ± SEM of three independent experiments in triplicate. Statistical analysis was performed by one-way ANOVA and Newman-Keuls post hoc test; compared with control group, # P
Figure Legend Snippet: Effect of LXA4 on the expression of aquaporin 5(AQP5) in LPS-induced acute lung injury. (A,B,C,D) The immunohistochemistry staining was performed to detect AQP5 protein expression (brown) in lung tissue section from each group. The representative images of each group were shown. (E) The protein expression of AQP5 in lung tissue from each group measured by western blot analysis. (F) The densitometry values were normalized to β-actin respectively for AQP5 protein. All data represent the means ± SEM of three independent experiments in triplicate. Statistical analysis was performed by one-way ANOVA and Newman-Keuls post hoc test; compared with control group, # P

Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot

7) Product Images from "Acetylation: A Lysine Modification with Neuroprotective Effects in Ischemic Retinal Degeneration"

Article Title: Acetylation: A Lysine Modification with Neuroprotective Effects in Ischemic Retinal Degeneration

Journal: Experimental eye research

doi: 10.1016/j.exer.2014.07.012

Effect of HDAC inhibition on acetylation levels of histone-H3 and cleaved caspase-3, 24 hours after ischemia induction, using two structurally distinct HDAC inhibitors, TSA (2.5 mg/kg; i.p) and VPA (100 mg/kg; i.p.). ( A ) Ratio of acetyl histone-H3/β-actin
Figure Legend Snippet: Effect of HDAC inhibition on acetylation levels of histone-H3 and cleaved caspase-3, 24 hours after ischemia induction, using two structurally distinct HDAC inhibitors, TSA (2.5 mg/kg; i.p) and VPA (100 mg/kg; i.p.). ( A ) Ratio of acetyl histone-H3/β-actin

Techniques Used: Inhibition

Effect of 45 minutes of acute ischemia on retinal cleaved caspase-3 levels 2, 4, 8, and 24 hours from ischemia initiation. ( A ) Representative Western blot of retinal lysates for cleaved caspase-3 and β-actin at 2, 4, 8, and 24 hours after initiation
Figure Legend Snippet: Effect of 45 minutes of acute ischemia on retinal cleaved caspase-3 levels 2, 4, 8, and 24 hours from ischemia initiation. ( A ) Representative Western blot of retinal lysates for cleaved caspase-3 and β-actin at 2, 4, 8, and 24 hours after initiation

Techniques Used: Western Blot

8) Product Images from "Molecular Determinants of Sensitivity or Resistance of Cancer Cells Toward Sanguinarine"

Article Title: Molecular Determinants of Sensitivity or Resistance of Cancer Cells Toward Sanguinarine

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00136

Western blot analysis of the effect of Sanguinarine on CEM/ADR5000 leukemia cells. Evaluation of the P-gp, NFκB, and IκBα expressions. β-actin was used as loading control. Bands were normalized to β-actin in order to obtain numerical values (Mean ± SD ).
Figure Legend Snippet: Western blot analysis of the effect of Sanguinarine on CEM/ADR5000 leukemia cells. Evaluation of the P-gp, NFκB, and IκBα expressions. β-actin was used as loading control. Bands were normalized to β-actin in order to obtain numerical values (Mean ± SD ).

Techniques Used: Western Blot

9) Product Images from "Synergistic Inhibition of Thalidomide and Icotinib on Human Non-Small Cell Lung Carcinomas Through ERK and AKT Signaling"

Article Title: Synergistic Inhibition of Thalidomide and Icotinib on Human Non-Small Cell Lung Carcinomas Through ERK and AKT Signaling

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.909977

Thalidomide and icotinib inhibited the EGFR and VEGF-R2 pathways in PC9 cells. Western blot analysis showed the expression of pEGFR, pVEGF-R2, AKT, pAKT, ERK, and pERK in PC9 cells in the control group, thalidomide group, icotinib group, and combination group. β-actin was used as a loading control. Data are represented as the means ±SD, n=3, * P
Figure Legend Snippet: Thalidomide and icotinib inhibited the EGFR and VEGF-R2 pathways in PC9 cells. Western blot analysis showed the expression of pEGFR, pVEGF-R2, AKT, pAKT, ERK, and pERK in PC9 cells in the control group, thalidomide group, icotinib group, and combination group. β-actin was used as a loading control. Data are represented as the means ±SD, n=3, * P

Techniques Used: Western Blot, Expressing

10) Product Images from "IRE-1α promotes viral infection by conferring resistance to apoptosis"

Article Title: IRE-1α promotes viral infection by conferring resistance to apoptosis

Journal: Science signaling

doi: 10.1126/scisignal.aai7814

IRE1α mediates reduction in pro-apoptotic miR-125a ( A ) BMDMs were cultured from Xbp1 flox/flox ESR Cre+ ( Xbp1 Δ), or Cre- littermate (WT) mice in the presence of tamoxifen. Volcano plot demonstrating distribution of microRNAs between WT and Xbp1 Δ BMDMs measured using the NanoString nCounter assay. Data are from one experiment with quadruplicates. ( B ) BMDMs were cultured from Xbp1 flox/flox ESR Cre+ ( Xbp1 Δ), Xbp1 flox/flox Ern1 flox/flox ESR Cre+ ( Xbp1 Δ Ire1α Δ) or Cre- littermate (WT) mice in the presence of tamoxifen. The relative abundance of Bcl-xL, Mcl-1 and β-actin in the cell lysates was determined by Western blotting and densitometry. The ratio of Bcl-xL or Mcl-1 to β-actin is shown, normalized to WT. Data are means ± SD from three independent experiments. a.u., arbitrary units.( C and D ) WT and Xbp1 −/− MEFs were transfected with negative control microRNA mimetic (miR-ctrl) or miR-125a mimetic. Cells were left untreated (mock) or treated with staurosporine. Seven hours later, caspase-3 activity was assessed by measuring fluorometric substrate cleavage, and is shown relative to untreated WT cells (C). Twenty-four hours after treatment, viability was assessed by measuring MTS reduction (D). Data are means ± SD of three replicates and are representative of two experiments. ( E ) MicroRNA transfected MEFs were infected with VSV-GFP for 24 hours. Cell death was then assessed with a membrane impermeant, amine-reactive fluorescent dye, which was measured by flow cytometry. The extent of infection was determined by measuring the relative abundance of GFP by flow cytometry. Data are from one experiment representative of two independent experiments. * P
Figure Legend Snippet: IRE1α mediates reduction in pro-apoptotic miR-125a ( A ) BMDMs were cultured from Xbp1 flox/flox ESR Cre+ ( Xbp1 Δ), or Cre- littermate (WT) mice in the presence of tamoxifen. Volcano plot demonstrating distribution of microRNAs between WT and Xbp1 Δ BMDMs measured using the NanoString nCounter assay. Data are from one experiment with quadruplicates. ( B ) BMDMs were cultured from Xbp1 flox/flox ESR Cre+ ( Xbp1 Δ), Xbp1 flox/flox Ern1 flox/flox ESR Cre+ ( Xbp1 Δ Ire1α Δ) or Cre- littermate (WT) mice in the presence of tamoxifen. The relative abundance of Bcl-xL, Mcl-1 and β-actin in the cell lysates was determined by Western blotting and densitometry. The ratio of Bcl-xL or Mcl-1 to β-actin is shown, normalized to WT. Data are means ± SD from three independent experiments. a.u., arbitrary units.( C and D ) WT and Xbp1 −/− MEFs were transfected with negative control microRNA mimetic (miR-ctrl) or miR-125a mimetic. Cells were left untreated (mock) or treated with staurosporine. Seven hours later, caspase-3 activity was assessed by measuring fluorometric substrate cleavage, and is shown relative to untreated WT cells (C). Twenty-four hours after treatment, viability was assessed by measuring MTS reduction (D). Data are means ± SD of three replicates and are representative of two experiments. ( E ) MicroRNA transfected MEFs were infected with VSV-GFP for 24 hours. Cell death was then assessed with a membrane impermeant, amine-reactive fluorescent dye, which was measured by flow cytometry. The extent of infection was determined by measuring the relative abundance of GFP by flow cytometry. Data are from one experiment representative of two independent experiments. * P

Techniques Used: Cell Culture, Electron Paramagnetic Resonance, Mouse Assay, Western Blot, Transfection, Negative Control, Activity Assay, Infection, Flow Cytometry, Cytometry

11) Product Images from "Antimicrobial Peptide Epinecidin-1 Modulates MyD88 Protein Levels via the Proteasome Degradation Pathway"

Article Title: Antimicrobial Peptide Epinecidin-1 Modulates MyD88 Protein Levels via the Proteasome Degradation Pathway

Journal: Marine Drugs

doi: 10.3390/md15110362

Epinecidin-1 attenuates LPS-induced upregulation of A20, IRAK-M, and SOCS-1. ( A ) Cells were preincubated with Epi (6 μg/mL) for 30 min, followed by treatment with LPS (100 ng/mL) for additional 30 min. Cell lysates were collected after stimulation with Epi/LPS and probed with indicated antibodies. β-actin served as a loading control. Quantification of results for A20 ( B ), IRAK-M ( C ), and SOCS-1 ( D ). * p
Figure Legend Snippet: Epinecidin-1 attenuates LPS-induced upregulation of A20, IRAK-M, and SOCS-1. ( A ) Cells were preincubated with Epi (6 μg/mL) for 30 min, followed by treatment with LPS (100 ng/mL) for additional 30 min. Cell lysates were collected after stimulation with Epi/LPS and probed with indicated antibodies. β-actin served as a loading control. Quantification of results for A20 ( B ), IRAK-M ( C ), and SOCS-1 ( D ). * p

Techniques Used:

LPS-induced upregulation of MyD88 is abolished by epinecidin-1. Cells were preincubated with Epi (6 μg/mL) for 30 min followed by treatment with 100 ng/mL LPS ( A ) or 5 μg/mL lipoteichoic acid (LTA; C ) for an additional 30 min. Cell lysates were collected and probed with MyD88 and β-actin antibodies. ( B , D ) Quantification of MyD88 level. * p
Figure Legend Snippet: LPS-induced upregulation of MyD88 is abolished by epinecidin-1. Cells were preincubated with Epi (6 μg/mL) for 30 min followed by treatment with 100 ng/mL LPS ( A ) or 5 μg/mL lipoteichoic acid (LTA; C ) for an additional 30 min. Cell lysates were collected and probed with MyD88 and β-actin antibodies. ( B , D ) Quantification of MyD88 level. * p

Techniques Used:

Epinecidin-1 suppresses protein levels of MyD88. ( A ) Cells were treated with LPS (100 ng/mL) for 0, 5, 15, 30, 60, or 180 min, and lysates were immunoblotted with MyD88 and β-actin. ( B ) Quantification of MyD88 protein levels. ( C ) Cells were treated with 6 μg/mL Epi for 0, 5, 10, 15, 20, or 30 min. Cell lysates were harvested after stimulation and immunoblotted with indicated antibodies. Quantification of TLR4 ( D ) and MyD88 ( E ) levels. * p
Figure Legend Snippet: Epinecidin-1 suppresses protein levels of MyD88. ( A ) Cells were treated with LPS (100 ng/mL) for 0, 5, 15, 30, 60, or 180 min, and lysates were immunoblotted with MyD88 and β-actin. ( B ) Quantification of MyD88 protein levels. ( C ) Cells were treated with 6 μg/mL Epi for 0, 5, 10, 15, 20, or 30 min. Cell lysates were harvested after stimulation and immunoblotted with indicated antibodies. Quantification of TLR4 ( D ) and MyD88 ( E ) levels. * p

Techniques Used:

Epinecidin-1 alone does not affect protein levels of A20, IRAK-M, and SOCS-1. ( A , B ) Raw264.7 cells were incubated with epinecidin-1 (Epi; 6 μg/mL) for short-term (0–30 min) or long-term (0–24 h) exposure. After treatment, cell lysates were collected and immunoblotted with indicated antibodies. β-actin served as a loading control.
Figure Legend Snippet: Epinecidin-1 alone does not affect protein levels of A20, IRAK-M, and SOCS-1. ( A , B ) Raw264.7 cells were incubated with epinecidin-1 (Epi; 6 μg/mL) for short-term (0–30 min) or long-term (0–24 h) exposure. After treatment, cell lysates were collected and immunoblotted with indicated antibodies. β-actin served as a loading control.

Techniques Used: Incubation

Lipopolysaccharide (LPS) elevates A20, IRAK-M and SOCS-1. ( A ) Raw264.7 cells were treated with LPS (100 ng/mL) for 0, 5, 15, 30, 60, or 180 min. Cell lysates were immunoblotted with A20, IRAK-M, and SOCS-1 antibodies, and band intensities were analyzed with ImageJ. Quantification of results for A20 ( B ), IRAK-M ( C ), and SOCS-1 ( D ). β-actin served as a loading control. * p
Figure Legend Snippet: Lipopolysaccharide (LPS) elevates A20, IRAK-M and SOCS-1. ( A ) Raw264.7 cells were treated with LPS (100 ng/mL) for 0, 5, 15, 30, 60, or 180 min. Cell lysates were immunoblotted with A20, IRAK-M, and SOCS-1 antibodies, and band intensities were analyzed with ImageJ. Quantification of results for A20 ( B ), IRAK-M ( C ), and SOCS-1 ( D ). β-actin served as a loading control. * p

Techniques Used:

MG132 and Heclin abolish epinecidin-1-mediated degradation of MyD88. Cells were preincubated with 10 μM MG132 ( A ) or 7 μM Heclin ( C ) for 1 h followed by treatment with 6 μg/mL Epi ( A ) or for an additional 30 min. Cell lysates were collected and probed with MyD88 and β-actin antibodies. ( B , D ) Quantification of MyD88 levels. Vehicle: 0.5% DMSO. * p
Figure Legend Snippet: MG132 and Heclin abolish epinecidin-1-mediated degradation of MyD88. Cells were preincubated with 10 μM MG132 ( A ) or 7 μM Heclin ( C ) for 1 h followed by treatment with 6 μg/mL Epi ( A ) or for an additional 30 min. Cell lysates were collected and probed with MyD88 and β-actin antibodies. ( B , D ) Quantification of MyD88 levels. Vehicle: 0.5% DMSO. * p

Techniques Used:

12) Product Images from "Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System"

Article Title: Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System

Journal: PLoS Currents

doi: 10.1371/currents.hd.0242d2e7ad72225efa72f6964589369a

(a) To identify 1C2-reactive full-length HTT protein indicative of homologous recombination a western blot assay was utilized. Total HTT (MAB2166) and β-actin are also shown. (b) Quantitated levels of 1C2 signal normalized to β-actin are significantly increased in cells treated with WT Cas9 and HTT specific gRNAs realtive to cells treated with a gRNA targeting a different (AAVS1) locus in the genome. (c) Cas9 D10A show significant increase in HTT gRNA2-mediated recombination relative to control, AAVS1 gRNA assisted cells. (d) 1C2 western blot analysis of individual clones (1-12) transfected with Cas9 D10A, HTT gRNA1, and donor compared with control gRNA and untransfected controls.
Figure Legend Snippet: (a) To identify 1C2-reactive full-length HTT protein indicative of homologous recombination a western blot assay was utilized. Total HTT (MAB2166) and β-actin are also shown. (b) Quantitated levels of 1C2 signal normalized to β-actin are significantly increased in cells treated with WT Cas9 and HTT specific gRNAs realtive to cells treated with a gRNA targeting a different (AAVS1) locus in the genome. (c) Cas9 D10A show significant increase in HTT gRNA2-mediated recombination relative to control, AAVS1 gRNA assisted cells. (d) 1C2 western blot analysis of individual clones (1-12) transfected with Cas9 D10A, HTT gRNA1, and donor compared with control gRNA and untransfected controls.

Techniques Used: Western Blot, Homologous Recombination, Clone Assay, Transfection

13) Product Images from "Molecular Determinants of Sensitivity or Resistance of Cancer Cells Toward Sanguinarine"

Article Title: Molecular Determinants of Sensitivity or Resistance of Cancer Cells Toward Sanguinarine

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00136

Western blot analysis of the effect of Sanguinarine on CEM/ADR5000 leukemia cells. Evaluation of the P-gp, NFκB, and IκBα expressions. β-actin was used as loading control. Bands were normalized to β-actin in order to obtain numerical values (Mean ± SD ).
Figure Legend Snippet: Western blot analysis of the effect of Sanguinarine on CEM/ADR5000 leukemia cells. Evaluation of the P-gp, NFκB, and IκBα expressions. β-actin was used as loading control. Bands were normalized to β-actin in order to obtain numerical values (Mean ± SD ).

Techniques Used: Western Blot

14) Product Images from "Upregulation of SALL4 by EGFR activation regulates the stemness of CD44-positive lung cancer"

Article Title: Upregulation of SALL4 by EGFR activation regulates the stemness of CD44-positive lung cancer

Journal: Oncogenesis

doi: 10.1038/s41389-018-0045-7

EGFR signaling activated by the ligand EGF stimulation and EGFR L858R point mutation induced the expression of SALL4 through ERK1/2 pathway. a qRT-PCR analysis of SALL4 expression in Beas-2B cells treated with different dose of EGF (0, 10, 20, and 40 ng/ml) for 30 min and the protein level of SALL4 was detected by Western blotting. b qRT-PCR and western blotting analysis of SALL4 expression in Bease-2B cells infected with Lenti-control and -EGFR L858R. c qRT-PCR and Western blot analysis of SALL4 expression in PC9 treated with different doses of Gefitinib (0, 5, 10, 100 nM) for 48 h respectively. d The protein level of SALL4, p-EGFR, p-ERK, ERK, p-AKT, and AKT in PC9 cells treated with Gefitinib (100, 500 nM) for 48 h, and β-actin was selected as a control. e The mRNA and protein level of SALL4 in PC9 cells, which were treated with 1 μM ERK inhibitor (SCH772984) and 2 μM AKT inhibitor (MK-22062HCL) for 72 h. Representative results from three independent experiments are shown (mean ± s.d.)
Figure Legend Snippet: EGFR signaling activated by the ligand EGF stimulation and EGFR L858R point mutation induced the expression of SALL4 through ERK1/2 pathway. a qRT-PCR analysis of SALL4 expression in Beas-2B cells treated with different dose of EGF (0, 10, 20, and 40 ng/ml) for 30 min and the protein level of SALL4 was detected by Western blotting. b qRT-PCR and western blotting analysis of SALL4 expression in Bease-2B cells infected with Lenti-control and -EGFR L858R. c qRT-PCR and Western blot analysis of SALL4 expression in PC9 treated with different doses of Gefitinib (0, 5, 10, 100 nM) for 48 h respectively. d The protein level of SALL4, p-EGFR, p-ERK, ERK, p-AKT, and AKT in PC9 cells treated with Gefitinib (100, 500 nM) for 48 h, and β-actin was selected as a control. e The mRNA and protein level of SALL4 in PC9 cells, which were treated with 1 μM ERK inhibitor (SCH772984) and 2 μM AKT inhibitor (MK-22062HCL) for 72 h. Representative results from three independent experiments are shown (mean ± s.d.)

Techniques Used: Mutagenesis, Expressing, Quantitative RT-PCR, Western Blot, Infection

15) Product Images from "Rapamycin-independent IGF2 expression in Tsc2-null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells"

Article Title: Rapamycin-independent IGF2 expression in Tsc2-null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0197105

IGF2 expression in LAM lungs and Tsc2 -/- MEFs. Representative images of IHC analysis show IGF2 expression in (A) LAM lesion and (B) LAM cluster detected with specific antibodies. Non-immune IgG was used as a control (see S1 Fig ). Igf2 expression in Tsc2 -/- MEFs was detected by (C) qPCR (D) Western blot and (E) ELISA. (F) Tsc2 -/- MEFs were transfected with 50nM Igf2 siRNA (siIGF2) or NT siRNA (siNT) for 48 hrs. Decreased levels of Igf2 protein expression were confirmed via western blot with β-actin as an internal loading control. (G) Decreased Igf2 protein secretion was confirmed via ELISA. Igf2 knockdown resulted in (H) increased cleaved caspase-3 levels as measured via immunocytostaining and flow for Alexa Fluor® 488 -Cleaved Caspase 3 where the population of positively stained MEFs was normalized to the control population, and (J) decreased cell viability as assessed by 0.4% Trypan Blue staining normalized to the control cell viability. Student's t-tests were used to determine the statistical significance of the differences, and p -values reflect a sample size of 3 replicates.
Figure Legend Snippet: IGF2 expression in LAM lungs and Tsc2 -/- MEFs. Representative images of IHC analysis show IGF2 expression in (A) LAM lesion and (B) LAM cluster detected with specific antibodies. Non-immune IgG was used as a control (see S1 Fig ). Igf2 expression in Tsc2 -/- MEFs was detected by (C) qPCR (D) Western blot and (E) ELISA. (F) Tsc2 -/- MEFs were transfected with 50nM Igf2 siRNA (siIGF2) or NT siRNA (siNT) for 48 hrs. Decreased levels of Igf2 protein expression were confirmed via western blot with β-actin as an internal loading control. (G) Decreased Igf2 protein secretion was confirmed via ELISA. Igf2 knockdown resulted in (H) increased cleaved caspase-3 levels as measured via immunocytostaining and flow for Alexa Fluor® 488 -Cleaved Caspase 3 where the population of positively stained MEFs was normalized to the control population, and (J) decreased cell viability as assessed by 0.4% Trypan Blue staining normalized to the control cell viability. Student's t-tests were used to determine the statistical significance of the differences, and p -values reflect a sample size of 3 replicates.

Techniques Used: Expressing, Laser Capture Microdissection, Immunohistochemistry, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Staining

16) Product Images from "Porcine circovirus type 2 increases interleukin-1beta and interleukin-10 production via the MyD88–NF-kappa B signaling pathway in porcine alveolar macrophages in vitro"

Article Title: Porcine circovirus type 2 increases interleukin-1beta and interleukin-10 production via the MyD88–NF-kappa B signaling pathway in porcine alveolar macrophages in vitro

Journal: Journal of Veterinary Science

doi: 10.4142/jvs.2017.18.2.183

Changes in MyD88 protein expression after porcine circovirus type 2 (PCV2) infection. Western blotting was used to measure the expression of MyD88 protein in the cytoplasm at 6, 12, 24, and 48 h in control, PCV2, MyD88 siRNA, and PCV2 +MyD88 small interfering (si)RNA groups. Expression of β-actin was used as a positive control; * p
Figure Legend Snippet: Changes in MyD88 protein expression after porcine circovirus type 2 (PCV2) infection. Western blotting was used to measure the expression of MyD88 protein in the cytoplasm at 6, 12, 24, and 48 h in control, PCV2, MyD88 siRNA, and PCV2 +MyD88 small interfering (si)RNA groups. Expression of β-actin was used as a positive control; * p

Techniques Used: Expressing, Infection, Western Blot, Positive Control

Changes in nuclear factor kappa B (NF-κB) p65 protein expression in the cytoplasm after porcine circovirus type 2 (PCV2) infection. Western blotting was used to measure cytoplasmic expression of NF-κB p65 at 6, 12, 24, and 48 h in the control, PCV2, MyD88 small interfering (si)RNA, and PCV2 + MyD88 siRNA groups. Expression of β-actin was used as a positive control; * p
Figure Legend Snippet: Changes in nuclear factor kappa B (NF-κB) p65 protein expression in the cytoplasm after porcine circovirus type 2 (PCV2) infection. Western blotting was used to measure cytoplasmic expression of NF-κB p65 at 6, 12, 24, and 48 h in the control, PCV2, MyD88 small interfering (si)RNA, and PCV2 + MyD88 siRNA groups. Expression of β-actin was used as a positive control; * p

Techniques Used: Expressing, Infection, Western Blot, Positive Control

17) Product Images from "Attenuation of choroidal neovascularization by dietary intake of ω-3 long-chain polyunsaturated fatty acids and lutein in mice"

Article Title: Attenuation of choroidal neovascularization by dietary intake of ω-3 long-chain polyunsaturated fatty acids and lutein in mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0196037

Effects of ω-3 LCPUFAs and lutein on Nox4 expression in the choroid-retina. ( A ) Representative images of Nox4 immunofluorescence (blue) in RPE-choroid sections prepared from mice of the four experimental groups at 7 days after laser photocoagulation. Red ovals enclose the lesion area. Scale bar, 100 μm. ( B ) The amount of Nox4 in the retina isolated from mice of the four experimental groups at 7 days after laser photocoagulation was determined by densitometric scanning of immunoblots. Data were normalized by the abundance of β-actin and are means ± SEM of triplicate determinations for a pooled sample. ** P
Figure Legend Snippet: Effects of ω-3 LCPUFAs and lutein on Nox4 expression in the choroid-retina. ( A ) Representative images of Nox4 immunofluorescence (blue) in RPE-choroid sections prepared from mice of the four experimental groups at 7 days after laser photocoagulation. Red ovals enclose the lesion area. Scale bar, 100 μm. ( B ) The amount of Nox4 in the retina isolated from mice of the four experimental groups at 7 days after laser photocoagulation was determined by densitometric scanning of immunoblots. Data were normalized by the abundance of β-actin and are means ± SEM of triplicate determinations for a pooled sample. ** P

Techniques Used: Expressing, Immunofluorescence, Mouse Assay, Isolation, Western Blot

18) Product Images from "Everolimus Plus Ku0063794 Regimen Promotes Anticancer Effects against Hepatocellular Carcinoma Cells through the Paradoxical Inhibition of Autophagy"

Article Title: Everolimus Plus Ku0063794 Regimen Promotes Anticancer Effects against Hepatocellular Carcinoma Cells through the Paradoxical Inhibition of Autophagy

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

doi: 10.4143/crt.2017.085

Determination of the role of SIRT-1 during autophagy of HepG2 cells. (A) Western blot analyses showing the effects of everolimus and Ku0063794, either individually or in combination, on the expression of SIRT1 (left). Relative densities of these markers in each group (right). Unlike the monotherapies, combination therapy significantly inhibited the expression of SIRT1. (B) Identification of successful transfection of pcDNA-SIRT1 into HepG2 cells as detected by Western blot analysis. Successful integration was identified by the higher expression of SIRT1. Transfection with pcDNA-SIRT1 promoted autophagy, as demonstrated by higher expression of LC3B and lower expression of p62. (C) SIRT1 overexpression assay to evaluate whether combination therapy increases HCC cell apoptosis by decreasing SIRT1 (left). Relative densities of these markers in each group (right). Overexpression of SIRT1 abrogated both autophagy-inhibiting and pro-apoptotic effects of combination therapy, which was manifested by higher expression of LC3B and lower expression of p62, and lower expression of pro-apoptotic markers (c-PARP, c-Cas3, and Bim) and higher expression of Mcl-1. These data suggest that combination therapy promotes apoptosis of HepG2 cells by downregulating SIRT1 expression. The Band Analysis tools of ImageLab software (Bio-Rad) were used to determine the density of the bands in all blots. β-Actin was used as normalization control. Values represent mean±standard deviation of three independent experiments. *p
Figure Legend Snippet: Determination of the role of SIRT-1 during autophagy of HepG2 cells. (A) Western blot analyses showing the effects of everolimus and Ku0063794, either individually or in combination, on the expression of SIRT1 (left). Relative densities of these markers in each group (right). Unlike the monotherapies, combination therapy significantly inhibited the expression of SIRT1. (B) Identification of successful transfection of pcDNA-SIRT1 into HepG2 cells as detected by Western blot analysis. Successful integration was identified by the higher expression of SIRT1. Transfection with pcDNA-SIRT1 promoted autophagy, as demonstrated by higher expression of LC3B and lower expression of p62. (C) SIRT1 overexpression assay to evaluate whether combination therapy increases HCC cell apoptosis by decreasing SIRT1 (left). Relative densities of these markers in each group (right). Overexpression of SIRT1 abrogated both autophagy-inhibiting and pro-apoptotic effects of combination therapy, which was manifested by higher expression of LC3B and lower expression of p62, and lower expression of pro-apoptotic markers (c-PARP, c-Cas3, and Bim) and higher expression of Mcl-1. These data suggest that combination therapy promotes apoptosis of HepG2 cells by downregulating SIRT1 expression. The Band Analysis tools of ImageLab software (Bio-Rad) were used to determine the density of the bands in all blots. β-Actin was used as normalization control. Values represent mean±standard deviation of three independent experiments. *p

Techniques Used: Western Blot, Expressing, Transfection, Over Expression, Software, Standard Deviation

Effects of everolimus and Ku0063794, either individually or in combination, on apoptosis in HepG2 cells. (A) Western blot analyses showing the expression of markers reflecting apoptosis (c-PARP and c-Cas3) and anti-apoptosis (Mcl-1) depending on the treatment with everolimus (left) and Ku0063794 (middle), and their combination (right). Everolimus or Ku0063794 monotherapies increased the expression of c-PARP and c-Cas3, and reduced the expression of Mcl-1, mostly dose-dependently. The combination therapy appeared to potentiate the pro-apoptotic effects of individual monotherapies. ) (B) Representative expression of the markers related to pro-apoptosis (c-PARP, c-Cas9, c-Cas3, and Bim) and anti-apoptosis (Bcl-xL and Mcl-1) at the concentrations of everolimus (100 nM) and Ku0063794 (1 μM) used in this experiment. (C) Effects of everolimus and Ku0063794, either individually or in combination, on apoptosis using annexin V/PI staining and flow cytometry (top). Relative densities of these markers in each group (bottom). Apoptotic cells were expressed as the total percentage of annexin V–positive/PI-negative cells. The number of annexin V–positive cells (early and late apoptotic cells) was significantly higher in HepG2 cells treated with the combination compared to cells treated with the monotherapies. Band Analysis tools in ImageLab software (Bio-Rad) were used to determine the density of the bands in all blots. β-Actin was used as a normalization control. Values represent mean±standard deviation of three independent experiments. *p
Figure Legend Snippet: Effects of everolimus and Ku0063794, either individually or in combination, on apoptosis in HepG2 cells. (A) Western blot analyses showing the expression of markers reflecting apoptosis (c-PARP and c-Cas3) and anti-apoptosis (Mcl-1) depending on the treatment with everolimus (left) and Ku0063794 (middle), and their combination (right). Everolimus or Ku0063794 monotherapies increased the expression of c-PARP and c-Cas3, and reduced the expression of Mcl-1, mostly dose-dependently. The combination therapy appeared to potentiate the pro-apoptotic effects of individual monotherapies. ) (B) Representative expression of the markers related to pro-apoptosis (c-PARP, c-Cas9, c-Cas3, and Bim) and anti-apoptosis (Bcl-xL and Mcl-1) at the concentrations of everolimus (100 nM) and Ku0063794 (1 μM) used in this experiment. (C) Effects of everolimus and Ku0063794, either individually or in combination, on apoptosis using annexin V/PI staining and flow cytometry (top). Relative densities of these markers in each group (bottom). Apoptotic cells were expressed as the total percentage of annexin V–positive/PI-negative cells. The number of annexin V–positive cells (early and late apoptotic cells) was significantly higher in HepG2 cells treated with the combination compared to cells treated with the monotherapies. Band Analysis tools in ImageLab software (Bio-Rad) were used to determine the density of the bands in all blots. β-Actin was used as a normalization control. Values represent mean±standard deviation of three independent experiments. *p

Techniques Used: Western Blot, Expressing, Staining, Flow Cytometry, Cytometry, Software, Standard Deviation

Effects of everolimus and Ku0063794, either individually or in combination, on the expression of mTOR downstream molecules in HepG2 cells. (A-C) Western blot analyses of the dose-dependent effects of everolimus (A) and Ku0063794 (B), and their combination (C) on the expression of mTOR downstream molecules. Higher concentrations of everolimus and Ku0063794 reduced the expression of p-mTOR and p-p70S6K. However, combining both agents resulted in a stronger dosedependent inhibition of p-mTOR and p-p70S6K. (D) Representative expression of mTOR downstream molecules at the concentrations of everolimus (100 nM) and Ku0063794 (1 μM) used in this experiment (left). Relative densities of these markers in each group (right). The Band Analysis tools of ImageLab software (Bio-Rad) were used to determine the density of the bands in all blots. β-Actin was used as a normalization control. Values represent mean±standard deviation of three independent experiments. *p
Figure Legend Snippet: Effects of everolimus and Ku0063794, either individually or in combination, on the expression of mTOR downstream molecules in HepG2 cells. (A-C) Western blot analyses of the dose-dependent effects of everolimus (A) and Ku0063794 (B), and their combination (C) on the expression of mTOR downstream molecules. Higher concentrations of everolimus and Ku0063794 reduced the expression of p-mTOR and p-p70S6K. However, combining both agents resulted in a stronger dosedependent inhibition of p-mTOR and p-p70S6K. (D) Representative expression of mTOR downstream molecules at the concentrations of everolimus (100 nM) and Ku0063794 (1 μM) used in this experiment (left). Relative densities of these markers in each group (right). The Band Analysis tools of ImageLab software (Bio-Rad) were used to determine the density of the bands in all blots. β-Actin was used as a normalization control. Values represent mean±standard deviation of three independent experiments. *p

Techniques Used: Expressing, Western Blot, Inhibition, Software, Standard Deviation

Effects of everolimus and Ku0063794, either individually or in combination, on autophagy in HepG2 cells. (A) Western blot analyses of the dose-dependent effects of everolimus (left), Ku0063794 (middle), and their combination (right) on the expression of autophagy markers (LC3B and p62). Whereas the everolimus and Ku0063794 monotherapies increased autophagy (higher expression of LC3B and lower expression of p62), the combination of both agents paradoxically decreased autophagy (lower expression of LC3B and higher expression of p62). An arrow indicates the band for LC3B form-II, and autophagosome marker. (B) Representative expression of autophagy markers at the concentrations of everolimus (100 nM) and Ku0063794 (1 μM) used in this experiment. Note the paradoxical reduction in autophagy (lower expression of LC3B and higher expression of p62) in the combination group. (C) Autophagy studies demonstrating autophagy levels according to treatment with everolimus and Ku0063794, either individually or in combination. To precisely quantify the levels of autophagy in each group, we performed immunofluorescence combined with flow cytometry with MDC (left), quantitative analysis of autophagy using acridine orange staining and flow cytometry (middle), and GFP-LC3 puncta staining (right) of HepG2 cells in each group. The levels of autophagy correlate with numbers of MDC-positive cells, acridine orange-positive cells, and LC3B punta-positive cells. Relative densities of these markers in each group (bottom) (magnification, ×600). The autophagy studies consistently indicated that, unlike the individual monotherapies, combination therapy significantly decreased the levels of autophagy. (D) Western blot analysis showing the effects of autophagy suppression by bafilomycin A1 on the expression of autophagy (LC3B and p62), pro-apoptosis (c-PARP, c-Cas3, and Bim), and anti-apoptosis (Mcl-1 and Bcl-xL) markers (left). Relative densities of these markers in each group (right). Successful autophagy inhibition by Baf A1 was demonstrated by the higher expression of both LC3B and p63. Autophagy inhibition promoted the pro-apoptotic effects of combination therapy, as demonstrated by the higher expression of pro-apoptotic markers and by the lower expression of anti-apoptotic markers. Arrows indicate the bands for the cleaved forms of PARP and caspase 3, respectively. The Band Analysis tools of ImageLab software (Bio-Rad) were used to determine the density of the bands in all blots. β-Actin was used as normalization control. Values represent mean±standard deviation of three independent experiments. *p
Figure Legend Snippet: Effects of everolimus and Ku0063794, either individually or in combination, on autophagy in HepG2 cells. (A) Western blot analyses of the dose-dependent effects of everolimus (left), Ku0063794 (middle), and their combination (right) on the expression of autophagy markers (LC3B and p62). Whereas the everolimus and Ku0063794 monotherapies increased autophagy (higher expression of LC3B and lower expression of p62), the combination of both agents paradoxically decreased autophagy (lower expression of LC3B and higher expression of p62). An arrow indicates the band for LC3B form-II, and autophagosome marker. (B) Representative expression of autophagy markers at the concentrations of everolimus (100 nM) and Ku0063794 (1 μM) used in this experiment. Note the paradoxical reduction in autophagy (lower expression of LC3B and higher expression of p62) in the combination group. (C) Autophagy studies demonstrating autophagy levels according to treatment with everolimus and Ku0063794, either individually or in combination. To precisely quantify the levels of autophagy in each group, we performed immunofluorescence combined with flow cytometry with MDC (left), quantitative analysis of autophagy using acridine orange staining and flow cytometry (middle), and GFP-LC3 puncta staining (right) of HepG2 cells in each group. The levels of autophagy correlate with numbers of MDC-positive cells, acridine orange-positive cells, and LC3B punta-positive cells. Relative densities of these markers in each group (bottom) (magnification, ×600). The autophagy studies consistently indicated that, unlike the individual monotherapies, combination therapy significantly decreased the levels of autophagy. (D) Western blot analysis showing the effects of autophagy suppression by bafilomycin A1 on the expression of autophagy (LC3B and p62), pro-apoptosis (c-PARP, c-Cas3, and Bim), and anti-apoptosis (Mcl-1 and Bcl-xL) markers (left). Relative densities of these markers in each group (right). Successful autophagy inhibition by Baf A1 was demonstrated by the higher expression of both LC3B and p63. Autophagy inhibition promoted the pro-apoptotic effects of combination therapy, as demonstrated by the higher expression of pro-apoptotic markers and by the lower expression of anti-apoptotic markers. Arrows indicate the bands for the cleaved forms of PARP and caspase 3, respectively. The Band Analysis tools of ImageLab software (Bio-Rad) were used to determine the density of the bands in all blots. β-Actin was used as normalization control. Values represent mean±standard deviation of three independent experiments. *p

Techniques Used: Western Blot, Expressing, Marker, Immunofluorescence, Flow Cytometry, Cytometry, Staining, Inhibition, Software, Standard Deviation

19) Product Images from "Proapoptotic Ginsenosides Compound K and Rh2 Enhance Fas-induced Cell Death of Human Astrocytoma Cells Through Distinct Apoptotic Signaling Pathways"

Article Title: Proapoptotic Ginsenosides Compound K and Rh2 Enhance Fas-induced Cell Death of Human Astrocytoma Cells Through Distinct Apoptotic Signaling Pathways

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

doi: 10.4143/crt.2009.41.1.36

The involvement of p38MAPK in C-K-mediated cell death. (A) CRT-MG cells were incubated in the absence or presence of various MAPK inhibitors for 1 h, and then they were treated with C-K or Rh 2 for an additional 24 h, and cell death was measured by MTT assay. (B) The cells were treated with C-K (25 mg/L) for varying time periods, and the total cell lysates were subjected to western blot analysis for p38 MAPK, phospho-p38 MAPK and β-actin.
Figure Legend Snippet: The involvement of p38MAPK in C-K-mediated cell death. (A) CRT-MG cells were incubated in the absence or presence of various MAPK inhibitors for 1 h, and then they were treated with C-K or Rh 2 for an additional 24 h, and cell death was measured by MTT assay. (B) The cells were treated with C-K (25 mg/L) for varying time periods, and the total cell lysates were subjected to western blot analysis for p38 MAPK, phospho-p38 MAPK and β-actin.

Techniques Used: Incubation, MTT Assay, Western Blot

20) Product Images from "Long Non Coding RNA H19: A New Player in Hypoxia-Induced Multiple Myeloma Cell Dissemination"

Article Title: Long Non Coding RNA H19: A New Player in Hypoxia-Induced Multiple Myeloma Cell Dissemination

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20040801

qRT-PCR ( A ) and Western blot analysis on total protein extract ( B ) of HIF-1α expression in MM cells silenced or not for lncH19. Densitometric analysis with Image J software was done with respect to total protein level of β-actin, used as loading control. ( C ) Western blot analysis on nuclear extract of MM hypoxic cells silenced or not for lncH19. Densitometric analysis with Image J software was done with respect to nuclear protein level of histone H3, used as loading control. Values are presented as mean ± SD.
Figure Legend Snippet: qRT-PCR ( A ) and Western blot analysis on total protein extract ( B ) of HIF-1α expression in MM cells silenced or not for lncH19. Densitometric analysis with Image J software was done with respect to total protein level of β-actin, used as loading control. ( C ) Western blot analysis on nuclear extract of MM hypoxic cells silenced or not for lncH19. Densitometric analysis with Image J software was done with respect to nuclear protein level of histone H3, used as loading control. Values are presented as mean ± SD.

Techniques Used: Quantitative RT-PCR, Western Blot, Expressing, Software

Western blot analysis of IPO7 and VHL on total extract of MM hypoxic cells silenced, or not, for lncH19. Densitometric analysis with Image J software was done with respect to total protein level of β-actin, used as loading control. Values are presented as mean ± SD.
Figure Legend Snippet: Western blot analysis of IPO7 and VHL on total extract of MM hypoxic cells silenced, or not, for lncH19. Densitometric analysis with Image J software was done with respect to total protein level of β-actin, used as loading control. Values are presented as mean ± SD.

Techniques Used: Western Blot, Software

21) Product Images from "Drug-Tolerant Cancer Cells Show Reduced Tumor-Initiating Capacity: Depletion of CD44+ Cells and Evidence for Epigenetic Mechanisms"

Article Title: Drug-Tolerant Cancer Cells Show Reduced Tumor-Initiating Capacity: Depletion of CD44+ Cells and Evidence for Epigenetic Mechanisms

Journal: PLoS ONE

doi: 10.1371/journal.pone.0024397

Molecular changes and reduced CD44 + cells in drug-tolerant Du145 cell cultures. A. Whole cell lysate from the cell types indicated was used in Western blotting of p21, p27, Bcl-2, and hTERT and the blot was reprobed for β-actin. NS, non-specific. B. Western blotting of CD44 and ABCG2. The blot was reprobed for β-actin and GAPDH. C–D. Du145 and drug-tolerant Du145 cells were plated on glass coverslips and stained for CD44 using monoclonal antibody. Shown are representative images (C) and quantification of CD44 + cells (D; mean ± S.D, *, P
Figure Legend Snippet: Molecular changes and reduced CD44 + cells in drug-tolerant Du145 cell cultures. A. Whole cell lysate from the cell types indicated was used in Western blotting of p21, p27, Bcl-2, and hTERT and the blot was reprobed for β-actin. NS, non-specific. B. Western blotting of CD44 and ABCG2. The blot was reprobed for β-actin and GAPDH. C–D. Du145 and drug-tolerant Du145 cells were plated on glass coverslips and stained for CD44 using monoclonal antibody. Shown are representative images (C) and quantification of CD44 + cells (D; mean ± S.D, *, P

Techniques Used: Western Blot, Staining

22) Product Images from "Hepatic mRNA, microRNA, and miR-34a-Target responses in mice after 28 days exposure to doses of benzo(a)pyrene that elicit DNA damage and mutation"

Article Title: Hepatic mRNA, microRNA, and miR-34a-Target responses in mice after 28 days exposure to doses of benzo(a)pyrene that elicit DNA damage and mutation

Journal: Environmental and Molecular Mutagenesis

doi: 10.1002/em.20668

Western blot analysis of Bcl2, Ccne2, Ccnd1, Cdk6, and E2f3, protein expression in control, 25, 50, and 75 mg/kg/day BaP treated livers. (A) Representative Western immunoblot images showing levels of Bcl2, Ccne2, Ccnd1, Cdk6, E2f3, and β-actin in mouse liver using three independent samples. (B) Quantitative analysis of Western blots and summary histogram showing the expression of Bcl2, Ccne2, Ccnd1, Cdk6, and E2f3. Data are means ± S.E.M., n = 3 independent trials on tissue from x different animals. * Significantly different from the corresponding control value using the Student's t-test, P -value
Figure Legend Snippet: Western blot analysis of Bcl2, Ccne2, Ccnd1, Cdk6, and E2f3, protein expression in control, 25, 50, and 75 mg/kg/day BaP treated livers. (A) Representative Western immunoblot images showing levels of Bcl2, Ccne2, Ccnd1, Cdk6, E2f3, and β-actin in mouse liver using three independent samples. (B) Quantitative analysis of Western blots and summary histogram showing the expression of Bcl2, Ccne2, Ccnd1, Cdk6, and E2f3. Data are means ± S.E.M., n = 3 independent trials on tissue from x different animals. * Significantly different from the corresponding control value using the Student's t-test, P -value

Techniques Used: Western Blot, Expressing

23) Product Images from "Lactobacillus rhamnosus GG improves glucose tolerance through alleviating ER stress and suppressing macrophage activation in db/db mice"

Article Title: Lactobacillus rhamnosus GG improves glucose tolerance through alleviating ER stress and suppressing macrophage activation in db/db mice

Journal: Journal of Clinical Biochemistry and Nutrition

doi: 10.3164/jcbn.14-116

LGG alleviates ER stress by decreasing lipotoxicity in skeletal muscle of db/db mice. (A) Effect of LGG treatment on mRNA expression of ER stress genes in skeletal muscle. Total RNA were extracted from skeletal muscle of mice ( n = 9 or 10 per group) and gene expression levels were analyzed. (B) BiP protein levels in skeletal muscle of PBS- and LGG-treated mice ( n = 4 or 5 per group). Proteins were extracted from the tissue for SDS-PAGE-immunoblot analysis. (C) Effect of LGG treatment on mRNA expression of genes associated with lipid metabolism in skeletal muscle (9 or 10 mice per group). All genes are normalized with mRNA expression level of β-actin. Data represent means ± SEM. * p
Figure Legend Snippet: LGG alleviates ER stress by decreasing lipotoxicity in skeletal muscle of db/db mice. (A) Effect of LGG treatment on mRNA expression of ER stress genes in skeletal muscle. Total RNA were extracted from skeletal muscle of mice ( n = 9 or 10 per group) and gene expression levels were analyzed. (B) BiP protein levels in skeletal muscle of PBS- and LGG-treated mice ( n = 4 or 5 per group). Proteins were extracted from the tissue for SDS-PAGE-immunoblot analysis. (C) Effect of LGG treatment on mRNA expression of genes associated with lipid metabolism in skeletal muscle (9 or 10 mice per group). All genes are normalized with mRNA expression level of β-actin. Data represent means ± SEM. * p

Techniques Used: Mouse Assay, Expressing, SDS Page

24) Product Images from "Src Is a Prime Target Inhibited by Celtis choseniana Methanol Extract in Its Anti-Inflammatory Action"

Article Title: Src Is a Prime Target Inhibited by Celtis choseniana Methanol Extract in Its Anti-Inflammatory Action

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/3909038

Effects of Cc-ME on mRNA and protein expression of inflammatory genes. RAW264.7 cells or peritoneal macrophages pretreated with the indicated doses of Cc-ME or standard drug (Pred) for 30 min were treated with LPS (1 μ g/ml) for 6 h. (a, b, d, e, f, and g) mRNA and protein expression levels of iNOS, COX-2, TGF- β , IL-10, IL-6, IL-1 β , and TNF- α were determined by semiquantitative RT-PCR (a) or by quantitative real-time PCR (b, d, e, f, and g). (c and h) RAW264.7 cells pretreated with Cc-ME (50 and 100 μ g/ml) for 30 min were treated with LPS (1 μ g/ml) for the indicated time, and total levels of iNOS and COX-2 in whole cell lysates or p65 and p50 in nuclear fractions were examined by Western blot analysis. β -Actin and Lamin A/C were used as internal controls for whole cell lysate and nuclear fractions, respectively. The data presented in (a), (c), and (h) are a representative of three experiments. The data presented in (b), (d), (e), (f), and (g) are expressed as the means ± SD of experiments that were performed with six samples. Relative intensity (bottom panel of (a)) were expressed as means ± SD, calculated with data observed by three experiments using the DNR Bioimaging system. NF: nuclear fraction. # P
Figure Legend Snippet: Effects of Cc-ME on mRNA and protein expression of inflammatory genes. RAW264.7 cells or peritoneal macrophages pretreated with the indicated doses of Cc-ME or standard drug (Pred) for 30 min were treated with LPS (1 μ g/ml) for 6 h. (a, b, d, e, f, and g) mRNA and protein expression levels of iNOS, COX-2, TGF- β , IL-10, IL-6, IL-1 β , and TNF- α were determined by semiquantitative RT-PCR (a) or by quantitative real-time PCR (b, d, e, f, and g). (c and h) RAW264.7 cells pretreated with Cc-ME (50 and 100 μ g/ml) for 30 min were treated with LPS (1 μ g/ml) for the indicated time, and total levels of iNOS and COX-2 in whole cell lysates or p65 and p50 in nuclear fractions were examined by Western blot analysis. β -Actin and Lamin A/C were used as internal controls for whole cell lysate and nuclear fractions, respectively. The data presented in (a), (c), and (h) are a representative of three experiments. The data presented in (b), (d), (e), (f), and (g) are expressed as the means ± SD of experiments that were performed with six samples. Relative intensity (bottom panel of (a)) were expressed as means ± SD, calculated with data observed by three experiments using the DNR Bioimaging system. NF: nuclear fraction. # P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

The effect of Cc-ME on activation of the NF-κB signaling pathway. (a, b, and c) RAW264.7 cells pretreated with Cc-ME (100 μ g/ml) for 30 min were treated with LPS (1 μ g/ml) for the indicated time, and phosphorylated and total forms of Src, p85, AKT, IKK α / β , I κ B α , and β -actin were examined by Western blot analysis using their specific antibodies. (d) HEK293 cells were transfected with HA-Src for 48 h, and phosphorylated and total forms of Src, p85, AKT, IKK α / β , I κ B α , and β -actin were examined by Western blot analysis using their specific antibodies. (e) RAW264.7 cells pretreated with Cc-ME (100 μ g/ml) for 30 min were treated with LPS (1 μ g/ml) for 5 min. Phosphorylated Src was immunoprecipitated in the total cell lysates of the cells, and phosphorylated p85 was examined by Western blot analysis using its specific antibody. The data presented in (a), (b), (c), (d), and (e) are a representative of three experiments. Relative intensity (right panels of (a), (c), and (d), and bottom panel of (e)) was expressed as means ± SD, calculated with data observed by three experiments using the DNR Bioimaging system. WCL: whole cell lysate. HC: heavy chain. ∗ P
Figure Legend Snippet: The effect of Cc-ME on activation of the NF-κB signaling pathway. (a, b, and c) RAW264.7 cells pretreated with Cc-ME (100 μ g/ml) for 30 min were treated with LPS (1 μ g/ml) for the indicated time, and phosphorylated and total forms of Src, p85, AKT, IKK α / β , I κ B α , and β -actin were examined by Western blot analysis using their specific antibodies. (d) HEK293 cells were transfected with HA-Src for 48 h, and phosphorylated and total forms of Src, p85, AKT, IKK α / β , I κ B α , and β -actin were examined by Western blot analysis using their specific antibodies. (e) RAW264.7 cells pretreated with Cc-ME (100 μ g/ml) for 30 min were treated with LPS (1 μ g/ml) for 5 min. Phosphorylated Src was immunoprecipitated in the total cell lysates of the cells, and phosphorylated p85 was examined by Western blot analysis using its specific antibody. The data presented in (a), (b), (c), (d), and (e) are a representative of three experiments. Relative intensity (right panels of (a), (c), and (d), and bottom panel of (e)) was expressed as means ± SD, calculated with data observed by three experiments using the DNR Bioimaging system. WCL: whole cell lysate. HC: heavy chain. ∗ P

Techniques Used: Activation Assay, Western Blot, Transfection, Immunoprecipitation

25) Product Images from "Endoplasmic reticulum stress and cell death in mTORC1‐overactive cells is induced by nelfinavir and enhanced by chloroquine), Endoplasmic reticulum stress and cell death in mTORC1‐overactive cells is induced by nelfinavir and enhanced by chloroquine"

Article Title: Endoplasmic reticulum stress and cell death in mTORC1‐overactive cells is induced by nelfinavir and enhanced by chloroquine), Endoplasmic reticulum stress and cell death in mTORC1‐overactive cells is induced by nelfinavir and enhanced by chloroquine

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2014.11.005

Combined treatment of nelfinavir and chloroquine kills NCI‐H460 lung cancer cells. NCI‐H460 cells were treated with either DMSO vehicle alone, 20 μM nelfinavir (NFV), 20 μM chloroquine (CQ), or both NFV and CQ and subjected to western blot and death assays. 100 nm rapamycin was used to inhibit mTORC1 signalling. A) Following 3 h drug treatment, protein lysates were subjected to western blot analysis to determine levels of total and phosphorylated rpS6, total and phosphorylated S6K1, LC3‐II, SQSTM1 and β‐actin. B) For quantification of cell death following 48 h of treatment, NCI‐H460 cells were subjected to flow cytometry analysis following DRAQ7 staining. DRAQ7 exclusion (below line) represents the viable cell population, whilst positive DRAQ7 staining (above line) indicates cell death. The number of DRAQ7‐stained cells are graphed in C. n = 3.
Figure Legend Snippet: Combined treatment of nelfinavir and chloroquine kills NCI‐H460 lung cancer cells. NCI‐H460 cells were treated with either DMSO vehicle alone, 20 μM nelfinavir (NFV), 20 μM chloroquine (CQ), or both NFV and CQ and subjected to western blot and death assays. 100 nm rapamycin was used to inhibit mTORC1 signalling. A) Following 3 h drug treatment, protein lysates were subjected to western blot analysis to determine levels of total and phosphorylated rpS6, total and phosphorylated S6K1, LC3‐II, SQSTM1 and β‐actin. B) For quantification of cell death following 48 h of treatment, NCI‐H460 cells were subjected to flow cytometry analysis following DRAQ7 staining. DRAQ7 exclusion (below line) represents the viable cell population, whilst positive DRAQ7 staining (above line) indicates cell death. The number of DRAQ7‐stained cells are graphed in C. n = 3.

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Staining

Nelfinavir‐induced ER stress is more pronounced in Tsc2−/− MEFs. Tsc2−/− and Tsc2+/+ MEFs were treated with either DMSO vehicle alone, 20 μM nelfinavir (NFV), or 1 μM thapsigargin (TPG) for 3 h. Cells were then appropriately harvested to analyse A) CHOP mRNA, which was standardised against β‐actin mRNA (n = 3, *p
Figure Legend Snippet: Nelfinavir‐induced ER stress is more pronounced in Tsc2−/− MEFs. Tsc2−/− and Tsc2+/+ MEFs were treated with either DMSO vehicle alone, 20 μM nelfinavir (NFV), or 1 μM thapsigargin (TPG) for 3 h. Cells were then appropriately harvested to analyse A) CHOP mRNA, which was standardised against β‐actin mRNA (n = 3, *p

Techniques Used:

Nelfinavir inhibits mTORC1, induces autophagy and selectively kills Tsc2−/− MEFs. Tsc2−/− and Tsc2+/+ MEFs were treated with either DMSO vehicle alone, 20 μM nelfinavir (NFV), or 1 μM thapsigargin (TPG) for 3 h. A) Total and phosphorylated ‘(P)‐rpS6’, TSC2 and β‐actin were determined by western blot analysis from total protein lysates. 48 h DMSO and 20 μM nelfinavir‐treated Tsc2−/− and Tsc2+/+ MEFs were subjected to B) cell viability and C) cell death assays. D) Samples (prepared as for A) were subjected to western blot analysis to determine total and phosphorylated ‘(P)‐AMPK’, total and phosphorylated ‘(P)‐ACC’, and LC3 (‘LC3‐I’ and ‘LC3‐II’). E) HEK293 cells were pre‐treated with either 20 μM nelfinavir or 1 μM thapsigargin as indicated for 30 min prior to 100 nM insulin stimulation for 30 min. Total and phosphorylated ‘(P)‐rpS6’, LC3 (‘LC3‐I’ and ‘LC3‐II’) and β‐actin were then determined by western blot analysis from prepared cell lysates. n = 3, *p =
Figure Legend Snippet: Nelfinavir inhibits mTORC1, induces autophagy and selectively kills Tsc2−/− MEFs. Tsc2−/− and Tsc2+/+ MEFs were treated with either DMSO vehicle alone, 20 μM nelfinavir (NFV), or 1 μM thapsigargin (TPG) for 3 h. A) Total and phosphorylated ‘(P)‐rpS6’, TSC2 and β‐actin were determined by western blot analysis from total protein lysates. 48 h DMSO and 20 μM nelfinavir‐treated Tsc2−/− and Tsc2+/+ MEFs were subjected to B) cell viability and C) cell death assays. D) Samples (prepared as for A) were subjected to western blot analysis to determine total and phosphorylated ‘(P)‐AMPK’, total and phosphorylated ‘(P)‐ACC’, and LC3 (‘LC3‐I’ and ‘LC3‐II’). E) HEK293 cells were pre‐treated with either 20 μM nelfinavir or 1 μM thapsigargin as indicated for 30 min prior to 100 nM insulin stimulation for 30 min. Total and phosphorylated ‘(P)‐rpS6’, LC3 (‘LC3‐I’ and ‘LC3‐II’) and β‐actin were then determined by western blot analysis from prepared cell lysates. n = 3, *p =

Techniques Used: Western Blot

Combination treatments of nelfinavir and bafilomycin‐A1 or nelfinavir and 3‐methyladenine fail to kill Tsc2−/− MEFs. Tsc2−/− and Tsc2+/+ MEFs were treated with either DMSO vehicle alone, 2.5 μM baflilomycin‐A1 (BAF), 10 mM 3‐methyladenine (3‐MA), 10 μM mefloquine (MQ) or combined nelfinavir (NFV) BAF/3‐MA/MQ. A) Following 3 h drug treatment, protein lysates were subjected to western blot analysis to determine levels of LC3‐II, SQSTM1 and β‐actin. B) For quantification of cell death following 48 h of treatment, Tsc2−/− and Tsc2+/+ MEFs were subjected to flow cytometry analysis following DRAQ7 staining. DRAQ7 exclusion (below line) represents the viable cell population, whilst positive DRAQ7 staining (above line) indicates cell death. The number of DRAQ7‐stained cells are graphed in C. n = 3, *p =
Figure Legend Snippet: Combination treatments of nelfinavir and bafilomycin‐A1 or nelfinavir and 3‐methyladenine fail to kill Tsc2−/− MEFs. Tsc2−/− and Tsc2+/+ MEFs were treated with either DMSO vehicle alone, 2.5 μM baflilomycin‐A1 (BAF), 10 mM 3‐methyladenine (3‐MA), 10 μM mefloquine (MQ) or combined nelfinavir (NFV) BAF/3‐MA/MQ. A) Following 3 h drug treatment, protein lysates were subjected to western blot analysis to determine levels of LC3‐II, SQSTM1 and β‐actin. B) For quantification of cell death following 48 h of treatment, Tsc2−/− and Tsc2+/+ MEFs were subjected to flow cytometry analysis following DRAQ7 staining. DRAQ7 exclusion (below line) represents the viable cell population, whilst positive DRAQ7 staining (above line) indicates cell death. The number of DRAQ7‐stained cells are graphed in C. n = 3, *p =

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Staining

Chloroquine further sensitises Tsc2−/− MEFs to nelfinavir‐induced ER stress. Tsc2−/− and Tsc2+/+ MEFs were treated with DMSO vehicle alone, 20 μM nelfinavir (NFV), and/or chloroquine (CQ, 10 μM and 20 μM) as indicated. A) Following 3 h drug treatment, protein lysates were subjected to western blot analysis to determine levels of total and phosphorylated ‘(P)‐rpS6’, total and phosphorylated ‘(P)‐S6K1’, LC3‐II, TSC2 and β‐actin. B) CHOP mRNA was analysed and standardised against β‐actin (n = 3, *p
Figure Legend Snippet: Chloroquine further sensitises Tsc2−/− MEFs to nelfinavir‐induced ER stress. Tsc2−/− and Tsc2+/+ MEFs were treated with DMSO vehicle alone, 20 μM nelfinavir (NFV), and/or chloroquine (CQ, 10 μM and 20 μM) as indicated. A) Following 3 h drug treatment, protein lysates were subjected to western blot analysis to determine levels of total and phosphorylated ‘(P)‐rpS6’, total and phosphorylated ‘(P)‐S6K1’, LC3‐II, TSC2 and β‐actin. B) CHOP mRNA was analysed and standardised against β‐actin (n = 3, *p

Techniques Used: Western Blot

Combined treatment of nelfinavir and chloroquine selectively kills Tsc2−/− ELT3 cells. Tsc2−/− and Tsc2+/+ ELT3 cells were treated with either DMSO vehicle alone, 20 μM nelfinavir (NFV), 20 μM chloroquine (CQ), or both NFV and CQ and subjected to western blot and death assays. A) Following 3 h drug treatment, protein lysates were subjected to western blot analysis to determine levels of total and phosphorylated ‘(P)‐rpS6’, total and phosphorylated ‘(P)‐S6K1’, LC3‐II, SQSTM1, TSC2 and β‐actin. B) Quantification of cell viability. C) For quantification of cell death following 48 h of treatment, Tsc2+/+ and Tsc2−/− ELT3 cells were subjected to flow cytometry analysis following DRAQ7 staining. DRAQ7 exclusion (below line) represents the viable cell population, whilst positive DRAQ7 staining (above line) indicates cell death. The number of DRAQ7‐stained cells are graphed in D. E) Relative DNA fragmentation as determined by cell death ELISA. n = 3, *p =
Figure Legend Snippet: Combined treatment of nelfinavir and chloroquine selectively kills Tsc2−/− ELT3 cells. Tsc2−/− and Tsc2+/+ ELT3 cells were treated with either DMSO vehicle alone, 20 μM nelfinavir (NFV), 20 μM chloroquine (CQ), or both NFV and CQ and subjected to western blot and death assays. A) Following 3 h drug treatment, protein lysates were subjected to western blot analysis to determine levels of total and phosphorylated ‘(P)‐rpS6’, total and phosphorylated ‘(P)‐S6K1’, LC3‐II, SQSTM1, TSC2 and β‐actin. B) Quantification of cell viability. C) For quantification of cell death following 48 h of treatment, Tsc2+/+ and Tsc2−/− ELT3 cells were subjected to flow cytometry analysis following DRAQ7 staining. DRAQ7 exclusion (below line) represents the viable cell population, whilst positive DRAQ7 staining (above line) indicates cell death. The number of DRAQ7‐stained cells are graphed in D. E) Relative DNA fragmentation as determined by cell death ELISA. n = 3, *p =

Techniques Used: Western Blot, Flow Cytometry, Cytometry, Staining, Enzyme-linked Immunosorbent Assay

26) Product Images from "Structure of hRpn10 bound to UBQLN2 UBL illustrates basis for complementarity between shuttle factors and substrates at the proteasome"

Article Title: Structure of hRpn10 bound to UBQLN2 UBL illustrates basis for complementarity between shuttle factors and substrates at the proteasome

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2019.01.021

UBQLN proteins interact with hRpn10 in HCT116 cells. ( a ) A 10–40 % linear glycerol gradient was loaded with HCT116 cell lysate and subjected to ultracentrifugation. Gradient fractions were subjected to SDS-PAGE and immunoprobed for proteasome markers hRpn2 and β5 as well as β-actin. An antibody against UBQLN1/2/4 was used to locate these UBQLN proteins while anti-hRpn10 antibody revealed the presence of hRpn10. ( b ) HCT116 cell was treated with crosslinker DSP or DMSO (as a control). Cell lysates were immunoprecipitated with anti-UBQLN1/2/4 antibody, subjected to SDS-PAGE, and immunoprobed with anti-hRpn10 or anti-UBQLN1/2/4 antibodies, as indicated (right panel). Immunoblots of the cell lysates prior to immunoprecipitation for the DSP- and DMSO-treated cells is also included (left panel).
Figure Legend Snippet: UBQLN proteins interact with hRpn10 in HCT116 cells. ( a ) A 10–40 % linear glycerol gradient was loaded with HCT116 cell lysate and subjected to ultracentrifugation. Gradient fractions were subjected to SDS-PAGE and immunoprobed for proteasome markers hRpn2 and β5 as well as β-actin. An antibody against UBQLN1/2/4 was used to locate these UBQLN proteins while anti-hRpn10 antibody revealed the presence of hRpn10. ( b ) HCT116 cell was treated with crosslinker DSP or DMSO (as a control). Cell lysates were immunoprecipitated with anti-UBQLN1/2/4 antibody, subjected to SDS-PAGE, and immunoprobed with anti-hRpn10 or anti-UBQLN1/2/4 antibodies, as indicated (right panel). Immunoblots of the cell lysates prior to immunoprecipitation for the DSP- and DMSO-treated cells is also included (left panel).

Techniques Used: SDS Page, Immunoprecipitation, Western Blot

27) Product Images from "Erythropoietin Derived Peptide Improved Endoplasmic Reticulum Stress and Ischemia-Reperfusion Related Cellular and Renal Injury"

Article Title: Erythropoietin Derived Peptide Improved Endoplasmic Reticulum Stress and Ischemia-Reperfusion Related Cellular and Renal Injury

Journal: Frontiers in Medicine

doi: 10.3389/fmed.2020.00005

The dynamic change of cell viability and the expression of CHOP mRNA and protein in TCMK-1 cells and kidneys. (A) The cell viability was gradually decreased with the time of H 2 O 2 treatment, reached the significance from 1 to 12 h, but was significantly improved in the H 2 O 2 + CHBP group after 4 h. (B–D) The mRNA and protein of CHOP were significantly increased in the TCMK-1 cells stimulated by H 2 O 2 , but reversed by CHBP. (E,F) CHOP protein was also significantly increased in the IR group at 2 and 8 weeks, but was decreased by CHBP only at 2 weeks. Data are expressed as mean ± SEM ( n = 3–6). The volume density of western blots was corrected by against 42 kD β-actin or 54 kD α-Tubulin as a loading control. (G,H) CHOP protein was positively correlated with the level of TID and active caspase-3 protein at 2 weeks. *, # P
Figure Legend Snippet: The dynamic change of cell viability and the expression of CHOP mRNA and protein in TCMK-1 cells and kidneys. (A) The cell viability was gradually decreased with the time of H 2 O 2 treatment, reached the significance from 1 to 12 h, but was significantly improved in the H 2 O 2 + CHBP group after 4 h. (B–D) The mRNA and protein of CHOP were significantly increased in the TCMK-1 cells stimulated by H 2 O 2 , but reversed by CHBP. (E,F) CHOP protein was also significantly increased in the IR group at 2 and 8 weeks, but was decreased by CHBP only at 2 weeks. Data are expressed as mean ± SEM ( n = 3–6). The volume density of western blots was corrected by against 42 kD β-actin or 54 kD α-Tubulin as a loading control. (G,H) CHOP protein was positively correlated with the level of TID and active caspase-3 protein at 2 weeks. *, # P

Techniques Used: Expressing, Western Blot

Cell apoptosis analyzed by flow cytometry and CHOP protein detected by western blot. ( A,B : Annexin V-FITC-/PI-) The percentage of living cells was decreased by H 2 O 2 , but reversed by CHBP. ( A,C : Annexin V +/PI-; A,D : Annexin V +/PI+) The early apoptotic cells, as well as later apoptotic cells were increased, respectively, by H 2 O 2 , but reduced by CHBP. (E–G) The dynamic expression of CHOP at mRNA and protein level. (E,F) CHOP protein was significantly raised and picked at 12 h. (G) CHOP mRNA was also significantly increased at 12 h and peaked at 24 h after IR injury. Both CHOP protein and mRNA were decreased at 1 week. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. * P
Figure Legend Snippet: Cell apoptosis analyzed by flow cytometry and CHOP protein detected by western blot. ( A,B : Annexin V-FITC-/PI-) The percentage of living cells was decreased by H 2 O 2 , but reversed by CHBP. ( A,C : Annexin V +/PI-; A,D : Annexin V +/PI+) The early apoptotic cells, as well as later apoptotic cells were increased, respectively, by H 2 O 2 , but reduced by CHBP. (E–G) The dynamic expression of CHOP at mRNA and protein level. (E,F) CHOP protein was significantly raised and picked at 12 h. (G) CHOP mRNA was also significantly increased at 12 h and peaked at 24 h after IR injury. Both CHOP protein and mRNA were decreased at 1 week. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. * P

Techniques Used: Flow Cytometry, Western Blot, Expressing

CHOP siRNA downregulated its mRNA, protein and caspase-3 in TCMK-1 cells. (A) The expression of CHOP mRNA in the TCMK-1 cells transfected with CHOP siRNA was reduced by 53% and 56% compared to the cells treated by the H 2 O 2 and the NC siRNA. (B,C,E,G) The expression of CHOP protein as well as caspase-3 mRNA (D) and 17 kD caspase-3 protein was increased at the H 2 O 2 and the NC siRNA group but reversed by CHOP siRNA. (E,F) The expression of 32 kD caspase 3 protein was decreased by H 2 O 2 or NC siRNA and further decreased by CHOP siRNA. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. * P
Figure Legend Snippet: CHOP siRNA downregulated its mRNA, protein and caspase-3 in TCMK-1 cells. (A) The expression of CHOP mRNA in the TCMK-1 cells transfected with CHOP siRNA was reduced by 53% and 56% compared to the cells treated by the H 2 O 2 and the NC siRNA. (B,C,E,G) The expression of CHOP protein as well as caspase-3 mRNA (D) and 17 kD caspase-3 protein was increased at the H 2 O 2 and the NC siRNA group but reversed by CHOP siRNA. (E,F) The expression of 32 kD caspase 3 protein was decreased by H 2 O 2 or NC siRNA and further decreased by CHOP siRNA. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. * P

Techniques Used: Expressing, Transfection, Western Blot

The renoprotection of CHBP associated with ERS improvement, apoptosis and inflammation. (A,B) HMGB-1 protein was significantly up-regulated by H 2 O 2 , but was down-regulated by CHOP siRNA compared to the H 2 O 2 and NC siRNA groups. (C,D) The percentage of living cells was decreased by H 2 O 2 and H 2 O 2 + NC siRNA but reversed by H 2 O 2 + CHOP siRNA. (E,F) The both early and later apoptotic cells were increased by H 2 O 2 and H 2 O 2 + NC siRNA, but only the early apoptotic cells were reduced by H 2 O 2 + CHOP siRNA. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. * P
Figure Legend Snippet: The renoprotection of CHBP associated with ERS improvement, apoptosis and inflammation. (A,B) HMGB-1 protein was significantly up-regulated by H 2 O 2 , but was down-regulated by CHOP siRNA compared to the H 2 O 2 and NC siRNA groups. (C,D) The percentage of living cells was decreased by H 2 O 2 and H 2 O 2 + NC siRNA but reversed by H 2 O 2 + CHOP siRNA. (E,F) The both early and later apoptotic cells were increased by H 2 O 2 and H 2 O 2 + NC siRNA, but only the early apoptotic cells were reduced by H 2 O 2 + CHOP siRNA. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. * P

Techniques Used: Western Blot

The expression of PERK, JNK and HMGB-1 protein and/or mRNA. The mRNA (A) and protein (B,C) of PERK, as well as JNK protein (D,E) and HMGB-1 protein (F,G) , were significantly increased by H 2 O 2 in the TCMK-1 cells. However, these effects were reversed by CHBP. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin or 54 kD α-Tubulin as a loading control. * P
Figure Legend Snippet: The expression of PERK, JNK and HMGB-1 protein and/or mRNA. The mRNA (A) and protein (B,C) of PERK, as well as JNK protein (D,E) and HMGB-1 protein (F,G) , were significantly increased by H 2 O 2 in the TCMK-1 cells. However, these effects were reversed by CHBP. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin or 54 kD α-Tubulin as a loading control. * P

Techniques Used: Expressing, Western Blot

The dynamic change of CHOP and its regulator PERK. (A,B) The mRNA level of CHOP and PERK was gradually increased and reached the significance at 8 h and peaked at 12 h. (C,D) The expression of CHOP protein was significantly increased at 12 h. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. * P
Figure Legend Snippet: The dynamic change of CHOP and its regulator PERK. (A,B) The mRNA level of CHOP and PERK was gradually increased and reached the significance at 8 h and peaked at 12 h. (C,D) The expression of CHOP protein was significantly increased at 12 h. Data are expressed as mean ± SEM ( n = 3). The volume density of western blots was corrected by against 42 kD β-actin as a loading control. * P

Techniques Used: Expressing, Western Blot

28) Product Images from "The food intake-suppressive effects of glucagon-like peptide-1 receptor signaling in the ventral tegmental area are mediated by AMPA/kainate receptors"

Article Title: The food intake-suppressive effects of glucagon-like peptide-1 receptor signaling in the ventral tegmental area are mediated by AMPA/kainate receptors

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00413.2013

A : intra-VTA injection of Ex-4 (0.05 μg; vehicle, 100 nl of aCSF) increased VTA tyrosine hydrolase (TH; normalized to β-actin) 15 min after drug administration. B : representative immunoblots for TH and β-actin are shown. * P ≤
Figure Legend Snippet: A : intra-VTA injection of Ex-4 (0.05 μg; vehicle, 100 nl of aCSF) increased VTA tyrosine hydrolase (TH; normalized to β-actin) 15 min after drug administration. B : representative immunoblots for TH and β-actin are shown. * P ≤

Techniques Used: Injection, Western Blot

29) Product Images from "Dietary apple pectic oligosaccharide improves gut barrier function of rotavirus-challenged weaned pigs by increasing antioxidant capacity of enterocytes"

Article Title: Dietary apple pectic oligosaccharide improves gut barrier function of rotavirus-challenged weaned pigs by increasing antioxidant capacity of enterocytes

Journal: Oncotarget

doi: 10.18632/oncotarget.21367

The effect of dietary APOS supplementation and/or RV challenge on the levels of ZO-1, occludin, claudin 1 and claudin 3 in the jejunal mucosa of weaned pigs Representative Western blots for ZO-1, occludin, claudin 1, claudin 3 and β-actin in the jejunal mucosa of weaned pigs were shown. Results were expressed as the amount of ZO-1, occludin, claudin 1 and claudin 3 to β-actin in each treatment as a ratio of the other pigs to the unchallenging pigs fed basal diet. Values are means ± SE; n = 7. Values with different letters are significantly different ( P
Figure Legend Snippet: The effect of dietary APOS supplementation and/or RV challenge on the levels of ZO-1, occludin, claudin 1 and claudin 3 in the jejunal mucosa of weaned pigs Representative Western blots for ZO-1, occludin, claudin 1, claudin 3 and β-actin in the jejunal mucosa of weaned pigs were shown. Results were expressed as the amount of ZO-1, occludin, claudin 1 and claudin 3 to β-actin in each treatment as a ratio of the other pigs to the unchallenging pigs fed basal diet. Values are means ± SE; n = 7. Values with different letters are significantly different ( P

Techniques Used: Western Blot

The effect of dietary APOS supplementation and/or RV challenge on the levels of p-mTOR, mTOR, Beclin 1, CHOP, Bax and Bcl-2 in the jejunal mucosa of weaned pigs Representative Western blots for p-mTOR, mTOR, Beclin 1, CHOP, Bax, Bcl-2 and β-actin in the jejunal mucosa of weaned pigs were shown. Results were expressed as the amount of p-mTOR, mTOR, Beclin 1, CHOP, Bax and Bcl-2 to β-actin in each treatment as a ratio of the other pigs to the unchallenging pigs fed basal diet. Values are means ± SE; n = 7. Values with different letters are significantly different ( P
Figure Legend Snippet: The effect of dietary APOS supplementation and/or RV challenge on the levels of p-mTOR, mTOR, Beclin 1, CHOP, Bax and Bcl-2 in the jejunal mucosa of weaned pigs Representative Western blots for p-mTOR, mTOR, Beclin 1, CHOP, Bax, Bcl-2 and β-actin in the jejunal mucosa of weaned pigs were shown. Results were expressed as the amount of p-mTOR, mTOR, Beclin 1, CHOP, Bax and Bcl-2 to β-actin in each treatment as a ratio of the other pigs to the unchallenging pigs fed basal diet. Values are means ± SE; n = 7. Values with different letters are significantly different ( P

Techniques Used: Western Blot

30) Product Images from "Combination of SLC administration and Tregs depletion is an attractive strategy for targeting hepatocellular carcinoma"

Article Title: Combination of SLC administration and Tregs depletion is an attractive strategy for targeting hepatocellular carcinoma

Journal: Molecular Cancer

doi: 10.1186/1476-4598-12-153

Dynamic changes of CCR7 and Foxp3 in tumors after the treatments. (A) Expression of CCR7 in Hepa 1–6 cells, tumors and immune organs. (B) Expression of CCR7 and Foxp3 in tumors on day 1 to 9 post-treatment. (C) Representative staining of CCR7 (dark brown) in tumors on day 1 to 9 post-treatment. Control: control group; SLC: SLC group; SLC + anti-CD25: combination therapy group. β-actin was used as a quantitative control. Densitometer quantitation was relative to the first data set in each case (indicated by a value of 1). All data are representative of at least two independent experiments. Bar = 50 μm.
Figure Legend Snippet: Dynamic changes of CCR7 and Foxp3 in tumors after the treatments. (A) Expression of CCR7 in Hepa 1–6 cells, tumors and immune organs. (B) Expression of CCR7 and Foxp3 in tumors on day 1 to 9 post-treatment. (C) Representative staining of CCR7 (dark brown) in tumors on day 1 to 9 post-treatment. Control: control group; SLC: SLC group; SLC + anti-CD25: combination therapy group. β-actin was used as a quantitative control. Densitometer quantitation was relative to the first data set in each case (indicated by a value of 1). All data are representative of at least two independent experiments. Bar = 50 μm.

Techniques Used: Expressing, Staining, Quantitation Assay

31) Product Images from "FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer"

Article Title: FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-78

FOXA1 affects the expression of AR in human EC cells. A : FOXA1 and AR expression in the indicated EC cell lines as determined were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). β-actin was used as a loading control. B : Stable transfection of MFE-296 cells with negative control vector (MFE-296-NC) or shFOXA1 (MFE-296-shFOXA1). By comparing the cells in white light (the upper panels) with the cells in green fluorescence (the lower panels), the percentage of transfected/fluorescing cells was estimated at > 85%. Magnification, ×400. C : Quantification of FOXA1 mRNA by qRT-PCR in untransfected MFE-296 (MFE-296), MFE-296 transfected with shRNA control plasmid (MFE-296/NC), and MFE-296 transfected with shFOXA1 (MFE-296/shFOXA1). D : Quantification of AR mRNA by qRT-PCR in MFE-296, MFE-296/NC, and MFE-296/shFOXA1 cells. E : FOXA1 and AR expression in MFE-296, MFE-296/NC and MFE-296/shFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). F : Quantification of FOXA1 mRNA by qRT-PCR in untransfected AN3CA (AN3CA), AN3CA transfected with control plasmid (AN3CA/NC), and AN3CA transfected with FOXA1 expression plasmid (AN3CA/exFOXA1). G : Quantification of AR mRNA by qRT-PCR in AN3CA, AN3CA/NC, and AN3CA/exFOXA1 cells. H : AR and FOXA1 expression in AN3CA, AN3CA/NC and AN3CA/exFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). *p
Figure Legend Snippet: FOXA1 affects the expression of AR in human EC cells. A : FOXA1 and AR expression in the indicated EC cell lines as determined were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). β-actin was used as a loading control. B : Stable transfection of MFE-296 cells with negative control vector (MFE-296-NC) or shFOXA1 (MFE-296-shFOXA1). By comparing the cells in white light (the upper panels) with the cells in green fluorescence (the lower panels), the percentage of transfected/fluorescing cells was estimated at > 85%. Magnification, ×400. C : Quantification of FOXA1 mRNA by qRT-PCR in untransfected MFE-296 (MFE-296), MFE-296 transfected with shRNA control plasmid (MFE-296/NC), and MFE-296 transfected with shFOXA1 (MFE-296/shFOXA1). D : Quantification of AR mRNA by qRT-PCR in MFE-296, MFE-296/NC, and MFE-296/shFOXA1 cells. E : FOXA1 and AR expression in MFE-296, MFE-296/NC and MFE-296/shFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). F : Quantification of FOXA1 mRNA by qRT-PCR in untransfected AN3CA (AN3CA), AN3CA transfected with control plasmid (AN3CA/NC), and AN3CA transfected with FOXA1 expression plasmid (AN3CA/exFOXA1). G : Quantification of AR mRNA by qRT-PCR in AN3CA, AN3CA/NC, and AN3CA/exFOXA1 cells. H : AR and FOXA1 expression in AN3CA, AN3CA/NC and AN3CA/exFOXA1 cells were measured by western blotting (Left), and further quantified by densitometry of triplicate experiments (Right). *p

Techniques Used: Expressing, Western Blot, Stable Transfection, Negative Control, Plasmid Preparation, Fluorescence, Transfection, Quantitative RT-PCR, shRNA

FOXA1 affects AR-mediated transcription. A : MFE-296 cells were treated with DHT (10 −9 to 10 −7 M) or vehicle (control) for 24 h. qRT-PCR was used to assess the levels of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. The levels of each mRNA are shown relative to the level expressed in the vehicle sample. B : Quantification of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA by qRT-PCR in MFE-296 cells treated with 10 −7 M DHT for 0–48 h. C : Western blotting analysis of AR in MFE-296 cells treated with vehicle, 10 −7 M DHT, or 10 −7 M DHT plus 10 −6 M flutamide (DHT + FLU) for 24 h. β-actin was used as a loading control. D : MFE-296/NC and MFE-296/shFOXA1 cells were treated with 10 −7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. E : AN3CA/NC and AN3CA/exFOXA1 cells were treated with 10 −7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. F : Quantification of AR expression by qRT-PCR and western blotting in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or exAR (MFE-296/exAR). G : Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected MFE-296 cells and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR was measured by qRT-PCR. H : Quantification of AR expression by qRT-PCR and western blotting in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) or siAR (AN3CA/siAR). I : Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected AN3CA cells and AN3CA cells transfected with NC, exFOXA1, or exFOXA1 and siAR was measured by qRT-PCR. *p
Figure Legend Snippet: FOXA1 affects AR-mediated transcription. A : MFE-296 cells were treated with DHT (10 −9 to 10 −7 M) or vehicle (control) for 24 h. qRT-PCR was used to assess the levels of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. The levels of each mRNA are shown relative to the level expressed in the vehicle sample. B : Quantification of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA by qRT-PCR in MFE-296 cells treated with 10 −7 M DHT for 0–48 h. C : Western blotting analysis of AR in MFE-296 cells treated with vehicle, 10 −7 M DHT, or 10 −7 M DHT plus 10 −6 M flutamide (DHT + FLU) for 24 h. β-actin was used as a loading control. D : MFE-296/NC and MFE-296/shFOXA1 cells were treated with 10 −7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. E : AN3CA/NC and AN3CA/exFOXA1 cells were treated with 10 −7 M DHT or vehicle for 24 h followed by qRT-PCR analysis of AR, XBP1, MYC, ZBTB16, and UHRF1 mRNA. F : Quantification of AR expression by qRT-PCR and western blotting in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC) or exAR (MFE-296/exAR). G : Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected MFE-296 cells and MFE-296 cells transfected with NC, shFOXA1, or shFOXA1 and exAR was measured by qRT-PCR. H : Quantification of AR expression by qRT-PCR and western blotting in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) or siAR (AN3CA/siAR). I : Expression of XBP1, MYC, ZBTB16, and UHRF1 mRNA in untransfected AN3CA cells and AN3CA cells transfected with NC, exFOXA1, or exFOXA1 and siAR was measured by qRT-PCR. *p

Techniques Used: Quantitative RT-PCR, Western Blot, Expressing, Transfection

FOXA1 affects AR-mediated transcription via binding with AR and activates the Notch pathway. A : Co-immunoprecipitation (IP) of FOXA1 with AR in MFE-296 cells. WB: western blotting. B : Co-immunoprecipitation of FOXA1 with AR in AN3CA cells treated with 10 −7 M DHT or vehicle. C : Schematic representation of the MYC locus. FOXA1-binding sites and AR-binding sites upstream of the TSS of MYC were predicted by ChIP-seq analysis. ChIP-PCR assays were performed using anti-FOXA1 antibody or anti-AR antibody. Pro: promoter region; Enh-1: enhancer 1 region; End-2: enhancer 2 region; TSS: transcription starting sites. D : Immunoprecipitated DNA fragments in ChIP-PCR assays were examined by qRT-PCR. Each sample was assayed in triplicate in each of three independent experiments. IgG was used as negative control. Primers were designed specifically for the promoter region (Pro), the enhancer 1 region (Enh-1), and the three putative FOXA1-AR binding sites within enhancer 2 region (Enh-2a, Enh-2b, and Enh-2c) according to the study [ 19 ]. E : Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC), shFOXA1 (MFE-296/shFOXA1), or shFOXA1 and exAR (MFE-296/shFOXA1 + exAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). F : Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) , exFOXA1 (AN3CA/exFOXA1), or exFOXA1 and siAR (AN3CA/exFOXA1 + siAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). β-actin was used as a loading control. *p
Figure Legend Snippet: FOXA1 affects AR-mediated transcription via binding with AR and activates the Notch pathway. A : Co-immunoprecipitation (IP) of FOXA1 with AR in MFE-296 cells. WB: western blotting. B : Co-immunoprecipitation of FOXA1 with AR in AN3CA cells treated with 10 −7 M DHT or vehicle. C : Schematic representation of the MYC locus. FOXA1-binding sites and AR-binding sites upstream of the TSS of MYC were predicted by ChIP-seq analysis. ChIP-PCR assays were performed using anti-FOXA1 antibody or anti-AR antibody. Pro: promoter region; Enh-1: enhancer 1 region; End-2: enhancer 2 region; TSS: transcription starting sites. D : Immunoprecipitated DNA fragments in ChIP-PCR assays were examined by qRT-PCR. Each sample was assayed in triplicate in each of three independent experiments. IgG was used as negative control. Primers were designed specifically for the promoter region (Pro), the enhancer 1 region (Enh-1), and the three putative FOXA1-AR binding sites within enhancer 2 region (Enh-2a, Enh-2b, and Enh-2c) according to the study [ 19 ]. E : Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected MFE-296 cells (MFE-296) and MFE-296 cells transfected with NC (MFE-296/NC), shFOXA1 (MFE-296/shFOXA1), or shFOXA1 and exAR (MFE-296/shFOXA1 + exAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). F : Protein levels of FOXA1, AR, Notch1, and Hes1 in untransfected AN3CA cells (AN3CA) and AN3CA cells transfected with NC (AN3CA/NC) , exFOXA1 (AN3CA/exFOXA1), or exFOXA1 and siAR (AN3CA/exFOXA1 + siAR) were measured by western blotting (Right), and further quantified by densitometry of triplicate experiments (Left). β-actin was used as a loading control. *p

Techniques Used: Binding Assay, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control, Transfection

32) Product Images from "Identification of Steroidogenic Components Derived From Gardenia jasminoides Ellis Potentially Useful for Treating Postmenopausal Syndrome"

Article Title: Identification of Steroidogenic Components Derived From Gardenia jasminoides Ellis Potentially Useful for Treating Postmenopausal Syndrome

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00390

Effects of rutin, CGA, and GA on levels of FSHR, aromatase and StAR in ovarian granulosa cells after 12 h of treatment. (A,C,D) Effects of rutin, CGA, and GA on FSHR, aromatase and StAR expression in ovarian granulosa cells. Ovarian granulosa cells of SD rats were treated with various concentrations of rutin, CGA, and GA respectively for 12 h, respectively, and then total cell lysates were extracted for Western blot analysis by using antibodies specific to FSHR, aromatase and StAR. The representative image and the relative expression levels of FSHR, aromatase and StAR are shown. (B) Estrogen stimulating effect of rutin, CGA, and GA on ovarian granulosa cells with antibody-blocked FSHR diminished. The data were normalized with the internal control β-actin, each value is the mean ± SEM ( n = 4), with * p
Figure Legend Snippet: Effects of rutin, CGA, and GA on levels of FSHR, aromatase and StAR in ovarian granulosa cells after 12 h of treatment. (A,C,D) Effects of rutin, CGA, and GA on FSHR, aromatase and StAR expression in ovarian granulosa cells. Ovarian granulosa cells of SD rats were treated with various concentrations of rutin, CGA, and GA respectively for 12 h, respectively, and then total cell lysates were extracted for Western blot analysis by using antibodies specific to FSHR, aromatase and StAR. The representative image and the relative expression levels of FSHR, aromatase and StAR are shown. (B) Estrogen stimulating effect of rutin, CGA, and GA on ovarian granulosa cells with antibody-blocked FSHR diminished. The data were normalized with the internal control β-actin, each value is the mean ± SEM ( n = 4), with * p

Techniques Used: Expressing, Western Blot

Effects of rutin, CGA, and GA on estrogen receptor expression in ovarian granulosa cells after treatment for 12 h. Ovarian granulosa cells of SD rats were treated with various concentrations of rutin, CGA, and GA for 12 h, respectively, and then total cell lysates were extracted for Western blot analysis by using antibodies specific to ERα and ERβ. The representative image and the relative expression levels analyzed of (A) ERα and (B) ERβ are shown. The loading control of ERβ is reused from that of StAR since they were from the same membrane. The data were normalized with the internal control β-actin, and each value is the mean ± SEM ( n = 4), with * p
Figure Legend Snippet: Effects of rutin, CGA, and GA on estrogen receptor expression in ovarian granulosa cells after treatment for 12 h. Ovarian granulosa cells of SD rats were treated with various concentrations of rutin, CGA, and GA for 12 h, respectively, and then total cell lysates were extracted for Western blot analysis by using antibodies specific to ERα and ERβ. The representative image and the relative expression levels analyzed of (A) ERα and (B) ERβ are shown. The loading control of ERβ is reused from that of StAR since they were from the same membrane. The data were normalized with the internal control β-actin, and each value is the mean ± SEM ( n = 4), with * p

Techniques Used: Expressing, Western Blot

33) Product Images from "Novel downstream molecular targets of SIRT1 in melanoma: A quantitative proteomics approach"

Article Title: Novel downstream molecular targets of SIRT1 in melanoma: A quantitative proteomics approach

Journal: Oncotarget

doi:

Effect of SIRT1 and SIRT2 knockdown on BUB family proteins Following SIRT1 and SIRT2 knockdown, immunoblot analyses were performed as described in ‘Materials and Methods’. Knockdown was confirmed by probing with SIRT1 or SIRT2 antibodies (A), and effect on BUB3, BUB1 and BUBR1 protein levels were assessed following SIRT1 and SIRT2 knockdown (B). Equal loading was confirmed by re-probing the blots with β-actin.
Figure Legend Snippet: Effect of SIRT1 and SIRT2 knockdown on BUB family proteins Following SIRT1 and SIRT2 knockdown, immunoblot analyses were performed as described in ‘Materials and Methods’. Knockdown was confirmed by probing with SIRT1 or SIRT2 antibodies (A), and effect on BUB3, BUB1 and BUBR1 protein levels were assessed following SIRT1 and SIRT2 knockdown (B). Equal loading was confirmed by re-probing the blots with β-actin.

Techniques Used:

34) Product Images from "Low-intensity pulsed ultrasound stimulation facilitates in vitro osteogenic differentiation of human adipose-derived stem cells via up-regulation of heat shock protein (HSP)70, HSP90, and bone morphogenetic protein (BMP) signaling pathway"

Article Title: Low-intensity pulsed ultrasound stimulation facilitates in vitro osteogenic differentiation of human adipose-derived stem cells via up-regulation of heat shock protein (HSP)70, HSP90, and bone morphogenetic protein (BMP) signaling pathway

Journal: Bioscience Reports

doi: 10.1042/BSR20180087

Expression levels of Runx2, OPN, and OCN during ASCs osteogenic differentiation hASCs were treated with LIPUS at 20 and 30 mW cm −2 for 30 min daily, and the expression of Runx2, OPN, and OCN was analyzed on day 14. ( A ) The mRNA levels of Runx2, OPN and OCN were determined by real-time PCR, and normalized to GAPDH, and presented as fold increase relative to the controls (value set at 1). ( B ) Representative images of Western blotting. ( C ) The protein levels of Runx2, OPN, and OCN were quantified by normalization to β-actin. Data are presented as mean ± SD ( n =3); * P
Figure Legend Snippet: Expression levels of Runx2, OPN, and OCN during ASCs osteogenic differentiation hASCs were treated with LIPUS at 20 and 30 mW cm −2 for 30 min daily, and the expression of Runx2, OPN, and OCN was analyzed on day 14. ( A ) The mRNA levels of Runx2, OPN and OCN were determined by real-time PCR, and normalized to GAPDH, and presented as fold increase relative to the controls (value set at 1). ( B ) Representative images of Western blotting. ( C ) The protein levels of Runx2, OPN, and OCN were quantified by normalization to β-actin. Data are presented as mean ± SD ( n =3); * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Effect of LIPUS stimulation on BMPs hASCs were treated by LIPUS at 20 and 30 mW cm −2 for 30 min daily, and the protein levels of BMP-2, BMP-6, BMP-7, and BMP-9 were detected using Western blot analysis. ( A ) Representative Western blot bands that displayed the protein expression of BMP-2, BMP-6, BMP-7, and BMP-9. ( B ) The protein levels of BMP-2, BMP-6, BMP-7, and BMP-9 relative to β-actin used as a loading control. Data are presented as mean ± SD ( n =3); * P
Figure Legend Snippet: Effect of LIPUS stimulation on BMPs hASCs were treated by LIPUS at 20 and 30 mW cm −2 for 30 min daily, and the protein levels of BMP-2, BMP-6, BMP-7, and BMP-9 were detected using Western blot analysis. ( A ) Representative Western blot bands that displayed the protein expression of BMP-2, BMP-6, BMP-7, and BMP-9. ( B ) The protein levels of BMP-2, BMP-6, BMP-7, and BMP-9 relative to β-actin used as a loading control. Data are presented as mean ± SD ( n =3); * P

Techniques Used: Western Blot, Expressing

Analyses of HSP27, HSP40, HSP70, and HSP90 hASCs were treated with LIPUS at and 30 mW cm −2 for 30 min, and the temperature in the culture medium increased from 37 to 40°C during 30 min ultrasound exposure. Cell lysates were then extracted and subject to SDS/PAGE analysis. ( A ) Representative Western blot bands that exhibited the protein expression of HSP27, HSP40, HSP70, and HSP90. ( B ) The protein levels of HSP27, HSP40, HSP70, and HSP90 relative to β-actin that was used as a loading control. Data are presented as mean ± SD ( n =3); * P
Figure Legend Snippet: Analyses of HSP27, HSP40, HSP70, and HSP90 hASCs were treated with LIPUS at and 30 mW cm −2 for 30 min, and the temperature in the culture medium increased from 37 to 40°C during 30 min ultrasound exposure. Cell lysates were then extracted and subject to SDS/PAGE analysis. ( A ) Representative Western blot bands that exhibited the protein expression of HSP27, HSP40, HSP70, and HSP90. ( B ) The protein levels of HSP27, HSP40, HSP70, and HSP90 relative to β-actin that was used as a loading control. Data are presented as mean ± SD ( n =3); * P

Techniques Used: SDS Page, Western Blot, Expressing

Evaluation of BMP signaling and Noggin expression hASCs were treated with LIPUS at 20 and 30 mW cm −2 for 30 min daily, and on day 14, the expression of BMP downstream genes Smad and antagonist Noggin was evaluated. ( A ) Real-time PCR was used to determine the mRNA levels of Smad 1, Smad 5, and Noggin. ( B ) Representative images of Western blotting. ( C ) The protein levels of p-Smad 1/5, Smad5, and Noggin were determined by normalization to β-actin. Data are presented as mean ± SD ( n =3); * P
Figure Legend Snippet: Evaluation of BMP signaling and Noggin expression hASCs were treated with LIPUS at 20 and 30 mW cm −2 for 30 min daily, and on day 14, the expression of BMP downstream genes Smad and antagonist Noggin was evaluated. ( A ) Real-time PCR was used to determine the mRNA levels of Smad 1, Smad 5, and Noggin. ( B ) Representative images of Western blotting. ( C ) The protein levels of p-Smad 1/5, Smad5, and Noggin were determined by normalization to β-actin. Data are presented as mean ± SD ( n =3); * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

35) Product Images from "Rapamycin-independent IGF2 expression in Tsc2-null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells"

Article Title: Rapamycin-independent IGF2 expression in Tsc2-null mouse embryo fibroblasts and human lymphangioleiomyomatosis cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0197105

IGF2 expression in LAM lungs and Tsc2 -/- MEFs. ). Igf2 expression in Tsc2 -/- MEFs was detected by (C) qPCR (D) Western blot and (E) ELISA. (F) Tsc2 -/- MEFs were transfected with 50nM Igf2 siRNA (siIGF2) or NT siRNA (siNT) for 48 hrs. Decreased levels of Igf2 protein expression were confirmed via western blot with β-actin as an internal loading control. (G) Decreased Igf2 protein secretion was confirmed via ELISA. Igf2 knockdown resulted in (H) increased cleaved caspase-3 levels as measured via immunocytostaining and flow for Alexa Fluor® 488 -Cleaved Caspase 3 where the population of positively stained MEFs was normalized to the control population, and (J) decreased cell viability as assessed by 0.4% Trypan Blue staining normalized to the control cell viability. Student's t-tests were used to determine the statistical significance of the differences, and p -values reflect a sample size of 3 replicates.
Figure Legend Snippet: IGF2 expression in LAM lungs and Tsc2 -/- MEFs. ). Igf2 expression in Tsc2 -/- MEFs was detected by (C) qPCR (D) Western blot and (E) ELISA. (F) Tsc2 -/- MEFs were transfected with 50nM Igf2 siRNA (siIGF2) or NT siRNA (siNT) for 48 hrs. Decreased levels of Igf2 protein expression were confirmed via western blot with β-actin as an internal loading control. (G) Decreased Igf2 protein secretion was confirmed via ELISA. Igf2 knockdown resulted in (H) increased cleaved caspase-3 levels as measured via immunocytostaining and flow for Alexa Fluor® 488 -Cleaved Caspase 3 where the population of positively stained MEFs was normalized to the control population, and (J) decreased cell viability as assessed by 0.4% Trypan Blue staining normalized to the control cell viability. Student's t-tests were used to determine the statistical significance of the differences, and p -values reflect a sample size of 3 replicates.

Techniques Used: Expressing, Laser Capture Microdissection, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Flow Cytometry, Staining

36) Product Images from "Integrin α1-null Mice Exhibit Improved Fatty Liver When Fed a High Fat Diet Despite Severe Hepatic Insulin Resistance *"

Article Title: Integrin α1-null Mice Exhibit Improved Fatty Liver When Fed a High Fat Diet Despite Severe Hepatic Insulin Resistance *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.615716

Collagen I protein expression is increased in integrin α1-null mice. A , Western blot analysis of collagen I ( Col I ) performed on liver homogenates from basal 5-h fasted mice ( n = 4/group). Integrated intensities were obtained using ImageJ software, and collagen I protein expression was normalized to β-actin. A.U. , arbitrary units. Data are presented as means ± S.E. §, p
Figure Legend Snippet: Collagen I protein expression is increased in integrin α1-null mice. A , Western blot analysis of collagen I ( Col I ) performed on liver homogenates from basal 5-h fasted mice ( n = 4/group). Integrated intensities were obtained using ImageJ software, and collagen I protein expression was normalized to β-actin. A.U. , arbitrary units. Data are presented as means ± S.E. §, p

Techniques Used: Expressing, Mouse Assay, Western Blot, Software

37) Product Images from "Deficiency of Sphingosine-1-Phosphate Reduces the Expression of Prohibitin and Causes β-Cell Impairment via Mitochondrial Dysregulation"

Article Title: Deficiency of Sphingosine-1-Phosphate Reduces the Expression of Prohibitin and Causes β-Cell Impairment via Mitochondrial Dysregulation

Journal: Endocrinology and Metabolism

doi: 10.3803/EnM.2018.33.3.403

Knockdown of the sphingosine kinase 2 ( SPHK2 ) gene decreases prohibitin (PHB) and induces mitochondrial impairment in β-cells. (A) PHB expression was measured by an immunoblotting assay in mouse insulinoma 6 (MIN6) cells that were incubated with a sphingosine kinase inhibitor (SphKi) and/or sphingosine-1-phosphate (S1P) for 48 hours. (B) MIN6 cells were transfected with silencing genes targeting SPHK1 or SPHK2 . After incubation for 8 hours, the transcription of PHB was determined by real-time reverse-transcription polymerase chain reaction. (C, D) MIN6 cells were transfected with small interfering RNA (siRNA) for SPHK2 for 8 hours, and a portion of the cells were incubated with S1P for 48 hours. Expression of PHB was analyzed by an immunoblotting assay, and quantitative levels were represented by normalization to β-actin expression. (E) After knockdown of SPHK2 and/or S1P treatment, MIN6 cells were exposed to 100 ng/mL tetramethylrhodamine ethyl ester (TMRE) and 10 ng/mL Hoechst. Mitochondrial membrane potentials were probed by TMRE showing red fluorescence, and the nucleus was visualized by blue fluorescence. Bars=20 µm. (F, G) Adenosine triphosphate (ATP) levels and the oxygen consumption rate were analyzed in MIN6 cells after transfection of SPHK2 siRNA and/or S1P treatment. (H) Glucose-stimulated (5.5 or 25 mM) insulin secretion was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit in MIN6 cells after transfection of SPHK2 siRNA and S1P treatment. Each value represents the mean of three experiments. Veh, vehicle; si, small interfering; Scr, scramble. a P
Figure Legend Snippet: Knockdown of the sphingosine kinase 2 ( SPHK2 ) gene decreases prohibitin (PHB) and induces mitochondrial impairment in β-cells. (A) PHB expression was measured by an immunoblotting assay in mouse insulinoma 6 (MIN6) cells that were incubated with a sphingosine kinase inhibitor (SphKi) and/or sphingosine-1-phosphate (S1P) for 48 hours. (B) MIN6 cells were transfected with silencing genes targeting SPHK1 or SPHK2 . After incubation for 8 hours, the transcription of PHB was determined by real-time reverse-transcription polymerase chain reaction. (C, D) MIN6 cells were transfected with small interfering RNA (siRNA) for SPHK2 for 8 hours, and a portion of the cells were incubated with S1P for 48 hours. Expression of PHB was analyzed by an immunoblotting assay, and quantitative levels were represented by normalization to β-actin expression. (E) After knockdown of SPHK2 and/or S1P treatment, MIN6 cells were exposed to 100 ng/mL tetramethylrhodamine ethyl ester (TMRE) and 10 ng/mL Hoechst. Mitochondrial membrane potentials were probed by TMRE showing red fluorescence, and the nucleus was visualized by blue fluorescence. Bars=20 µm. (F, G) Adenosine triphosphate (ATP) levels and the oxygen consumption rate were analyzed in MIN6 cells after transfection of SPHK2 siRNA and/or S1P treatment. (H) Glucose-stimulated (5.5 or 25 mM) insulin secretion was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit in MIN6 cells after transfection of SPHK2 siRNA and S1P treatment. Each value represents the mean of three experiments. Veh, vehicle; si, small interfering; Scr, scramble. a P

Techniques Used: Expressing, Incubation, Transfection, Reverse Transcription Polymerase Chain Reaction, Small Interfering RNA, Fluorescence, Enzyme-linked Immunosorbent Assay

Lack of sphingosine-1-phosphate (S1P) induces mitochondrial impairment in β-cells. (A) After incubation with a sphingosine kinase inhibitor (SphKi) and/or S1P for 48 hours, mouse insulinoma 6 (MIN6) cells were incubated with tetramethylrhodamine ethyl ester (TMRE) to evaluate the mitochondrial membrane potential. After washing with phosphate-buffered saline, the intensity of red fluorescence in the TMRE-stained cells was detected, and (B) represented by quantitative analysis. Bar=20 µm. (C) Expression of mitochondrial dynamin-like GTPase (OPA1) and mitofusin 1 (Mfn1), key molecules regulating mitochondrial fusion and fission, was analyzed by an immunoblotting assay, and (D) the ratio to β-actin was determined. (E, F) Mitochondrial proteins regulating oxidative phosphorylation (OxPHOS) were detected using an OxPHOS complex antibody cocktail by an immunoblotting assay, and the ratio to β-actin was determined. (G, H) Adenosine triphosphate (ATP) levels and the oxygen consumption rate in MIN6 cells treated with SphKi and/or S1P were compared. The amount of ATP is represented as the percent of control, and the rate of oxygen consumption is described as picomoles per minute. Each value represents the mean of three experiments. Veh, vehicle; M, markers of size. a P
Figure Legend Snippet: Lack of sphingosine-1-phosphate (S1P) induces mitochondrial impairment in β-cells. (A) After incubation with a sphingosine kinase inhibitor (SphKi) and/or S1P for 48 hours, mouse insulinoma 6 (MIN6) cells were incubated with tetramethylrhodamine ethyl ester (TMRE) to evaluate the mitochondrial membrane potential. After washing with phosphate-buffered saline, the intensity of red fluorescence in the TMRE-stained cells was detected, and (B) represented by quantitative analysis. Bar=20 µm. (C) Expression of mitochondrial dynamin-like GTPase (OPA1) and mitofusin 1 (Mfn1), key molecules regulating mitochondrial fusion and fission, was analyzed by an immunoblotting assay, and (D) the ratio to β-actin was determined. (E, F) Mitochondrial proteins regulating oxidative phosphorylation (OxPHOS) were detected using an OxPHOS complex antibody cocktail by an immunoblotting assay, and the ratio to β-actin was determined. (G, H) Adenosine triphosphate (ATP) levels and the oxygen consumption rate in MIN6 cells treated with SphKi and/or S1P were compared. The amount of ATP is represented as the percent of control, and the rate of oxygen consumption is described as picomoles per minute. Each value represents the mean of three experiments. Veh, vehicle; M, markers of size. a P

Techniques Used: Incubation, Fluorescence, Staining, Expressing

Lack of sphingosine-1-phosphate (S1P) induces β-cell dysfunction and apoptosis. Mouse insulinoma 6 (MIN6) cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) media (containing 25 mM glucose and 10% fetal bovine serum). At 50% cell confluence, the cells were incubated in serum-free media with a sphingosine kinase inhibitor (15 µM) and/or S1P (D18:1, 10 µM) for 48 hours. (A, B) Cellular levels of S1P and apoptotic proteins, including cleaved caspase-3 and phosphorylated c-Jun N-terminal kinase (p-JNK), were evaluated by an immunoblotting assay. Quantitative levels of p-JNK and cleaved caspase-3 were normalized to total JNK and caspase-3 expression. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is presented as the percent of relative absorbance compared to the vehicle (Veh) group. (D) Transcription of insulin 1 ( INS1 ) was measured by real-time reverse-transcription polymerase chain reaction and normalized to β-actin. (E) Nuclear expression of pancreatic and duodenal homeobox 1 (Pdx1) was analyzed by an immunoblotting assay, and normalized to lamin B1. Each value represents the mean of 3 experiments. (F) The glucose-stimulated (5.5 or 25 mM) insulin secretion of MIN6 cells was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit. The values are representative of four independent experiments. t-JNK, total c-Jun N-terminal kinase; c-casp3, cleaved caspase-3; t-casp3, total caspase-3; SphKi, sphingosine kinase inhibitor. a P
Figure Legend Snippet: Lack of sphingosine-1-phosphate (S1P) induces β-cell dysfunction and apoptosis. Mouse insulinoma 6 (MIN6) cells were maintained in Dulbecco's Modified Eagle's medium (DMEM) media (containing 25 mM glucose and 10% fetal bovine serum). At 50% cell confluence, the cells were incubated in serum-free media with a sphingosine kinase inhibitor (15 µM) and/or S1P (D18:1, 10 µM) for 48 hours. (A, B) Cellular levels of S1P and apoptotic proteins, including cleaved caspase-3 and phosphorylated c-Jun N-terminal kinase (p-JNK), were evaluated by an immunoblotting assay. Quantitative levels of p-JNK and cleaved caspase-3 were normalized to total JNK and caspase-3 expression. (C) Poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) activity is presented as the percent of relative absorbance compared to the vehicle (Veh) group. (D) Transcription of insulin 1 ( INS1 ) was measured by real-time reverse-transcription polymerase chain reaction and normalized to β-actin. (E) Nuclear expression of pancreatic and duodenal homeobox 1 (Pdx1) was analyzed by an immunoblotting assay, and normalized to lamin B1. Each value represents the mean of 3 experiments. (F) The glucose-stimulated (5.5 or 25 mM) insulin secretion of MIN6 cells was evaluated by a mouse insulin enzyme-linked immunosorbent assay kit. The values are representative of four independent experiments. t-JNK, total c-Jun N-terminal kinase; c-casp3, cleaved caspase-3; t-casp3, total caspase-3; SphKi, sphingosine kinase inhibitor. a P

Techniques Used: Modification, Incubation, Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

38) Product Images from "Anti-tumor effect of bisphosphonate (YM529) on non-small cell lung cancer cell lines"

Article Title: Anti-tumor effect of bisphosphonate (YM529) on non-small cell lung cancer cell lines

Journal: BMC Cancer

doi: 10.1186/1471-2407-7-8

Concentration- course changes of Ras, Rap1A, Akt, phospho-Akt, ERK1/2, phospho-ERK1/2 activation in NCI-H1819 cells treated with YM529 by using Western blot. Cells were incubated in CM with 10 % FBS. Equal amounts of protein were loaded in each lane. β-Actin was used as internal control.
Figure Legend Snippet: Concentration- course changes of Ras, Rap1A, Akt, phospho-Akt, ERK1/2, phospho-ERK1/2 activation in NCI-H1819 cells treated with YM529 by using Western blot. Cells were incubated in CM with 10 % FBS. Equal amounts of protein were loaded in each lane. β-Actin was used as internal control.

Techniques Used: Concentration Assay, Activation Assay, Western Blot, Incubation

39) Product Images from "Acanthus ebracteatus leaf extract provides neuronal cell protection against oxidative stress injury induced by glutamate"

Article Title: Acanthus ebracteatus leaf extract provides neuronal cell protection against oxidative stress injury induced by glutamate

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-018-2340-4

Effect of AE leaf extracts on expression of Nrf2-regulated antioxidant genes in glutamate-treated HT22 cells. ( a ) Quantitative real-time RT PCR analysis of EAAT3, Nqo1, and Gclm mRNA expression in untreated HT22 cells (control) or cells exposed to 5 mM glutamate alone or glutamate combined with either AEH or AEE at 50 μg/mL for 20 h. β-actin served as an internal control to normalize the mRNA expression levels. ( b ) Western blot analysis of EAAT3 protein in whole cell lysates isolated from untreated HT22 cells (control) or cells exposed to 5 mM glutamate alone or glutamate combined with 50 μg/mL AEE for 22 h. ( c ) Western blot analysis of Nrf2 protein in nuclear and cytoplasmic fractions isolated from HT22 cells exposed for 1 h to the similar treatment conditions as in ( b ). Lamin B1 and β-actin were used as endogenous loading controls to normalize the protein expression levels from nuclear and cytoplasmic fractions/whole cell lysates, respectively. Data are expressed as the means ± SD, # P
Figure Legend Snippet: Effect of AE leaf extracts on expression of Nrf2-regulated antioxidant genes in glutamate-treated HT22 cells. ( a ) Quantitative real-time RT PCR analysis of EAAT3, Nqo1, and Gclm mRNA expression in untreated HT22 cells (control) or cells exposed to 5 mM glutamate alone or glutamate combined with either AEH or AEE at 50 μg/mL for 20 h. β-actin served as an internal control to normalize the mRNA expression levels. ( b ) Western blot analysis of EAAT3 protein in whole cell lysates isolated from untreated HT22 cells (control) or cells exposed to 5 mM glutamate alone or glutamate combined with 50 μg/mL AEE for 22 h. ( c ) Western blot analysis of Nrf2 protein in nuclear and cytoplasmic fractions isolated from HT22 cells exposed for 1 h to the similar treatment conditions as in ( b ). Lamin B1 and β-actin were used as endogenous loading controls to normalize the protein expression levels from nuclear and cytoplasmic fractions/whole cell lysates, respectively. Data are expressed as the means ± SD, # P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Isolation

Effect of AE leaf extract on subcellular distribution of AIF in glutamate-treated HT22 cells. ( a ) Representative confocal photographs of immunofluorescence staining with an antibody specific for AIF (red) and nuclei counterstaining with DAPI (blue) of untreated HT22 cells (control; top panel) or cells exposed to 5 mM glutamate alone (middle panel) or glutamate combined with 50 μg/mL AEE (bottom panel) for 16 h. ( b ) Western blot analysis of AIF protein in nuclear and cytoplasmic fractions isolated from HT22 cells exposed to the similar treatment conditions as in ( a ). Lamin B1 and β-actin were used as endogenous loading controls to normalize the expression level of AIF protein from nuclear and cytoplasmic fractions, respectively. Data are expressed as the means ± SD, ## P
Figure Legend Snippet: Effect of AE leaf extract on subcellular distribution of AIF in glutamate-treated HT22 cells. ( a ) Representative confocal photographs of immunofluorescence staining with an antibody specific for AIF (red) and nuclei counterstaining with DAPI (blue) of untreated HT22 cells (control; top panel) or cells exposed to 5 mM glutamate alone (middle panel) or glutamate combined with 50 μg/mL AEE (bottom panel) for 16 h. ( b ) Western blot analysis of AIF protein in nuclear and cytoplasmic fractions isolated from HT22 cells exposed to the similar treatment conditions as in ( a ). Lamin B1 and β-actin were used as endogenous loading controls to normalize the expression level of AIF protein from nuclear and cytoplasmic fractions, respectively. Data are expressed as the means ± SD, ## P

Techniques Used: Immunofluorescence, Staining, Western Blot, Isolation, Expressing

40) Product Images from "Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation"

Article Title: Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation

Journal: Journal of hepatology

doi: 10.1016/j.jhep.2018.01.036

Prolonged ischaemia inhibits AMP-activated protein kinase (AMPK) activities in human livers. Human liver tissues peripheral of tumours were collected during hepatic tumour resection after various lengths of ischaemia (0, 10, 30, and 50 min) from five patients with hepatocellular carcinoma, as described in “Materials and methods”. Western blot analysis was performed to determine phosphorylated (Thr172) and total levels of AMPK, as well as phosphorylated acetyl-CoA carboxylase (Ser79) (p-ACC (Ser79)), phosphorylated glycogen synthase kinase 3β (Ser9) [p-Gsk3β (S9)], and total small heterodimer protein (SHP). β-Actin was used as a control. Two representative results are shown.
Figure Legend Snippet: Prolonged ischaemia inhibits AMP-activated protein kinase (AMPK) activities in human livers. Human liver tissues peripheral of tumours were collected during hepatic tumour resection after various lengths of ischaemia (0, 10, 30, and 50 min) from five patients with hepatocellular carcinoma, as described in “Materials and methods”. Western blot analysis was performed to determine phosphorylated (Thr172) and total levels of AMPK, as well as phosphorylated acetyl-CoA carboxylase (Ser79) (p-ACC (Ser79)), phosphorylated glycogen synthase kinase 3β (Ser9) [p-Gsk3β (S9)], and total small heterodimer protein (SHP). β-Actin was used as a control. Two representative results are shown.

Techniques Used: Western Blot

Myeloid glycogen synthase kinase 3β (Gsk3β) deficiency protects livers from ischaemia–reperfusion (IR) injury. (A) Gsk3β expression in livers of myeloid Gsk3β wild-type (WT) and knockout (KO) mice. Protein lysates were prepared from liver tissues or hepatocytes or liver non-parenchymal cells (NPCs) and subjected to western blot analysis of Gsk3β and β-actin. (B) Myeloid Gsk3β WT or KO mice were subjected to liver partial warm ischaemia followed by 6 or 24 h of reperfusion, as described in “Materials and methods”. Average serum alanine aminotransferase (ALT) levels in different groups of mice are plotted, as well as average Suzuki scores and liver myeloperoxidase (MPO) activities. (C) Representative haematoxylin and eosin staining of liver tissue sections, sham livers, and ischaemic livers after 6 or 24 h of reperfusion. (D) Liver inflammatory gene expression was measured by quantitative reverse transcription PCR. Average target gene/ Hprt1 gene ratios of different experimental groups are plotted. All results are representative of at least two independent experiments. There was from four to six mice per group. Data were tested for normal distribution and analysed by one-way analysis of variance with post-test. * p
Figure Legend Snippet: Myeloid glycogen synthase kinase 3β (Gsk3β) deficiency protects livers from ischaemia–reperfusion (IR) injury. (A) Gsk3β expression in livers of myeloid Gsk3β wild-type (WT) and knockout (KO) mice. Protein lysates were prepared from liver tissues or hepatocytes or liver non-parenchymal cells (NPCs) and subjected to western blot analysis of Gsk3β and β-actin. (B) Myeloid Gsk3β WT or KO mice were subjected to liver partial warm ischaemia followed by 6 or 24 h of reperfusion, as described in “Materials and methods”. Average serum alanine aminotransferase (ALT) levels in different groups of mice are plotted, as well as average Suzuki scores and liver myeloperoxidase (MPO) activities. (C) Representative haematoxylin and eosin staining of liver tissue sections, sham livers, and ischaemic livers after 6 or 24 h of reperfusion. (D) Liver inflammatory gene expression was measured by quantitative reverse transcription PCR. Average target gene/ Hprt1 gene ratios of different experimental groups are plotted. All results are representative of at least two independent experiments. There was from four to six mice per group. Data were tested for normal distribution and analysed by one-way analysis of variance with post-test. * p

Techniques Used: Expressing, Knock-Out, Mouse Assay, Western Blot, Staining, Polymerase Chain Reaction

Glycogen synthase kinase 3β (Gsk3β) regulates macrophage AMP-activated protein kinase (AMPK) activation and small heterodimer partner (SHP) induction. (A) Gsk3β wild-type (WT) and knockout (KO) bone marrow-derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) for 0 min, 30 min, 1 h, and 6 h. Total cellular proteins were extracted and analysed by western blotting with anti-phosphorylated signal transducer and activator of transcription 1 (p-Stat1), signal transducer and activator of transcription 3 (p-Stat3), signal transducer and activator of transcription 6 (p-Stat6), NF-κBp65 (Ser536) (p-NF-kBp65), cyclic AMP response element-binding protein (Ser133) (p-CREB), AMPK (Thr172) (p-AMPK(T172)), AMPK (Thr479) (p-AMPK(T479)), and acetyl-CoA carboxylase (p-ACC) (Ser79), anti-SHP, anti-Gsk3β, and anti-β-actin. (B) Gsk3β WT (top) and KO BMMs (bottom) were treated with either vehicle or compound C before LPS stimulation. Cellular proteins were harvested after 0, 3, 6, 12, and 24 h of stimulation and analysed by western blotting with anti-SHP or anti-β-actin. (C) Gsk3β WT BMMs were treated with vehicle control or the Gsk3 inhibitor SB216763 before the addition of LPS. Cellular proteins were harvested after 0, 3, 6, and 12 h of stimulation and analysed by western blotting with anti-p AMPK(T172) and anti-SHP or anti-β-actin. (D). Gsk3β WT and KO BMMs were stimulated with LPS (top panel), or Gsk3β WT BMMs were treated with vehicle control or SB216763 before the addition of LPS (middle panel), or Gsk3β KO BMMs were treated with either vehicle or compound C before LPS stimulation (bottom panel). Total cellular RNA was prepared and reverse transcribed. Nr0b2 and Hprt1 gene expression levels were measured by quantitative reverse transcription PCR. Average Nr0b2/Hprt1 gene ratios of different experimental groups are plotted. All results are representative of at least two independent experiments. There was three mice per group. Data were tested for normal distribution and analysed by one-way analysis of variance with post-test. * p
Figure Legend Snippet: Glycogen synthase kinase 3β (Gsk3β) regulates macrophage AMP-activated protein kinase (AMPK) activation and small heterodimer partner (SHP) induction. (A) Gsk3β wild-type (WT) and knockout (KO) bone marrow-derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) for 0 min, 30 min, 1 h, and 6 h. Total cellular proteins were extracted and analysed by western blotting with anti-phosphorylated signal transducer and activator of transcription 1 (p-Stat1), signal transducer and activator of transcription 3 (p-Stat3), signal transducer and activator of transcription 6 (p-Stat6), NF-κBp65 (Ser536) (p-NF-kBp65), cyclic AMP response element-binding protein (Ser133) (p-CREB), AMPK (Thr172) (p-AMPK(T172)), AMPK (Thr479) (p-AMPK(T479)), and acetyl-CoA carboxylase (p-ACC) (Ser79), anti-SHP, anti-Gsk3β, and anti-β-actin. (B) Gsk3β WT (top) and KO BMMs (bottom) were treated with either vehicle or compound C before LPS stimulation. Cellular proteins were harvested after 0, 3, 6, 12, and 24 h of stimulation and analysed by western blotting with anti-SHP or anti-β-actin. (C) Gsk3β WT BMMs were treated with vehicle control or the Gsk3 inhibitor SB216763 before the addition of LPS. Cellular proteins were harvested after 0, 3, 6, and 12 h of stimulation and analysed by western blotting with anti-p AMPK(T172) and anti-SHP or anti-β-actin. (D). Gsk3β WT and KO BMMs were stimulated with LPS (top panel), or Gsk3β WT BMMs were treated with vehicle control or SB216763 before the addition of LPS (middle panel), or Gsk3β KO BMMs were treated with either vehicle or compound C before LPS stimulation (bottom panel). Total cellular RNA was prepared and reverse transcribed. Nr0b2 and Hprt1 gene expression levels were measured by quantitative reverse transcription PCR. Average Nr0b2/Hprt1 gene ratios of different experimental groups are plotted. All results are representative of at least two independent experiments. There was three mice per group. Data were tested for normal distribution and analysed by one-way analysis of variance with post-test. * p

Techniques Used: Activation Assay, Knock-Out, Derivative Assay, Western Blot, Binding Assay, Expressing, Polymerase Chain Reaction, Mouse Assay

Glycogen synthase kinase 3β (Gsk3β) regulated AMP-activated protein kinase (AMPK)/small heterodimer partner (SHP) independently of the phosphoinositide 3-kinase–Akt pathway. (A) Wild-type (WT) bone marrow-derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) for 0, 3, 6, 12, or 24 h. Total cellular proteins were extracted and analysed by western blotting with anti-phosphorylated Akt (Thr308) (p-AKT(T308)), Akt (Ser473) (pAKT(S473)), Gsk3β (Ser9) (p-Gsk3β(S9)), AMPK (Thr479) (p-AMPK(T479)), AMPK (Thr172) (p-AMPK(T172), and acetyl-CoA carboxylase (p-ACC) (Ser79), and β-actin. (B) Phosphatase and tensin homologue ( Pten ) WT or knockout (KO) BMMs (left panel) or Gsk3β S9A mutant knock-in (KI) BMMs (right panel) were stimulated with LPS for 6 or 24 h. Total cellular RNA was prepared and reverse transcribed. Tnf, Il10, Nr0b2 , and Hprt1 gene expression levels were measured by quantitative reverse transcription PCR. Average target gene/Hprt1 gene ratios of different experimental groups are plotted. All results are representative of at least two independent experiments. There were three mice per group. Data were tested for normal distribution and analysed by one-way analysis of variance with post-test. * p
Figure Legend Snippet: Glycogen synthase kinase 3β (Gsk3β) regulated AMP-activated protein kinase (AMPK)/small heterodimer partner (SHP) independently of the phosphoinositide 3-kinase–Akt pathway. (A) Wild-type (WT) bone marrow-derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) for 0, 3, 6, 12, or 24 h. Total cellular proteins were extracted and analysed by western blotting with anti-phosphorylated Akt (Thr308) (p-AKT(T308)), Akt (Ser473) (pAKT(S473)), Gsk3β (Ser9) (p-Gsk3β(S9)), AMPK (Thr479) (p-AMPK(T479)), AMPK (Thr172) (p-AMPK(T172), and acetyl-CoA carboxylase (p-ACC) (Ser79), and β-actin. (B) Phosphatase and tensin homologue ( Pten ) WT or knockout (KO) BMMs (left panel) or Gsk3β S9A mutant knock-in (KI) BMMs (right panel) were stimulated with LPS for 6 or 24 h. Total cellular RNA was prepared and reverse transcribed. Tnf, Il10, Nr0b2 , and Hprt1 gene expression levels were measured by quantitative reverse transcription PCR. Average target gene/Hprt1 gene ratios of different experimental groups are plotted. All results are representative of at least two independent experiments. There were three mice per group. Data were tested for normal distribution and analysed by one-way analysis of variance with post-test. * p

Techniques Used: Derivative Assay, Western Blot, Knock-Out, Mutagenesis, Knock-In, Expressing, Polymerase Chain Reaction, Mouse Assay

41) Product Images from "Intrauterine growth retardation promotes fetal intestinal autophagy in rats via the mechanistic target of rapamycin pathway"

Article Title: Intrauterine growth retardation promotes fetal intestinal autophagy in rats via the mechanistic target of rapamycin pathway

Journal: The Journal of Reproduction and Development

doi: 10.1262/jrd.2017-050

Protein levels of mTOR and mRNA expression levels of Beclin1 and ULK1 in the small intestine of IUGR and NW fetuses. Protein expression data are expressed relative to β-actin protein levels (n = 4), and mRNA expression data are expressed relative to the levels of the housekeeping gene GAPDH (n = 12) and normalized to the NW group. * P
Figure Legend Snippet: Protein levels of mTOR and mRNA expression levels of Beclin1 and ULK1 in the small intestine of IUGR and NW fetuses. Protein expression data are expressed relative to β-actin protein levels (n = 4), and mRNA expression data are expressed relative to the levels of the housekeeping gene GAPDH (n = 12) and normalized to the NW group. * P

Techniques Used: Expressing

Protein expression of p62 in the small intestine of IUGR and NW fetuses. Data are expressed relative to β-actin and normalized to the NW group (n = 4). ** P
Figure Legend Snippet: Protein expression of p62 in the small intestine of IUGR and NW fetuses. Data are expressed relative to β-actin and normalized to the NW group (n = 4). ** P

Techniques Used: Expressing

42) Product Images from "The IL-17B-IL-17 receptor B pathway promotes resistance to paclitaxel in breast tumors through activation of the ERK1/2 pathway"

Article Title: The IL-17B-IL-17 receptor B pathway promotes resistance to paclitaxel in breast tumors through activation of the ERK1/2 pathway

Journal: Oncotarget

doi: 10.18632/oncotarget.23008

IL-17B-induced resistance to paclitaxel is mediated by activation of the ERK pathway (A) Human phospho-kinase array of BT20 cultured in the presence of medium alone or rIL-17B 10 ng/ml for 30 min. Red squares identify reference spot/positive (full lane) and PBS/negative (dotted lane) controls. Arrows indicate proteins that are differentially phosphorylated between the 2 conditions. ( B) Western blot analysis of ERK1/2 phosphorylation (pErk) in BT20, MDA-MB-468 and cells MCF7 stimulated or not with 10 ng/mL rIL-17B for different times. Total ERK and β-actin were used as protein loading control. Results are representative of at least 3 experiments. ( C) Percentages of drug-induced cell cytotoxicity in BT20, MDA-MB-468 and cells MCF7 cultured in FCS-free medium completed or not with 10 ng/mL rIL-17B and/or 25 µM PD98059 (MAPK/ERK1/2 inhibitor) for 24 h before treatment with paclitaxel and treated with paclitaxel at concentrations ranging from 2.5 µg/mL to 40 µg/mL for 7 h. The Student’s t- test was used to compare control and treatment groups; * p
Figure Legend Snippet: IL-17B-induced resistance to paclitaxel is mediated by activation of the ERK pathway (A) Human phospho-kinase array of BT20 cultured in the presence of medium alone or rIL-17B 10 ng/ml for 30 min. Red squares identify reference spot/positive (full lane) and PBS/negative (dotted lane) controls. Arrows indicate proteins that are differentially phosphorylated between the 2 conditions. ( B) Western blot analysis of ERK1/2 phosphorylation (pErk) in BT20, MDA-MB-468 and cells MCF7 stimulated or not with 10 ng/mL rIL-17B for different times. Total ERK and β-actin were used as protein loading control. Results are representative of at least 3 experiments. ( C) Percentages of drug-induced cell cytotoxicity in BT20, MDA-MB-468 and cells MCF7 cultured in FCS-free medium completed or not with 10 ng/mL rIL-17B and/or 25 µM PD98059 (MAPK/ERK1/2 inhibitor) for 24 h before treatment with paclitaxel and treated with paclitaxel at concentrations ranging from 2.5 µg/mL to 40 µg/mL for 7 h. The Student’s t- test was used to compare control and treatment groups; * p

Techniques Used: Activation Assay, Cell Culture, Western Blot, Multiple Displacement Amplification

43) Product Images from "Raptor mediates the antiproliferation of cardamonin by mTORC1 inhibition in SKOV3 cells"

Article Title: Raptor mediates the antiproliferation of cardamonin by mTORC1 inhibition in SKOV3 cells

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S155065

Cardamonin downregulated the phosphorylation of mTORC1-specific binding proteins. Notes: SKOV3 cells were transfected with Raptor siRNA for 24 h and then treated with cardamonin (20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 24 h. Expression of protein extracted from total cells was assayed by Western blotting. ( A ) The level of phosphorylation and total protein of mTORC1-specific binding proteins in Raptor RNAi SKOV3 cells. ( B ) The density ratios of each band by mTOR S2481/β-actin and mTOR/β-actin in Raptor RNAi SKOV3 cells. ( C ) The density ratios of each band by Raptor-S792/β-actin and Raptor/β-actin in Raptor RNAi SKOV3 cells. ( D ) The density ratios of each band by PRAS40-S183/β-actin and PRAS40/β-actin in Raptor RNAi SKOV3 cells. n=3; # P
Figure Legend Snippet: Cardamonin downregulated the phosphorylation of mTORC1-specific binding proteins. Notes: SKOV3 cells were transfected with Raptor siRNA for 24 h and then treated with cardamonin (20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 24 h. Expression of protein extracted from total cells was assayed by Western blotting. ( A ) The level of phosphorylation and total protein of mTORC1-specific binding proteins in Raptor RNAi SKOV3 cells. ( B ) The density ratios of each band by mTOR S2481/β-actin and mTOR/β-actin in Raptor RNAi SKOV3 cells. ( C ) The density ratios of each band by Raptor-S792/β-actin and Raptor/β-actin in Raptor RNAi SKOV3 cells. ( D ) The density ratios of each band by PRAS40-S183/β-actin and PRAS40/β-actin in Raptor RNAi SKOV3 cells. n=3; # P

Techniques Used: Binding Assay, Transfection, Expressing, Western Blot

Analysis of Raptor interference. Notes: SKOV3 cells were transfected with Raptor siRNA for 24 h and then treated with cardamonin (20 μM) for 24 h. Cell extracts were analyzed by Western blotting for the expression of total Raptor. ( A ) Expression of total Raptor in Raptor RNAi SKOV3 cells. ( B ) The density ratios of each protein expression band by Raptor/β-actin in Raptor RNAi SKOV3 cells. n=5; # P
Figure Legend Snippet: Analysis of Raptor interference. Notes: SKOV3 cells were transfected with Raptor siRNA for 24 h and then treated with cardamonin (20 μM) for 24 h. Cell extracts were analyzed by Western blotting for the expression of total Raptor. ( A ) Expression of total Raptor in Raptor RNAi SKOV3 cells. ( B ) The density ratios of each protein expression band by Raptor/β-actin in Raptor RNAi SKOV3 cells. n=5; # P

Techniques Used: Transfection, Western Blot, Expressing

Cardamonin downregulated the expression of mTOR S2481. Notes: SKOV3 cells were incubated with cardamonin (5 and 20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 2, 16, and 24 h. Western blotting was used to detect the expression of mTOR S2481 and total mTOR. ( A ) Autoradiograph of expression of mTOR S2481 and total mTOR. The density ratios of each band by mTOR/β-actin ( B ) and mTOR S2481/β-actin ( C ). n=3; # P
Figure Legend Snippet: Cardamonin downregulated the expression of mTOR S2481. Notes: SKOV3 cells were incubated with cardamonin (5 and 20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 2, 16, and 24 h. Western blotting was used to detect the expression of mTOR S2481 and total mTOR. ( A ) Autoradiograph of expression of mTOR S2481 and total mTOR. The density ratios of each band by mTOR/β-actin ( B ) and mTOR S2481/β-actin ( C ). n=3; # P

Techniques Used: Expressing, Incubation, Western Blot, Autoradiography

Cardamonin downregulated the phosphorylation of S6K1. Notes: SKOV3 cells were transfected with Raptor siRNA for 24 h and then treated with cardamonin (20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 24 h. Protein expression of phosphorylation and total S6K1 extracted from the total cell extracts were assayed by Western blotting. ( A ) The level of phosphorylation and total S6K1 in Raptor RNAi SKOV3 cells. ( B ) The density ratios of each band by S6K1 T389/β-actin and S6K1/β-actin in Raptor knock-down SKOV3 cells. n=3; # P
Figure Legend Snippet: Cardamonin downregulated the phosphorylation of S6K1. Notes: SKOV3 cells were transfected with Raptor siRNA for 24 h and then treated with cardamonin (20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 24 h. Protein expression of phosphorylation and total S6K1 extracted from the total cell extracts were assayed by Western blotting. ( A ) The level of phosphorylation and total S6K1 in Raptor RNAi SKOV3 cells. ( B ) The density ratios of each band by S6K1 T389/β-actin and S6K1/β-actin in Raptor knock-down SKOV3 cells. n=3; # P

Techniques Used: Transfection, Expressing, Western Blot

Cardamonin downregulated the expression of Raptor-S792 and total Raptor. Notes: SKOV3 cells were incubated with cardamonin (5 and 20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 2, 16, and 24 h. Western blotting was used to detect the expression of Raptor-S792 and total Raptor. ( A ) Autoradiograph of expression of Raptor-S792 and total Raptor. The density ratios of each band by Raptor-S792/β-actin ( B ) and Raptor/β-actin ( C ). n=3; # P
Figure Legend Snippet: Cardamonin downregulated the expression of Raptor-S792 and total Raptor. Notes: SKOV3 cells were incubated with cardamonin (5 and 20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 2, 16, and 24 h. Western blotting was used to detect the expression of Raptor-S792 and total Raptor. ( A ) Autoradiograph of expression of Raptor-S792 and total Raptor. The density ratios of each band by Raptor-S792/β-actin ( B ) and Raptor/β-actin ( C ). n=3; # P

Techniques Used: Expressing, Incubation, Western Blot, Autoradiography

Cardamonin downregulated the expression of PRAS40-S183. Notes: SKOV3 cells were incubated with cardamonin (5 and 20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 2, 16, and 24 h. Western blotting was used to detect the expression of PRAS40-S183, PRAS40-T246, and total PRAS40. ( A ) Autoradiograph of expression of PRAS40-S183, PRAS40-T246, and total PRAS40. The density ratios of each band by PRAS40-S183/β-actin ( B ), PRAS40-T246/β-actin ( C ), and PRAS40/β-actin ( D ). n=3, # P
Figure Legend Snippet: Cardamonin downregulated the expression of PRAS40-S183. Notes: SKOV3 cells were incubated with cardamonin (5 and 20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 2, 16, and 24 h. Western blotting was used to detect the expression of PRAS40-S183, PRAS40-T246, and total PRAS40. ( A ) Autoradiograph of expression of PRAS40-S183, PRAS40-T246, and total PRAS40. The density ratios of each band by PRAS40-S183/β-actin ( B ), PRAS40-T246/β-actin ( C ), and PRAS40/β-actin ( D ). n=3, # P

Techniques Used: Expressing, Incubation, Western Blot, Autoradiography

Cardamonin downregulated the expression of p-S6K1. Notes: SKOV3 cells were incubated with cardamonin (5 and 20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 24 h. Western blotting was used to detect the expression of S6K1 T389 and total S6K1. ( A ) Autoradiograph of expression of S6K1 T389 and total S6K1. ( B ) The density ratios of each band by S6K1 T389/β-actin and S6K1/β-actin. n=3; # P
Figure Legend Snippet: Cardamonin downregulated the expression of p-S6K1. Notes: SKOV3 cells were incubated with cardamonin (5 and 20 μM), rapamycin (0.1 μM), and AZD8055 (0.1 μM) for 24 h. Western blotting was used to detect the expression of S6K1 T389 and total S6K1. ( A ) Autoradiograph of expression of S6K1 T389 and total S6K1. ( B ) The density ratios of each band by S6K1 T389/β-actin and S6K1/β-actin. n=3; # P

Techniques Used: Expressing, Incubation, Western Blot, Autoradiography

44) Product Images from "Rosuvastatin Reduces Neuroinflammation in the Hemorrhagic Transformation After rt-PA Treatment in a Mouse Model of Experimental Stroke"

Article Title: Rosuvastatin Reduces Neuroinflammation in the Hemorrhagic Transformation After rt-PA Treatment in a Mouse Model of Experimental Stroke

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2018.00225

Rosuvastatin inhibited astrocyte activation. (A) Representative images of DAPI and glial fibrillary acidic protein (GFAP) staining, as described above. Scale bars: 50 μm. (B) The quantified GFAP immunofluorescence intensity at 24 h after reperfusion; the results were normalized to DAPI. (C) Transmission electron microscopy image showing the ultrastructure of astrocytes. Scale bar: 200 nm. (D) GFAP expression levels, as determined by western blotting ( n = 6 in each group); the results were normalized to β-actin. The values were expressed as the mean ± SEM ( a–d for the SHAM, HT, HT+LRO and HT+HRO groups, respectively, as shown in Figure 1 ). * P
Figure Legend Snippet: Rosuvastatin inhibited astrocyte activation. (A) Representative images of DAPI and glial fibrillary acidic protein (GFAP) staining, as described above. Scale bars: 50 μm. (B) The quantified GFAP immunofluorescence intensity at 24 h after reperfusion; the results were normalized to DAPI. (C) Transmission electron microscopy image showing the ultrastructure of astrocytes. Scale bar: 200 nm. (D) GFAP expression levels, as determined by western blotting ( n = 6 in each group); the results were normalized to β-actin. The values were expressed as the mean ± SEM ( a–d for the SHAM, HT, HT+LRO and HT+HRO groups, respectively, as shown in Figure 1 ). * P

Techniques Used: Activation Assay, Staining, Immunofluorescence, Transmission Assay, Electron Microscopy, Expressing, Western Blot

Rosuvastatin protected against HT via inhibiting the mitogen-activated protein kinase (MAPK) pathway. Western blot analysis of p-c-Jun, phosphorylated c-Jun-N-terminal kinase (p-JNK) and p-p38 expression levels at 24 h after reperfusion ( n = 6 in each group); the results are normalized to β-actin. The values are expressed as the mean ± SEM. * P
Figure Legend Snippet: Rosuvastatin protected against HT via inhibiting the mitogen-activated protein kinase (MAPK) pathway. Western blot analysis of p-c-Jun, phosphorylated c-Jun-N-terminal kinase (p-JNK) and p-p38 expression levels at 24 h after reperfusion ( n = 6 in each group); the results are normalized to β-actin. The values are expressed as the mean ± SEM. * P

Techniques Used: Western Blot, Expressing

Rosuvastatin protected against HT via inhibiting the NF-κB pathway. Western blot analysis of phospho-NF-κB p65 (p-p65) and phospho-inhibitory subunit of NF-κBα (p-IκBα) expression levels in the NF-κB pathway at 24 h after reperfusion ( n = 6 in each group); the results are normalized to β-actin. The values are expressed as the mean ± SEM.** P
Figure Legend Snippet: Rosuvastatin protected against HT via inhibiting the NF-κB pathway. Western blot analysis of phospho-NF-κB p65 (p-p65) and phospho-inhibitory subunit of NF-κBα (p-IκBα) expression levels in the NF-κB pathway at 24 h after reperfusion ( n = 6 in each group); the results are normalized to β-actin. The values are expressed as the mean ± SEM.** P

Techniques Used: Western Blot, Expressing

Rosuvastatin attenuated inflammation levels. (A) Immunoblotting analysis of tumor necrosis factor alpha (TNF-α), cyclooxygenase 2 (Cox-2), inducible nitric oxide synthase (iNOS), interleukin 1 beta (IL-1β) and IL-6 expression levels at 24 h after reperfusion in each group ( n = 6 in each group). (B) The results are normalized to β-actin. Values are expressed as the mean ± SEM, the determination of which was followed by the LSD test (homogeneity of variance was determined). ** P
Figure Legend Snippet: Rosuvastatin attenuated inflammation levels. (A) Immunoblotting analysis of tumor necrosis factor alpha (TNF-α), cyclooxygenase 2 (Cox-2), inducible nitric oxide synthase (iNOS), interleukin 1 beta (IL-1β) and IL-6 expression levels at 24 h after reperfusion in each group ( n = 6 in each group). (B) The results are normalized to β-actin. Values are expressed as the mean ± SEM, the determination of which was followed by the LSD test (homogeneity of variance was determined). ** P

Techniques Used: Expressing

Rosuvastatin inhibited microglial activation. (A) Representative images of DAPI and Iba-1 staining, as described above. Scale bars: 50 μm. (B) The quantified Iba-1 immunofluorescence intensity at 24 h after reperfusion; the results were normalized to DAPI. (C) Transmission electron microscopy image showing the ultrastructure of microglia. Scale bar: 200 nm. (D) Iba-1 expression levels, as determined by western blotting ( n = 6 in each group); the results are normalized to β-actin. The values were expressed as the mean ± SEM, the determination of which was followed by the LSD test (homogeneity of variance was determined; a–d for the SHAM, HT, HT+LRO and HT+HRO groups, respectively, as shown in Figure 1 ). * P
Figure Legend Snippet: Rosuvastatin inhibited microglial activation. (A) Representative images of DAPI and Iba-1 staining, as described above. Scale bars: 50 μm. (B) The quantified Iba-1 immunofluorescence intensity at 24 h after reperfusion; the results were normalized to DAPI. (C) Transmission electron microscopy image showing the ultrastructure of microglia. Scale bar: 200 nm. (D) Iba-1 expression levels, as determined by western blotting ( n = 6 in each group); the results are normalized to β-actin. The values were expressed as the mean ± SEM, the determination of which was followed by the LSD test (homogeneity of variance was determined; a–d for the SHAM, HT, HT+LRO and HT+HRO groups, respectively, as shown in Figure 1 ). * P

Techniques Used: Activation Assay, Staining, Immunofluorescence, Transmission Assay, Electron Microscopy, Expressing, Western Blot

45) Product Images from "DEPTOR suppresses the progression of esophageal squamous cell carcinoma and predicts poor prognosis"

Article Title: DEPTOR suppresses the progression of esophageal squamous cell carcinoma and predicts poor prognosis

Journal: Oncotarget

doi: 10.18632/oncotarget.7420

DEPTOR expression is decreased in human ESCC tissues and predicts a poor prognosis of ESCC patients ( A ) DEPTOR expression in ESCC tissues and non-cancerous adjacent tissues derived from human patients was detected by Western blotting, β-actin was used as the internal control. P1 means patient number 1. Western blotting results were quantified by ImageJ software and summarized in right panel . ** p
Figure Legend Snippet: DEPTOR expression is decreased in human ESCC tissues and predicts a poor prognosis of ESCC patients ( A ) DEPTOR expression in ESCC tissues and non-cancerous adjacent tissues derived from human patients was detected by Western blotting, β-actin was used as the internal control. P1 means patient number 1. Western blotting results were quantified by ImageJ software and summarized in right panel . ** p

Techniques Used: Expressing, Derivative Assay, Western Blot, Software

46) Product Images from "Sensing of bacterial cyclic dinucleotides by the oxidoreductase RECON promotes NF-κB activation and shapes a proinflammatory antibacterial state"

Article Title: Sensing of bacterial cyclic dinucleotides by the oxidoreductase RECON promotes NF-κB activation and shapes a proinflammatory antibacterial state

Journal: Immunity

doi: 10.1016/j.immuni.2017.02.014

RECON is a negative regulator of NF-κB activation (A) Steady-state expression of Akr1c13 mRNA in WT and Akr1c13 −/− TIB73 cell lines. (B, C) Expression of Il6 (B) or Nos2 (C) mRNA at 2 h post-treatment with the indicated ligands or Lm infection of WT or Akr1c13 −/− TIB73 hepatocytes. T P = Tri-DAP + Pam 3 CSK 4 co-stimulation. (D) Expression of Nos2 mRNA in TIB73 cells pre-treated for 1 h with DMSO (ctrl) or the NF-κB inhibitor Celastrol (0.1 and 1 μM). (E) NF-κB luciferase activity measured from WT or Akr1c13 −/− TIB73 hepatocytes treated with the indicated ligands for 6 h. The data are presented as luciferase units divided by the eGFP transfection control fluorescence (LUC) ( N=6 ). (F) Microscopy analysis of WT (top) or Akr1c13 −/− (bottom) TIB73 hepatocytes infected with Lm for 1 h. DAPI (green), NF-κB p65 (red), and F-actin (blue). (G) Quantification of the nuclear/cytoplasmic ratio of NF-κB p65 fluorescence of data presented in (I) and (K). N=100–120 cells for each treatment. Error bars represent ± SD of biological replicates. (H) Western blot analyses of proteins involved in the NF-κB pathway; WT or Akr1c13 −/− TIB73 hepatocytes were co-stimulated with Tri-DAP + Pam 3 CSK 4 for the indicated times (5–120 min). β-actin is shown as a loading control. Data are representative of two or more independent experiments with similar results. qRT-PCR data were performed in technical duplicates or triplicates and were normalized to untreated (mock) WT TIB73 cells. Unless otherwise notes, error bars represent ± SEM of technical replicates (* P
Figure Legend Snippet: RECON is a negative regulator of NF-κB activation (A) Steady-state expression of Akr1c13 mRNA in WT and Akr1c13 −/− TIB73 cell lines. (B, C) Expression of Il6 (B) or Nos2 (C) mRNA at 2 h post-treatment with the indicated ligands or Lm infection of WT or Akr1c13 −/− TIB73 hepatocytes. T P = Tri-DAP + Pam 3 CSK 4 co-stimulation. (D) Expression of Nos2 mRNA in TIB73 cells pre-treated for 1 h with DMSO (ctrl) or the NF-κB inhibitor Celastrol (0.1 and 1 μM). (E) NF-κB luciferase activity measured from WT or Akr1c13 −/− TIB73 hepatocytes treated with the indicated ligands for 6 h. The data are presented as luciferase units divided by the eGFP transfection control fluorescence (LUC) ( N=6 ). (F) Microscopy analysis of WT (top) or Akr1c13 −/− (bottom) TIB73 hepatocytes infected with Lm for 1 h. DAPI (green), NF-κB p65 (red), and F-actin (blue). (G) Quantification of the nuclear/cytoplasmic ratio of NF-κB p65 fluorescence of data presented in (I) and (K). N=100–120 cells for each treatment. Error bars represent ± SD of biological replicates. (H) Western blot analyses of proteins involved in the NF-κB pathway; WT or Akr1c13 −/− TIB73 hepatocytes were co-stimulated with Tri-DAP + Pam 3 CSK 4 for the indicated times (5–120 min). β-actin is shown as a loading control. Data are representative of two or more independent experiments with similar results. qRT-PCR data were performed in technical duplicates or triplicates and were normalized to untreated (mock) WT TIB73 cells. Unless otherwise notes, error bars represent ± SEM of technical replicates (* P

Techniques Used: Activation Assay, Expressing, Infection, Luciferase, Activity Assay, Transfection, Fluorescence, Microscopy, Western Blot, Quantitative RT-PCR

47) Product Images from "Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter"

Article Title: Combined image guided monitoring the pharmacokinetics of rapamycin loaded human serum albumin nanoparticles with a split luciferase reporter

Journal: Nanoscale

doi: 10.1039/c5nr07308a

Time-dependent cellular uptake of Cy5 labeled HSA Rapa nanoparticles. (A) The Cy5 labeled HSA Rapa nanoparticles were incubated with HN12#2 cells. 4 h later, the cells were investigated with confocal microscopy. The lower right picture is a magnification. (B) The nanoparticles were incubated with cells for different time periods (0.5, 1, 2, 4, 6, 24, 48, 72 h), then the cells were harvested and subjected to flow cytometer analysis. (C) The HN12#2 stable cells received the HSA Rapa for different time periods (0.5, 1, 2, 4, 6, 24, 48, 72 h). At the end of time point, bioluminescence imaging was used to investigate the luciferase activity. The graph is the qualification of the bioluminescence imaging results. The error bars represent the mean ± SD for triplicate experiments. (D) The cells were treated with HSA Rapa for different time periods (1, 6, 24 h), then western blot was performed with primary antibodies for P-AKT, AKT and β-actin.
Figure Legend Snippet: Time-dependent cellular uptake of Cy5 labeled HSA Rapa nanoparticles. (A) The Cy5 labeled HSA Rapa nanoparticles were incubated with HN12#2 cells. 4 h later, the cells were investigated with confocal microscopy. The lower right picture is a magnification. (B) The nanoparticles were incubated with cells for different time periods (0.5, 1, 2, 4, 6, 24, 48, 72 h), then the cells were harvested and subjected to flow cytometer analysis. (C) The HN12#2 stable cells received the HSA Rapa for different time periods (0.5, 1, 2, 4, 6, 24, 48, 72 h). At the end of time point, bioluminescence imaging was used to investigate the luciferase activity. The graph is the qualification of the bioluminescence imaging results. The error bars represent the mean ± SD for triplicate experiments. (D) The cells were treated with HSA Rapa for different time periods (1, 6, 24 h), then western blot was performed with primary antibodies for P-AKT, AKT and β-actin.

Techniques Used: Labeling, Incubation, Confocal Microscopy, Flow Cytometry, Cytometry, Imaging, Luciferase, Activity Assay, Western Blot

48) Product Images from "Gadd45b deficiency promotes premature senescence and skin aging"

Article Title: Gadd45b deficiency promotes premature senescence and skin aging

Journal: Oncotarget

doi: 10.18632/oncotarget.8854

Signaling events involved in Gadd45b Senescence A. Western blotting analysis of p16 Ink4A , p19 ARF , p21, Phospho-p53 (Ser15), Total-p53, cdc2, Cyclin B1, Phospho-SAPK/JNK (Thr183/Tyr185) and Total-SAPK/JNK expression in cell extracts prepared from Gadd45b +/+ (WT) and Gadd45b −/− (KO) MEFs at different passages cultured at 21% oxygen. β-Actin was used as a loading control. B. Gadd45b +/+ (black) and Gadd45b −/− (grey) MEFs cultured at 21% oxygen (passage 6) were treated with vehicle (0.1% DMSO) or J NK inhibitor , SP600125 for 24 hours and stained for SA-β-gal 5 days later. SA-β-gal positive cells were counted in at least 10 fields from triplicate plates. A quantification of SA-β-gal positive MEFs is shown. * P
Figure Legend Snippet: Signaling events involved in Gadd45b Senescence A. Western blotting analysis of p16 Ink4A , p19 ARF , p21, Phospho-p53 (Ser15), Total-p53, cdc2, Cyclin B1, Phospho-SAPK/JNK (Thr183/Tyr185) and Total-SAPK/JNK expression in cell extracts prepared from Gadd45b +/+ (WT) and Gadd45b −/− (KO) MEFs at different passages cultured at 21% oxygen. β-Actin was used as a loading control. B. Gadd45b +/+ (black) and Gadd45b −/− (grey) MEFs cultured at 21% oxygen (passage 6) were treated with vehicle (0.1% DMSO) or J NK inhibitor , SP600125 for 24 hours and stained for SA-β-gal 5 days later. SA-β-gal positive cells were counted in at least 10 fields from triplicate plates. A quantification of SA-β-gal positive MEFs is shown. * P

Techniques Used: Western Blot, Expressing, Cell Culture, Staining

49) Product Images from "Acetylation impacts Fli-1 driven regulation of granulocyte colony stimulating factor (G-CSF)"

Article Title: Acetylation impacts Fli-1 driven regulation of granulocyte colony stimulating factor (G-CSF)

Journal: European journal of immunology

doi: 10.1002/eji.201646315

The role of acetylation in Fli-1 driven activation of the G-CSF promoter (A) Luciferase assay data illustrating activation from the G-CSF promoter for Fli-1 and a Fli-1 acetylation mutant (aa380). Equivalent amounts of Fli-1 and the Fli-1 acetylation mutant were transfected into NIH3T3 cells and luciferase activity measured after 48 hours. (B) Western blot, representative of three experiments illustrating Fli-1 protein concentrations after transfection into the NIH3T3 cells. Arrows are used to denote the Fli-1 and β-actin loading control bands on the left and the approximate location of the 50Kd and 37Kd molecular weight marker bands is labeled on the right. (C) Luciferase assay results from co-transfecting Fli-1 (0.5 µg) with the histone acetyltransferases (PCAF and p300) at concentrations of 1.5 µg. (D) Luciferase assay results from co-transfecting the Fli-1 acetylation mutant along with PCAF and p300. The concentrations transfected are the same as in panel (C). Equivalent amounts of DNA were transfected in all cases with the difference being made up by empty expression vectors. For panels A, C and D all values shown are fold activation over the empty vector control and are the mean + standard error for three replicate experiments (n=9). ** p-value of
Figure Legend Snippet: The role of acetylation in Fli-1 driven activation of the G-CSF promoter (A) Luciferase assay data illustrating activation from the G-CSF promoter for Fli-1 and a Fli-1 acetylation mutant (aa380). Equivalent amounts of Fli-1 and the Fli-1 acetylation mutant were transfected into NIH3T3 cells and luciferase activity measured after 48 hours. (B) Western blot, representative of three experiments illustrating Fli-1 protein concentrations after transfection into the NIH3T3 cells. Arrows are used to denote the Fli-1 and β-actin loading control bands on the left and the approximate location of the 50Kd and 37Kd molecular weight marker bands is labeled on the right. (C) Luciferase assay results from co-transfecting Fli-1 (0.5 µg) with the histone acetyltransferases (PCAF and p300) at concentrations of 1.5 µg. (D) Luciferase assay results from co-transfecting the Fli-1 acetylation mutant along with PCAF and p300. The concentrations transfected are the same as in panel (C). Equivalent amounts of DNA were transfected in all cases with the difference being made up by empty expression vectors. For panels A, C and D all values shown are fold activation over the empty vector control and are the mean + standard error for three replicate experiments (n=9). ** p-value of

Techniques Used: Activation Assay, Luciferase, Mutagenesis, Transfection, Activity Assay, Western Blot, Molecular Weight, Marker, Labeling, Expressing, Plasmid Preparation

Reduced G-CSF mRNA and protein expression in endothelial cells after treatment with Fli-1 siRNA (A–E) Endothelial cells (murine MS1(A–C) and HUVECs (D–E)) were transfected with Fli-1 specific or negative control siRNA and were stimulated with LPS.(A) Western blot results showing the specific knockdown of Fli-1 in MS1 cells. Arrows are used to denote Fli-1 and β-actin as a loading control. (B) G-CSF mRNA expression in MS1 cells after siRNA knockdown of Fli-1 and LPS stimulation (0, 6 and 24 hours). Relative expression was determined using the comparative Ct method for G-CSF compared to the GAPDH housekeeping gene prior to stimulation. Data presented are mean (2 −ΔΔCt ) + standard deviation range, samples performed in triplicate (n=3). (C). G-CSF concentrations in murine MS1cells were measured by ELISA 0, 6, 12, or 24 hours after stimulation. (D) Western blot results showing specific knockdown of Fli-1 in HUVECs. Arrows denote the Fli-1 and β-actin (loading control) bands. (E). The G-CSF concentrations in HUVECs were measured using ELISA 0, 6, and 24 hours after stimulation. (A and D) Blots are representative of three independent experiments. (B.) The experiment was performed in triplicate n=3. C and E) Data are shown as mean ± SD and are representative of three separate experiments (n=12).* p
Figure Legend Snippet: Reduced G-CSF mRNA and protein expression in endothelial cells after treatment with Fli-1 siRNA (A–E) Endothelial cells (murine MS1(A–C) and HUVECs (D–E)) were transfected with Fli-1 specific or negative control siRNA and were stimulated with LPS.(A) Western blot results showing the specific knockdown of Fli-1 in MS1 cells. Arrows are used to denote Fli-1 and β-actin as a loading control. (B) G-CSF mRNA expression in MS1 cells after siRNA knockdown of Fli-1 and LPS stimulation (0, 6 and 24 hours). Relative expression was determined using the comparative Ct method for G-CSF compared to the GAPDH housekeeping gene prior to stimulation. Data presented are mean (2 −ΔΔCt ) + standard deviation range, samples performed in triplicate (n=3). (C). G-CSF concentrations in murine MS1cells were measured by ELISA 0, 6, 12, or 24 hours after stimulation. (D) Western blot results showing specific knockdown of Fli-1 in HUVECs. Arrows denote the Fli-1 and β-actin (loading control) bands. (E). The G-CSF concentrations in HUVECs were measured using ELISA 0, 6, and 24 hours after stimulation. (A and D) Blots are representative of three independent experiments. (B.) The experiment was performed in triplicate n=3. C and E) Data are shown as mean ± SD and are representative of three separate experiments (n=12).* p

Techniques Used: Expressing, Transfection, Negative Control, Western Blot, Standard Deviation, Enzyme-linked Immunosorbent Assay

50) Product Images from "Melanin extract from Gallus gallus domesticus promotes proliferation and differentiation of osteoblastic MG-63 cells via bone morphogenetic protein-2 signaling"

Article Title: Melanin extract from Gallus gallus domesticus promotes proliferation and differentiation of osteoblastic MG-63 cells via bone morphogenetic protein-2 signaling

Journal: Nutrition Research and Practice

doi: 10.4162/nrp.2017.11.3.190

Effects of melanin extracted from Gallus gallus domesticus (GD) on bone morphogenetic protein-2 (BMP) signal pathway in MG-63 cells. Cells were differentiated for 4 days in differentiation medium in the presence or absence of melanin (250 µg/mL). (A) Protein expression of BMP signaling pathway-related markers such as BMP-2, runt-related transcription factor 2 (RUNX2), and small mothers against decapentaplegic homologs 5 (SMAD5) was detected by Western blot. (B) Relative expression was quantified by image J and calculated according to the reference bands of β-actin. Each value is expressed as mean ± SD (n = 3). Values with different letters were found to be significantly different using th e Duncan's multiple range test ( P
Figure Legend Snippet: Effects of melanin extracted from Gallus gallus domesticus (GD) on bone morphogenetic protein-2 (BMP) signal pathway in MG-63 cells. Cells were differentiated for 4 days in differentiation medium in the presence or absence of melanin (250 µg/mL). (A) Protein expression of BMP signaling pathway-related markers such as BMP-2, runt-related transcription factor 2 (RUNX2), and small mothers against decapentaplegic homologs 5 (SMAD5) was detected by Western blot. (B) Relative expression was quantified by image J and calculated according to the reference bands of β-actin. Each value is expressed as mean ± SD (n = 3). Values with different letters were found to be significantly different using th e Duncan's multiple range test ( P

Techniques Used: Expressing, Western Blot

51) Product Images from "Lactobacillus casei Zhang and vitamin K2 prevent intestinal tumorigenesis in mice via adiponectin-elevated different signaling pathways"

Article Title: Lactobacillus casei Zhang and vitamin K2 prevent intestinal tumorigenesis in mice via adiponectin-elevated different signaling pathways

Journal: Oncotarget

doi: 10.18632/oncotarget.15791

Liver and colonic expression of proteins involved in murine CRC ( A ) Immunoblot of hepatic CLCN3, CLCN4, NF-κB, AMPK, p-AMPK, VDR, GSK-3β and Caspase3 expression from NC, CC, ZA and VK mice. Equal amounts of protein extract 90 μg were loaded on each lane. ( B ) Immunoblot of colonic Claudin15, Clic4, TGF-β and β-actin from NC, CC, ZA and VK mice. ( C ) CLCN3 immunostaining in liver and colon tissues of representative CC and ZA mice. Immunostaining with anti-CLCN3 antibody (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) of fixed liver and colon sections.
Figure Legend Snippet: Liver and colonic expression of proteins involved in murine CRC ( A ) Immunoblot of hepatic CLCN3, CLCN4, NF-κB, AMPK, p-AMPK, VDR, GSK-3β and Caspase3 expression from NC, CC, ZA and VK mice. Equal amounts of protein extract 90 μg were loaded on each lane. ( B ) Immunoblot of colonic Claudin15, Clic4, TGF-β and β-actin from NC, CC, ZA and VK mice. ( C ) CLCN3 immunostaining in liver and colon tissues of representative CC and ZA mice. Immunostaining with anti-CLCN3 antibody (red), and 4′,6-diamidino-2-phenylindole (DAPI; blue) of fixed liver and colon sections.

Techniques Used: Expressing, Mouse Assay, Immunostaining

52) Product Images from "Suppression of Hepatic Epithelial-to-Mesenchymal Transition by Melittin via Blocking of TGFβ/Smad and MAPK-JNK Signaling Pathways"

Article Title: Suppression of Hepatic Epithelial-to-Mesenchymal Transition by Melittin via Blocking of TGFβ/Smad and MAPK-JNK Signaling Pathways

Journal: Toxins

doi: 10.3390/toxins9040138

MEL antagonizes the TGF-β1-stimulated non-Smad/MAPK signal pathway. ( A ) Immunoblot of mitogen-activated protein kinase (MAPK). MEL strongly inhibited the TGF-β1-stimulated pJNK1/2; ( B ) AML12 was transfected with control (Con) or specific c-Jun N-terminal kinase (JNK)1/2 siRNA and then treated with TGF-β1 for 24 h or 48 h; ( C ) Smad4 siRNA, Smad3 inhibitor (SIS) and MEL inhibited TGF-β1-stimulated pJNK1/2 expression; ( D ) JNK siRNA, JNK inhibitor (SP, SP600125) and MEL inhibited TGF-β1-stimulated Smad4 expression. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p
Figure Legend Snippet: MEL antagonizes the TGF-β1-stimulated non-Smad/MAPK signal pathway. ( A ) Immunoblot of mitogen-activated protein kinase (MAPK). MEL strongly inhibited the TGF-β1-stimulated pJNK1/2; ( B ) AML12 was transfected with control (Con) or specific c-Jun N-terminal kinase (JNK)1/2 siRNA and then treated with TGF-β1 for 24 h or 48 h; ( C ) Smad4 siRNA, Smad3 inhibitor (SIS) and MEL inhibited TGF-β1-stimulated pJNK1/2 expression; ( D ) JNK siRNA, JNK inhibitor (SP, SP600125) and MEL inhibited TGF-β1-stimulated Smad4 expression. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p

Techniques Used: Transfection, Expressing

Effect of MEL on Smad and non-Smad/MAPK signal pathway in vivo. ( A ) Typical examples of immunohistochemical staining of TGF-β1, pSmad2/3 and pJNK; ( B ) Immunoblot show that inhibition of TGF-β1, pSmad2/3, Smad4 and pJNK expression by MEL. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin; ( C ) Nuclear extracts were subjected to SBE DNA binding assay by EMSA. The data are representative of three similar experiments and quantified as mean values ± S.E. * p
Figure Legend Snippet: Effect of MEL on Smad and non-Smad/MAPK signal pathway in vivo. ( A ) Typical examples of immunohistochemical staining of TGF-β1, pSmad2/3 and pJNK; ( B ) Immunoblot show that inhibition of TGF-β1, pSmad2/3, Smad4 and pJNK expression by MEL. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin; ( C ) Nuclear extracts were subjected to SBE DNA binding assay by EMSA. The data are representative of three similar experiments and quantified as mean values ± S.E. * p

Techniques Used: In Vivo, Immunohistochemistry, Staining, Inhibition, Expressing, DNA Binding Assay

Effects of melittin (MEL) on TGF-β1-induced epithelial-to-mesenchymal transition (EMT) in vitro. Cells were pretreated for 1 h with MEL, followed by incubation with TGF-β1 for 48 h. ( A ) MEL inhibited the TGF-β1-stimulated EMT marker in AML12. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin; ( B ) E-cadherin, ZO-1, fibronectin and vimentin mRNA expression was analyzed by real-time PCR in vitro. The quantitative ratios are shown as relative optical density that are normalized to the expression of β-actin; ( C ) Immunofluorescence double staining for E-cadherin (green) and vimentin (red) in TGF-β1-stimulated AML12 after treatment of MEL. Cells was counterstained with Hoechst 33342 (blue). Magnifications ×200; ( D ) Real-time PCR of EMT related gene markers. The quantitative ratios are shown as relative optical density that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p
Figure Legend Snippet: Effects of melittin (MEL) on TGF-β1-induced epithelial-to-mesenchymal transition (EMT) in vitro. Cells were pretreated for 1 h with MEL, followed by incubation with TGF-β1 for 48 h. ( A ) MEL inhibited the TGF-β1-stimulated EMT marker in AML12. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin; ( B ) E-cadherin, ZO-1, fibronectin and vimentin mRNA expression was analyzed by real-time PCR in vitro. The quantitative ratios are shown as relative optical density that are normalized to the expression of β-actin; ( C ) Immunofluorescence double staining for E-cadherin (green) and vimentin (red) in TGF-β1-stimulated AML12 after treatment of MEL. Cells was counterstained with Hoechst 33342 (blue). Magnifications ×200; ( D ) Real-time PCR of EMT related gene markers. The quantitative ratios are shown as relative optical density that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p

Techniques Used: In Vitro, Incubation, Marker, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Double Staining

MEL suppressed CCl 4 -induced EMT in vivo. ( A ) Immunofluorescence double staining for E-cadherin (green) and vimentin (red) in CCl 4 induced changes in EMT markers. Cells was counterstained with Hoechst 33342 (blue). Magnifications ×200; ( B ) Immunoblot results show the effects of MEL on the inhibition of CCl 4 induced changes in EMT markers including E-cadherin and vimentin. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p
Figure Legend Snippet: MEL suppressed CCl 4 -induced EMT in vivo. ( A ) Immunofluorescence double staining for E-cadherin (green) and vimentin (red) in CCl 4 induced changes in EMT markers. Cells was counterstained with Hoechst 33342 (blue). Magnifications ×200; ( B ) Immunoblot results show the effects of MEL on the inhibition of CCl 4 induced changes in EMT markers including E-cadherin and vimentin. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p

Techniques Used: In Vivo, Immunofluorescence, Double Staining, Inhibition, Expressing

MET antagonizes the TGF-β1-stimulated Smad signal pathway in vitro. Cells were pretreated for 1 h with MEL, followed by incubation with TGF-β1 for 24 h. ( A ) Nuclear extracts were subjected to SBE DNA binding and Smad4 antibody (Ab) assay by EMSA supershift assay; ( B ) MEL inhibits TGF-β1-dependent transcriptional activity of the CAGA x12 -Luc reporter in a dose-dependent manner; ( C ) Immunoblot of the effect of MEL on the TGF-β1-stimulated pSmad2, pSamd3 and Smad4; ( D ) AML12 was transfected with control (Con) or specific Smad4 siRNA and then treated with TGF-β1 for 24 h or 48 h. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p
Figure Legend Snippet: MET antagonizes the TGF-β1-stimulated Smad signal pathway in vitro. Cells were pretreated for 1 h with MEL, followed by incubation with TGF-β1 for 24 h. ( A ) Nuclear extracts were subjected to SBE DNA binding and Smad4 antibody (Ab) assay by EMSA supershift assay; ( B ) MEL inhibits TGF-β1-dependent transcriptional activity of the CAGA x12 -Luc reporter in a dose-dependent manner; ( C ) Immunoblot of the effect of MEL on the TGF-β1-stimulated pSmad2, pSamd3 and Smad4; ( D ) AML12 was transfected with control (Con) or specific Smad4 siRNA and then treated with TGF-β1 for 24 h or 48 h. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p

Techniques Used: In Vitro, Incubation, Binding Assay, Activity Assay, Transfection, Expressing

53) Product Images from "Selective expression of Parkinson's disease-related Leucine-rich repeat kinase 2 G2019S missense mutation in midbrain dopaminergic neurons impairs dopamine release and dopaminergic gene expression"

Article Title: Selective expression of Parkinson's disease-related Leucine-rich repeat kinase 2 G2019S missense mutation in midbrain dopaminergic neurons impairs dopamine release and dopaminergic gene expression

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddv249

Transgenic expression of human LRRK2 selectively in mouse midbrain DA neurons. ( A ) Schematic diagram depicts the generation of LRRK2 bigenic mice using a tetracycline-regulated gene expression system. Sample images show staining of HA-tagged LRRK2 (green) and TH (red) in sagittal section of 1-month-old tetO -G2019S single transgenic and G2019S bigenic mice. Scale bars: 400 µm (top two panels) and 1 mm (bottom panel). ( B ) Subcellular distribution of LRRK2 in SNpc DA neurons. Immunofluorescent images show the distribution of transgenic human LRRK2 at soma, dendrites (den.), axons and axon terminal striatum of SNpc DA neurons in 1-month-old G2019S mice. Human LRRK2 (green) was revealed by HA antibody staining; DA neurons were visualized by TH immunostaining (red). Scale bar: 20 µm (top panel) and 10 µm (lower panels). ( C ) Western blots show the levels of transgenic and endogenous LRRK2 expression in the striatum (ST) and midbrain (MB) homogenates of 1-month-old nTg, WT and G2019S mice. The cerebral cortex (CX) of Lrrk2 homozygous knockout (KO) mice was used as a negative control for the specificity of LRRK2 antibody. TH, dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP32) and β-actin were used as the loading controls. Bar graph estimates the level of LRRK2 overexpression (normalized against β-actin expression) in the striatum and midbrain of nTg and bigenic mice. ( D ) Representative images show mouse and human LRRK2 expressions in SNpc DA neurons of 1-month-old G2019S and nTg mice. Scale bar: 20 µm. Scatter plot estimates the LRRK2 protein expression levels. Data were presented as mean ± SEM. ( E ) Western blots show the expression of transgenic and endogenous LRRK2 proteins in the midbrain of post-natal day 1 (P1), 1-month (1M) and 3-month (3M)-old nTg and G2019S bigenic mice treated with (+) or without (−) doxycycline (DOX). TH and β-actin were used as the loading controls.
Figure Legend Snippet: Transgenic expression of human LRRK2 selectively in mouse midbrain DA neurons. ( A ) Schematic diagram depicts the generation of LRRK2 bigenic mice using a tetracycline-regulated gene expression system. Sample images show staining of HA-tagged LRRK2 (green) and TH (red) in sagittal section of 1-month-old tetO -G2019S single transgenic and G2019S bigenic mice. Scale bars: 400 µm (top two panels) and 1 mm (bottom panel). ( B ) Subcellular distribution of LRRK2 in SNpc DA neurons. Immunofluorescent images show the distribution of transgenic human LRRK2 at soma, dendrites (den.), axons and axon terminal striatum of SNpc DA neurons in 1-month-old G2019S mice. Human LRRK2 (green) was revealed by HA antibody staining; DA neurons were visualized by TH immunostaining (red). Scale bar: 20 µm (top panel) and 10 µm (lower panels). ( C ) Western blots show the levels of transgenic and endogenous LRRK2 expression in the striatum (ST) and midbrain (MB) homogenates of 1-month-old nTg, WT and G2019S mice. The cerebral cortex (CX) of Lrrk2 homozygous knockout (KO) mice was used as a negative control for the specificity of LRRK2 antibody. TH, dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP32) and β-actin were used as the loading controls. Bar graph estimates the level of LRRK2 overexpression (normalized against β-actin expression) in the striatum and midbrain of nTg and bigenic mice. ( D ) Representative images show mouse and human LRRK2 expressions in SNpc DA neurons of 1-month-old G2019S and nTg mice. Scale bar: 20 µm. Scatter plot estimates the LRRK2 protein expression levels. Data were presented as mean ± SEM. ( E ) Western blots show the expression of transgenic and endogenous LRRK2 proteins in the midbrain of post-natal day 1 (P1), 1-month (1M) and 3-month (3M)-old nTg and G2019S bigenic mice treated with (+) or without (−) doxycycline (DOX). TH and β-actin were used as the loading controls.

Techniques Used: Transgenic Assay, Expressing, Mouse Assay, Staining, Immunostaining, Western Blot, Knock-Out, Negative Control, Over Expression

Alternations of DA gene expression in G2019S mice. ( A ) Western blots show TH, VMAT2, DAT and ALDH1A1 expression in striatum homogenates from 1-, 6- and 18-month-old nTg, WT and G2019S (GS) mice. β-actin was used as the loading control. ( B and C ) Scatter plots quantify the relative expression levels of TH, VMAT2, DAT and ALDH1A1 in striatum homogenates of nTg, WT and G2019S (GS) mice at 1 (B) and 18 months of age (C). N ≥ 3 animals per genotype per age point. Data were presented as mean ± SEM. One-way ANOVA plus Tukey's post hoc test. * P
Figure Legend Snippet: Alternations of DA gene expression in G2019S mice. ( A ) Western blots show TH, VMAT2, DAT and ALDH1A1 expression in striatum homogenates from 1-, 6- and 18-month-old nTg, WT and G2019S (GS) mice. β-actin was used as the loading control. ( B and C ) Scatter plots quantify the relative expression levels of TH, VMAT2, DAT and ALDH1A1 in striatum homogenates of nTg, WT and G2019S (GS) mice at 1 (B) and 18 months of age (C). N ≥ 3 animals per genotype per age point. Data were presented as mean ± SEM. One-way ANOVA plus Tukey's post hoc test. * P

Techniques Used: Expressing, Mouse Assay, Western Blot

54) Product Images from "Hypoxia induces H19 expression through direct and indirect Hif-1α activity, promoting oncogenic effects in glioblastoma"

Article Title: Hypoxia induces H19 expression through direct and indirect Hif-1α activity, promoting oncogenic effects in glioblastoma

Journal: Scientific Reports

doi: 10.1038/srep45029

Expression levels of Hif-1α and H19 in human glioblastoma (GBM) and adjacent normal brain tissues (NBT). ( A ) The levels of Hif-1α in GBM and NBT were detected by western blot. The expression of Hif-1α was normalized to β-actin. ( B ) The levels of H19 in GBM and NBT were detected by QPCR. The expression of H19 was normalized to β-actin. ( C ) The correlation between Hif-1α and H19 in GBM specimens or in the subgroup on the basis of PTEN status. Pearson’s correlation coefficient was applied. ( D ) Immunohistochemistry analysis of Hif-1α and H19 in two representative tissues (one NBT specimen and one GBM specimen).
Figure Legend Snippet: Expression levels of Hif-1α and H19 in human glioblastoma (GBM) and adjacent normal brain tissues (NBT). ( A ) The levels of Hif-1α in GBM and NBT were detected by western blot. The expression of Hif-1α was normalized to β-actin. ( B ) The levels of H19 in GBM and NBT were detected by QPCR. The expression of H19 was normalized to β-actin. ( C ) The correlation between Hif-1α and H19 in GBM specimens or in the subgroup on the basis of PTEN status. Pearson’s correlation coefficient was applied. ( D ) Immunohistochemistry analysis of Hif-1α and H19 in two representative tissues (one NBT specimen and one GBM specimen).

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemistry

H19 plays an important role in hypoxia-driven migration and invasion in U87 and U251 cells. H19 regulates EMT-related protein expression, affecting migration and invasion in U87 and U251 cells. U87 and U251 cells were transfected with control siRNA or H19 siRNA. The samples are derive from the same experiment and that blots were processed in parallel. ( A ) Increased H19 expression correlates with glioma grade and confers a poor prognosis in high grade glioma (HGG) and GBM patients. Levels of H19 were analyzed in different glioma tissues of TCGA data. Kaplan–Meier survival curves for H19 expression in HGG and GBM patients of TCGA data. ( B ) The scratch-wound gap was photographed before and after 24 h incubation under normoxia and hypoxia. After performing the wound healing test. The results were analyzed by measuring the range of migrating cells from 3 different fields for each wound. ( C ) Transwell migration assays were used to evaluate glioblastoma cell invasion. Migrated cells with normoxic or hypoxic treatment for 24 h were stained with crystal violet and counted for statistical analysis. ( D ) EMT-related proteins in the three cell groups were examined by western blot and normalized to β-actin. (*p
Figure Legend Snippet: H19 plays an important role in hypoxia-driven migration and invasion in U87 and U251 cells. H19 regulates EMT-related protein expression, affecting migration and invasion in U87 and U251 cells. U87 and U251 cells were transfected with control siRNA or H19 siRNA. The samples are derive from the same experiment and that blots were processed in parallel. ( A ) Increased H19 expression correlates with glioma grade and confers a poor prognosis in high grade glioma (HGG) and GBM patients. Levels of H19 were analyzed in different glioma tissues of TCGA data. Kaplan–Meier survival curves for H19 expression in HGG and GBM patients of TCGA data. ( B ) The scratch-wound gap was photographed before and after 24 h incubation under normoxia and hypoxia. After performing the wound healing test. The results were analyzed by measuring the range of migrating cells from 3 different fields for each wound. ( C ) Transwell migration assays were used to evaluate glioblastoma cell invasion. Migrated cells with normoxic or hypoxic treatment for 24 h were stained with crystal violet and counted for statistical analysis. ( D ) EMT-related proteins in the three cell groups were examined by western blot and normalized to β-actin. (*p

Techniques Used: Migration, Expressing, Transfection, Incubation, Staining, Western Blot

H19 expression is elevated under hypoxia and Hif-1α is a critical factor responsible for the induction of H19 RNA in U87 and U251 cells. The levels of H19 RNA normalized to β-actin were quantified by qPCR. Protein levels were detected by western blot and normalized to β-actin levels. ( A ) U87 and U251 cells were exposed to hypoxia (2% O 2 ) for the indicated times. H19 levels at each time point (bar graphs) were normalized to normoxic controls. ( B ) U87 and U251 cells were transfected with Hif-1α expression plasmid or empty vector before culture under hypoxia for 24 h. ( C ) U87 and U251 cells were transfected with a control siRNA, or Hif-1α siRNA and cultured under hypoxia for 24 h. All experiments were repeated three times with similar results. (*p
Figure Legend Snippet: H19 expression is elevated under hypoxia and Hif-1α is a critical factor responsible for the induction of H19 RNA in U87 and U251 cells. The levels of H19 RNA normalized to β-actin were quantified by qPCR. Protein levels were detected by western blot and normalized to β-actin levels. ( A ) U87 and U251 cells were exposed to hypoxia (2% O 2 ) for the indicated times. H19 levels at each time point (bar graphs) were normalized to normoxic controls. ( B ) U87 and U251 cells were transfected with Hif-1α expression plasmid or empty vector before culture under hypoxia for 24 h. ( C ) U87 and U251 cells were transfected with a control siRNA, or Hif-1α siRNA and cultured under hypoxia for 24 h. All experiments were repeated three times with similar results. (*p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Cell Culture

55) Product Images from "Activated ?-catenin Forces N2A Cell-derived Neurons Back to Tumor-like Neuroblasts and Positively Correlates with a Risk for Human Neuroblastoma"

Article Title: Activated ?-catenin Forces N2A Cell-derived Neurons Back to Tumor-like Neuroblasts and Positively Correlates with a Risk for Human Neuroblastoma

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.3520

Activated β-catenin expression in clinical variants of human neuroblastoma. (A) Representative data of tissue sample Western blot. The expression of β-catenin protein in cytosol and nuclei was indicated. β-actin protein was used as a positive control for cytosol and negative control for nuclei, and H3-histone was used as a positive control for nuclei and negative control for cytosol. The symbols (1, 2, 3) indicated low, intermediate and high risk neuroblastoma tissue samples, respectively. (B) Quantitative assessment of the relative β-catenin protein levels of all tissue samples with Image J. The relative amount of β-catenin protein in cytosol and nuclei was normalized with β-actin and H3-histone, respectively. (C) (a-c) 3 stroma-poor deteriorated neuroblastomas with a favorable clinical outcome (low risk, a), 4 mixed histology with stroma-poor and stroma-rich regions (intermediate risk, b) and 5 stroma-rich neuroblastomas with poor outcome (high risk, c) were checked for their nucleus β-catenin levels (d for low risk, e for intermediate risk and f for high risk). (d-l) The level of β-catenin in nucleus was higher in high risk neuroblastomas than in intermediate risk neuroblastomas and low risk neuroblastomas. Scale bar= 50 μm for a-c, =25 μm for d-l.
Figure Legend Snippet: Activated β-catenin expression in clinical variants of human neuroblastoma. (A) Representative data of tissue sample Western blot. The expression of β-catenin protein in cytosol and nuclei was indicated. β-actin protein was used as a positive control for cytosol and negative control for nuclei, and H3-histone was used as a positive control for nuclei and negative control for cytosol. The symbols (1, 2, 3) indicated low, intermediate and high risk neuroblastoma tissue samples, respectively. (B) Quantitative assessment of the relative β-catenin protein levels of all tissue samples with Image J. The relative amount of β-catenin protein in cytosol and nuclei was normalized with β-actin and H3-histone, respectively. (C) (a-c) 3 stroma-poor deteriorated neuroblastomas with a favorable clinical outcome (low risk, a), 4 mixed histology with stroma-poor and stroma-rich regions (intermediate risk, b) and 5 stroma-rich neuroblastomas with poor outcome (high risk, c) were checked for their nucleus β-catenin levels (d for low risk, e for intermediate risk and f for high risk). (d-l) The level of β-catenin in nucleus was higher in high risk neuroblastomas than in intermediate risk neuroblastomas and low risk neuroblastomas. Scale bar= 50 μm for a-c, =25 μm for d-l.

Techniques Used: Expressing, Western Blot, Positive Control, Negative Control

56) Product Images from "Activation of PKA, p38 MAPK and ERK1/2 by gonadotropins in cumulus cells is critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation of porcine COCs"

Article Title: Activation of PKA, p38 MAPK and ERK1/2 by gonadotropins in cumulus cells is critical for induction of EGF-like factor and TACE/ADAM17 gene expression during in vitro maturation of porcine COCs

Journal: Journal of Ovarian Research

doi: 10.1186/1757-2215-2-20

Effect of H89, SB203580 or U0126 on ERK1/2 phosphorylation in cumulus cells . For reference, the COC that were cultured without FSH and LH for 5 h value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 5 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 5 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH SB230580 and EGF for 5 h. The respective value of protein levels of ERK1/2 phosphorylation were normalized according to those of β-ACTIN to evaluate arbitrary units of the relative abundance. FSH(-): COCs were cultured without FSH and LH for 5 h; FSH(+): COCs were cultured with FSH and LH for 5 h; EGF(-): COCs were cultured without EGF for 5 h; EGF(+): COCs were cultured with EGF for 5 h; +H89: COCs were cultured with H89 for 5 h; +SB: COCs were cultured with SB203580 for 5 h; +U0126: COCs were cultured with U0126 for 5 h
Figure Legend Snippet: Effect of H89, SB203580 or U0126 on ERK1/2 phosphorylation in cumulus cells . For reference, the COC that were cultured without FSH and LH for 5 h value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 5 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 5 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH SB230580 and EGF for 5 h. The respective value of protein levels of ERK1/2 phosphorylation were normalized according to those of β-ACTIN to evaluate arbitrary units of the relative abundance. FSH(-): COCs were cultured without FSH and LH for 5 h; FSH(+): COCs were cultured with FSH and LH for 5 h; EGF(-): COCs were cultured without EGF for 5 h; EGF(+): COCs were cultured with EGF for 5 h; +H89: COCs were cultured with H89 for 5 h; +SB: COCs were cultured with SB203580 for 5 h; +U0126: COCs were cultured with U0126 for 5 h

Techniques Used: Cell Culture

Effect of H89, SB203580, LY294002 or U0126 on Areg (A), Ereg (B) or Tace/Adam17 (C) mRNA . For reference, the 0 h COC value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 2.5 h. The respective value of among Areg , Ereg , and Adam17 mRNA were normalized according to those of β-actin mRNA to evaluate arbitrary units of the relative abundance of the targets. Free: COCs were cultured without FSH and LH for 2.5 h; Cont: COCs were cultured with FSH and LH for 2.5 h; H89: COCs were cultured with FSH, LH and H89 for 2.5 h; SB: COCs were cultured with FSH, LH and SB203580 for 2.5 h; LY: COCs were cultured with FSH, LH and LY294002 for 2.5 h; U0126: COCs were cultured with FSH, LH and U0126 for 2.5 h
Figure Legend Snippet: Effect of H89, SB203580, LY294002 or U0126 on Areg (A), Ereg (B) or Tace/Adam17 (C) mRNA . For reference, the 0 h COC value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 2.5 h. The respective value of among Areg , Ereg , and Adam17 mRNA were normalized according to those of β-actin mRNA to evaluate arbitrary units of the relative abundance of the targets. Free: COCs were cultured without FSH and LH for 2.5 h; Cont: COCs were cultured with FSH and LH for 2.5 h; H89: COCs were cultured with FSH, LH and H89 for 2.5 h; SB: COCs were cultured with FSH, LH and SB203580 for 2.5 h; LY: COCs were cultured with FSH, LH and LY294002 for 2.5 h; U0126: COCs were cultured with FSH, LH and U0126 for 2.5 h

Techniques Used: Cell Culture

Effect of H89, SB203580 or U0126 on expression of Has2 (A), Tnfaip6 (B) or Ptgs2 (C) mRNA . For reference, the 0 h COC value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 10 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 10 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 10 h. The respective value of among Has2 , Tnfaip6 and Ptgs2 mRNA were normalized according to those of β-actin mRNA to evaluate arbitrary units of the relative abundance of the targets. FSH(-): COCs were cultured without FSH and LH for 10 h; FSH(+): COCs were cultured with FSH and LH for 10 h; EGF(-): COCs were cultured without EGF for 10 h; EGF(+): COCs were cultured with EGF for 10 h; +H89: COCs were cultured with H89 for 10 h; +SB: COCs were cultured with SB203580 for 10 h; +U0126: COCs were cultured with U0126 for 10 h
Figure Legend Snippet: Effect of H89, SB203580 or U0126 on expression of Has2 (A), Tnfaip6 (B) or Ptgs2 (C) mRNA . For reference, the 0 h COC value was set as 1 and the data presented as the fold strength. Values are mean +/- SEM of 3 replicates. *: The significant differences were observed as compared with that in COCs cultured with FSH and LH for 10 h. **: The significant differences were observed as compared with that in COCs cultured with FSH, LH, H89 and EGF for 10 h. ***: The significant differences were observed as compared with that in COCs cultured with FSH, LH, SB230580 and EGF for 10 h. The respective value of among Has2 , Tnfaip6 and Ptgs2 mRNA were normalized according to those of β-actin mRNA to evaluate arbitrary units of the relative abundance of the targets. FSH(-): COCs were cultured without FSH and LH for 10 h; FSH(+): COCs were cultured with FSH and LH for 10 h; EGF(-): COCs were cultured without EGF for 10 h; EGF(+): COCs were cultured with EGF for 10 h; +H89: COCs were cultured with H89 for 10 h; +SB: COCs were cultured with SB203580 for 10 h; +U0126: COCs were cultured with U0126 for 10 h

Techniques Used: Expressing, Cell Culture

57) Product Images from "Celastrol Protects against Antimycin A-Induced Insulin Resistance in Human Skeletal Muscle Cells"

Article Title: Celastrol Protects against Antimycin A-Induced Insulin Resistance in Human Skeletal Muscle Cells

Journal: Molecules

doi: 10.3390/molecules20058242

Effects of AMA and celastrol treatments on the protein expression of AMPK (Thr172) and PKC θ (Ser643/676) in human skeletal muscle-derived myoblast. Cells were cultured in media containing 30 µM AMA in the absence and presence of 15 nM celastrol for 48 h. Thereafter, cell lysates were subjected to western blot analysis ( A ). The percentage of specific and total protein phosphorylation was calculated to determine the relative level of exact amino acid residue phosphorylation ( A–C ). β-actin was used as internal protein loading control. Protein levels for each corresponding antibody obtained from densitometry were normalized to the β-actin signal. * p
Figure Legend Snippet: Effects of AMA and celastrol treatments on the protein expression of AMPK (Thr172) and PKC θ (Ser643/676) in human skeletal muscle-derived myoblast. Cells were cultured in media containing 30 µM AMA in the absence and presence of 15 nM celastrol for 48 h. Thereafter, cell lysates were subjected to western blot analysis ( A ). The percentage of specific and total protein phosphorylation was calculated to determine the relative level of exact amino acid residue phosphorylation ( A–C ). β-actin was used as internal protein loading control. Protein levels for each corresponding antibody obtained from densitometry were normalized to the β-actin signal. * p

Techniques Used: Expressing, Derivative Assay, Cell Culture, Western Blot

AMA treatment of human myotubes in the absence and presence of celastrol. Cells were grown in 6-well plates and treated with AMA and celastrol for 48 h. The representative images of western blot analysis ( A ) of the relative expression level of NF-κB ( B ) and IκBα ( C ) activity was measured and quantified. β-actin was used as a loading control. Protein levels calculated by densitometry were normalized relative to β-actin signals. * p
Figure Legend Snippet: AMA treatment of human myotubes in the absence and presence of celastrol. Cells were grown in 6-well plates and treated with AMA and celastrol for 48 h. The representative images of western blot analysis ( A ) of the relative expression level of NF-κB ( B ) and IκBα ( C ) activity was measured and quantified. β-actin was used as a loading control. Protein levels calculated by densitometry were normalized relative to β-actin signals. * p

Techniques Used: Western Blot, Expressing, Activity Assay

Effects of AMA and celastrol treatment on insulin signaling pathways and glucose transporters (GLUT4) of human myotubes. Cells were treated with AMA (30 µM) for 48 h before incubation with celastrol (15 nM). β-actin was used as an internal protein loading control. Protein levels for each corresponding antibody obtained from densitometry were normalized to the β-actin signal ( A–F ). * p
Figure Legend Snippet: Effects of AMA and celastrol treatment on insulin signaling pathways and glucose transporters (GLUT4) of human myotubes. Cells were treated with AMA (30 µM) for 48 h before incubation with celastrol (15 nM). β-actin was used as an internal protein loading control. Protein levels for each corresponding antibody obtained from densitometry were normalized to the β-actin signal ( A–F ). * p

Techniques Used: Incubation

Effects of AMA and celastrol treatments on mitochondrial fusion and fission of human myotubes. Representative images of western blot analysis ( A ) on the relative expression of ( B ) mfn1, ( C ) mfn2 and ( D ) drp1 proteins were quantified with the corresponding antibodies using a densitometer. β-actin was used as loading control. Protein levels calculated by densitometry were normalized relative to β-actin signals. * p
Figure Legend Snippet: Effects of AMA and celastrol treatments on mitochondrial fusion and fission of human myotubes. Representative images of western blot analysis ( A ) on the relative expression of ( B ) mfn1, ( C ) mfn2 and ( D ) drp1 proteins were quantified with the corresponding antibodies using a densitometer. β-actin was used as loading control. Protein levels calculated by densitometry were normalized relative to β-actin signals. * p

Techniques Used: Western Blot, Expressing

58) Product Images from "Anti-Diabetic Effects of Acankoreagenin from the Leaves of Acanthopanax Gracilistylus Herb in RIN-m5F Cells via Suppression of NF-κB Activation"

Article Title: Anti-Diabetic Effects of Acankoreagenin from the Leaves of Acanthopanax Gracilistylus Herb in RIN-m5F Cells via Suppression of NF-κB Activation

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules23040958

Effects of acankoreagenin on the expression of insulin secretion-related gene in RIN-m5F cells. Expression was determined after culturing the cells in six-well plate containing 20 mM glucose and then treated in the absence (vehicle) or various concentrations of acankoreagenin for 24 h. Expression levels were analyzed by real-time RT-PCR. β-actin mRNA was used as an internal control. ** p
Figure Legend Snippet: Effects of acankoreagenin on the expression of insulin secretion-related gene in RIN-m5F cells. Expression was determined after culturing the cells in six-well plate containing 20 mM glucose and then treated in the absence (vehicle) or various concentrations of acankoreagenin for 24 h. Expression levels were analyzed by real-time RT-PCR. β-actin mRNA was used as an internal control. ** p

Techniques Used: Expressing, Quantitative RT-PCR

Effects of acankoreagenin on the cytokine-induced activation of NF-κB in RIN-m5F cells. RIN-m5F cells were pretreated with the indicated concentrations of samples for 1 h and then IL-1β (10 ng/mL) and IFN-γ (100 ng/mL) were added for 24 h. Then iNOS protein expression, I-κBα degradation, and activation of NF-κB p65 in RIN-m5F cells were determined by western blotting. β-actin was used as loading controls for cytosilic. Three independent experiments were done and all gave similar results.
Figure Legend Snippet: Effects of acankoreagenin on the cytokine-induced activation of NF-κB in RIN-m5F cells. RIN-m5F cells were pretreated with the indicated concentrations of samples for 1 h and then IL-1β (10 ng/mL) and IFN-γ (100 ng/mL) were added for 24 h. Then iNOS protein expression, I-κBα degradation, and activation of NF-κB p65 in RIN-m5F cells were determined by western blotting. β-actin was used as loading controls for cytosilic. Three independent experiments were done and all gave similar results.

Techniques Used: Activation Assay, Expressing, Western Blot

59) Product Images from "Increased abundance of the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1) in patients with obesity and type 2 diabetes: evidence for altered adiponectin signalling"

Article Title: Increased abundance of the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1) in patients with obesity and type 2 diabetes: evidence for altered adiponectin signalling

Journal: Diabetologia

doi: 10.1007/s00125-011-2173-x

Human skeletal muscle APPL1 protein level changes. a Vastus lateralis muscle biopsies were homogenised, separated by SDS-PAGE and proteins transferred electrophorectically to nitrocellulose membrane. Membranes were exposed to anti-APPL1 or β-actin antibody and visualised by chemiluminescence. Density of each band was determined using Versadoc software. b Protein quantification, expressed as ratio of APPL1:β-actin, showing that APPL1 levels increased significantly in type 2 diabetic participants as compared with lean control and obese control participants. Values mean±SEM (error bars). c Whole skeletal muscle homogenates were used for peak area analysis. Biopsies were homogenised, separated by SDS-PAGE and stained with Coomassie blue. Bands were cut for APPL1 and β-actin, subjected to trypsin digestion and quantified by MS as described. Protein levels are expressed as ratio of APPL1: β-actin (×10 2 ). Values are mean±SEM (error bars); n = 8 or 9 per group. * p
Figure Legend Snippet: Human skeletal muscle APPL1 protein level changes. a Vastus lateralis muscle biopsies were homogenised, separated by SDS-PAGE and proteins transferred electrophorectically to nitrocellulose membrane. Membranes were exposed to anti-APPL1 or β-actin antibody and visualised by chemiluminescence. Density of each band was determined using Versadoc software. b Protein quantification, expressed as ratio of APPL1:β-actin, showing that APPL1 levels increased significantly in type 2 diabetic participants as compared with lean control and obese control participants. Values mean±SEM (error bars). c Whole skeletal muscle homogenates were used for peak area analysis. Biopsies were homogenised, separated by SDS-PAGE and stained with Coomassie blue. Bands were cut for APPL1 and β-actin, subjected to trypsin digestion and quantified by MS as described. Protein levels are expressed as ratio of APPL1: β-actin (×10 2 ). Values are mean±SEM (error bars); n = 8 or 9 per group. * p

Techniques Used: SDS Page, Software, Staining, Mass Spectrometry

Human skeletal muscle expression of ( a ) APPL1 and ( b ) ADIPOR1 . Quantitative real-time PCR was used to quantify mRNA. mRNA fold changes were calculated relative to average lean control value quantified relative to β-actin. mRNA expression and fold changes are expressed using the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {2^{{ - \Delta \Delta {{\text{C}}_{\text{t}}}}}} $$\end{document} method [ 32 ]. * p
Figure Legend Snippet: Human skeletal muscle expression of ( a ) APPL1 and ( b ) ADIPOR1 . Quantitative real-time PCR was used to quantify mRNA. mRNA fold changes were calculated relative to average lean control value quantified relative to β-actin. mRNA expression and fold changes are expressed using the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {2^{{ - \Delta \Delta {{\text{C}}_{\text{t}}}}}} $$\end{document} method [ 32 ]. * p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

60) Product Images from "Exosomal miR‐95‐5p regulates chondrogenesis and cartilage degradation via histone deacetylase 2/8, et al. Exosomal miR‐95‐5p regulates chondrogenesis and cartilage degradation via histone deacetylase 2/8"

Article Title: Exosomal miR‐95‐5p regulates chondrogenesis and cartilage degradation via histone deacetylase 2/8, et al. Exosomal miR‐95‐5p regulates chondrogenesis and cartilage degradation via histone deacetylase 2/8

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13808

AC ‐miR‐95‐5p‐Exos regulates the expression of HDAC 2/8, Col2A1, aggrecan and SOX 9 during chondrogenesis. HMSC s were treated with TGF ‐β3 to induce chondrogenesis and co‐cultured with AC ‐miR‐95‐5p‐Exos or AC ‐anti‐miR‐95‐5p‐Exos. (A, I) The expression level of miR‐95‐5p were estimated by qRTPCR , while the expression levels of HDAC 2 (B, J, Q), HDAC 8 (C, K, Q), SOX 9 (D, L, Q), Aggrecan (E, M), COL 2A1 (F, N, Q), RUNX 2 (G, O, Q) and MMP 13 (H, P, Q) were estimated by both qRT ‐ PCR and western blotting. U6, GAPDH and β‐actin were used as endogenous controls. Data were presented as means ± standard deviation of three samples. * P
Figure Legend Snippet: AC ‐miR‐95‐5p‐Exos regulates the expression of HDAC 2/8, Col2A1, aggrecan and SOX 9 during chondrogenesis. HMSC s were treated with TGF ‐β3 to induce chondrogenesis and co‐cultured with AC ‐miR‐95‐5p‐Exos or AC ‐anti‐miR‐95‐5p‐Exos. (A, I) The expression level of miR‐95‐5p were estimated by qRTPCR , while the expression levels of HDAC 2 (B, J, Q), HDAC 8 (C, K, Q), SOX 9 (D, L, Q), Aggrecan (E, M), COL 2A1 (F, N, Q), RUNX 2 (G, O, Q) and MMP 13 (H, P, Q) were estimated by both qRT ‐ PCR and western blotting. U6, GAPDH and β‐actin were used as endogenous controls. Data were presented as means ± standard deviation of three samples. * P

Techniques Used: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Standard Deviation

61) Product Images from "High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates"

Article Title: High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1514018113

Time course of cytokine production and release in newborn monocytes. Newborn monocytes cultured in RPMI medium supplemented with 10% autologous plasma ( A–D ) or 10% charcoal-stripped FCS ( E ) were stimulated with E. coli , GBS (10 8 bacteria/mL), LPS (100 ng/mL), or Pam 3 CSK 4 (PAM, 1 µg/mL) or were exposed to estradiol (E, 10 −9 –10 −7 M), progesterone (P, 10 −7 –10 −5 M), or hydrocortisone (HCZ, 10 −8 –10 −6 M). TNF ( A ), IL-6 ( B ), and MIF ( C and E ) were measured by ELISA in supernatants collected at the indicated time ( A–D ) or 24 h ( E ). Intracellular MIF was detected by Western blotting, and densitometric values are expressed in A.U. relative to the expression of β-actin ( D ). Data represent means ± SEMs of 3–5 independent experiments performed in triplicates. * P
Figure Legend Snippet: Time course of cytokine production and release in newborn monocytes. Newborn monocytes cultured in RPMI medium supplemented with 10% autologous plasma ( A–D ) or 10% charcoal-stripped FCS ( E ) were stimulated with E. coli , GBS (10 8 bacteria/mL), LPS (100 ng/mL), or Pam 3 CSK 4 (PAM, 1 µg/mL) or were exposed to estradiol (E, 10 −9 –10 −7 M), progesterone (P, 10 −7 –10 −5 M), or hydrocortisone (HCZ, 10 −8 –10 −6 M). TNF ( A ), IL-6 ( B ), and MIF ( C and E ) were measured by ELISA in supernatants collected at the indicated time ( A–D ) or 24 h ( E ). Intracellular MIF was detected by Western blotting, and densitometric values are expressed in A.U. relative to the expression of β-actin ( D ). Data represent means ± SEMs of 3–5 independent experiments performed in triplicates. * P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

62) Product Images from "Resveratrol Protects SR-B1 levels In keratinocytes Exposed to Cigarette Smoke"

Article Title: Resveratrol Protects SR-B1 levels In keratinocytes Exposed to Cigarette Smoke

Journal: Free radical biology & medicine

doi: 10.1016/j.freeradbiomed.2014.01.007

Effect of resveratrol with or without CS exposure on decreased HNE protein adducts. Representative Western blot in the upper panel. Quantification of the HNE bands is shown in the bottom panel. Data are expressed in arbitrary units (averages of five different experiments ± SEM, * vs. control; # vs. CS). β-actin was used as loading control.
Figure Legend Snippet: Effect of resveratrol with or without CS exposure on decreased HNE protein adducts. Representative Western blot in the upper panel. Quantification of the HNE bands is shown in the bottom panel. Data are expressed in arbitrary units (averages of five different experiments ± SEM, * vs. control; # vs. CS). β-actin was used as loading control.

Techniques Used: Western Blot

63) Product Images from "Regulation of Notch 1 signaling in THP-1 cells enhances M2 macrophage differentiation"

Article Title: Regulation of Notch 1 signaling in THP-1 cells enhances M2 macrophage differentiation

Journal: American Journal of Physiology - Heart and Circulatory Physiology

doi: 10.1152/ajpheart.00896.2013

siRNA knockdown of Notch1R augments Notch 1 activation and signaling. A , top : representative blots from all control and experimental groups showing Notch 1, cleaved Notch 1 (C-Notch 1), and β-actin control expression. A , middle : quantified Notch
Figure Legend Snippet: siRNA knockdown of Notch1R augments Notch 1 activation and signaling. A , top : representative blots from all control and experimental groups showing Notch 1, cleaved Notch 1 (C-Notch 1), and β-actin control expression. A , middle : quantified Notch

Techniques Used: Activation Assay, Expressing

Treatment with DAPT diminishes Notch 1 activation and signaling. A, top : representative blots from all control and experimental groups showing Notch 1, cleaved Notch 1 (C-Notch 1), and β-actin control expression. A , middle : quantified Notch 1
Figure Legend Snippet: Treatment with DAPT diminishes Notch 1 activation and signaling. A, top : representative blots from all control and experimental groups showing Notch 1, cleaved Notch 1 (C-Notch 1), and β-actin control expression. A , middle : quantified Notch 1

Techniques Used: Activation Assay, Expressing

64) Product Images from "Tumor necrosis factor-α promotes the expression of excitatory amino-acid transporter 2 in astrocytes: Optimal concentration and incubation time"

Article Title: Tumor necrosis factor-α promotes the expression of excitatory amino-acid transporter 2 in astrocytes: Optimal concentration and incubation time

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2014.2024

Expression levels of EAATs in astrocytes treated with TNF-α for different time-periods. (A) Representative western blot analysis of EAAT1, EAAT2, GFAP and β-actin. (B) Densitometric analysis showing the expression levels of the EAATs or GFAP normalized to β-actin. Data are presented as the mean ± standard deviation (n=6). ** P
Figure Legend Snippet: Expression levels of EAATs in astrocytes treated with TNF-α for different time-periods. (A) Representative western blot analysis of EAAT1, EAAT2, GFAP and β-actin. (B) Densitometric analysis showing the expression levels of the EAATs or GFAP normalized to β-actin. Data are presented as the mean ± standard deviation (n=6). ** P

Techniques Used: Expressing, Western Blot, Standard Deviation

Expression of EAATs in astrocytes treated with different doses of TNF-α. (A) Representative western blot analysis of EAAT1, EAAT2, GFAP and β-actin. (B) Densitometric analysis showing the expression of EAATs or GFAP normalized to β-actin. Data are presented as the mean ± standard deviation (n=5). * P
Figure Legend Snippet: Expression of EAATs in astrocytes treated with different doses of TNF-α. (A) Representative western blot analysis of EAAT1, EAAT2, GFAP and β-actin. (B) Densitometric analysis showing the expression of EAATs or GFAP normalized to β-actin. Data are presented as the mean ± standard deviation (n=5). * P

Techniques Used: Expressing, Western Blot, Standard Deviation

65) Product Images from "Anti-Tumor and Immune Enhancing Activities of Rice Bran Gramisterol on Acute Myelogenous Leukemia"

Article Title: Anti-Tumor and Immune Enhancing Activities of Rice Bran Gramisterol on Acute Myelogenous Leukemia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146869

Gramisterol and IFN-γ inhibited STAT3 phosphorylation in tumor cells. Western blot analysis of pSTAT3 signaling activation in WEHI-3 cells after treatment with i) gramisterol, ii) culture supernatant containing cytokines released from RBDS-treated leukemic mice spleen cells plus a minor supplementation of cytokine IFN-γ, iii) the combination of both gramisterol and the cytokines. β-actin was used as an internal control. The experiment was performed in triplicate. The relative expression of pSTAT3 was shown as mean±SEM.
Figure Legend Snippet: Gramisterol and IFN-γ inhibited STAT3 phosphorylation in tumor cells. Western blot analysis of pSTAT3 signaling activation in WEHI-3 cells after treatment with i) gramisterol, ii) culture supernatant containing cytokines released from RBDS-treated leukemic mice spleen cells plus a minor supplementation of cytokine IFN-γ, iii) the combination of both gramisterol and the cytokines. β-actin was used as an internal control. The experiment was performed in triplicate. The relative expression of pSTAT3 was shown as mean±SEM.

Techniques Used: Western Blot, Activation Assay, Mouse Assay, Expressing

Pathway analysis of gramisterol effect on immune cells. Western blot analysis of pSTAT1 level in spleen cells (lanes 1–4) and peritoneal cells (lanes 5–8) after treatments with i) gramisterol, ii) culture supernatant containing cytokines released from RBDS-treated leukemic mice spleen cells plus a minor supplementation of cytokine IFN-γ, iii) the combination of both gramisterol and the cytokines. β-actin was used as an internal control. The experiment was performed in triplicate. The relative expression of pSTAT1 is shown as mean±SEM.
Figure Legend Snippet: Pathway analysis of gramisterol effect on immune cells. Western blot analysis of pSTAT1 level in spleen cells (lanes 1–4) and peritoneal cells (lanes 5–8) after treatments with i) gramisterol, ii) culture supernatant containing cytokines released from RBDS-treated leukemic mice spleen cells plus a minor supplementation of cytokine IFN-γ, iii) the combination of both gramisterol and the cytokines. β-actin was used as an internal control. The experiment was performed in triplicate. The relative expression of pSTAT1 is shown as mean±SEM.

Techniques Used: Western Blot, Mouse Assay, Expressing

66) Product Images from "Berberine prevents progression from hepatic steatosis to steatohepatitis and fibrosis by reducing endoplasmic reticulum stress"

Article Title: Berberine prevents progression from hepatic steatosis to steatohepatitis and fibrosis by reducing endoplasmic reticulum stress

Journal: Scientific Reports

doi: 10.1038/srep20848

Molecular changes in liver tissue of db/db mice treated with or without BBR. ( A,B ) QPCR and Western blot analysis of liver tissue samples on mRNA ( A ) and protein ( B ) expression of lipogenesis-related genes. ( C ) Analysis of mRNA on hepatic fibrosis, inflammation and oxidative stress-related genes; β-actin was used as an internal control. ( D,E ) Liver mRNA ( D ) and protein ( E ) expression of ER stress-related genes. The data represent the mean ± SEM values. *P
Figure Legend Snippet: Molecular changes in liver tissue of db/db mice treated with or without BBR. ( A,B ) QPCR and Western blot analysis of liver tissue samples on mRNA ( A ) and protein ( B ) expression of lipogenesis-related genes. ( C ) Analysis of mRNA on hepatic fibrosis, inflammation and oxidative stress-related genes; β-actin was used as an internal control. ( D,E ) Liver mRNA ( D ) and protein ( E ) expression of ER stress-related genes. The data represent the mean ± SEM values. *P

Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing

BBR could function as a chemical chaperone and block TM challenge-induced ER stress and NAFLD. ( A ) Chaperone activity of BBR. The rate of aggregation of reduced-α-lactalbumin (r-LA) was measured in the presence or absence of drugs. n = 6. ( B ) in vivo refolding assay was performed as described in the Methods section. ( C–F ) C57BL/6J mice were treated with vehicle and BBR for 3 days and then were administered IP TM. After 24 hours, the liver tissues were analysed. ( C ) TG levels were examined in liver tissues. ( D ) The hydroxyproline content was measured. ( E,F ) Liver mRNA ( E ) and protein ( F ) expression of ER stress and lipogenesis-related genes. β-actin was used as internal control. The data are presented as the mean ± SEM values, which represent three independent experiments in triplicate. *P
Figure Legend Snippet: BBR could function as a chemical chaperone and block TM challenge-induced ER stress and NAFLD. ( A ) Chaperone activity of BBR. The rate of aggregation of reduced-α-lactalbumin (r-LA) was measured in the presence or absence of drugs. n = 6. ( B ) in vivo refolding assay was performed as described in the Methods section. ( C–F ) C57BL/6J mice were treated with vehicle and BBR for 3 days and then were administered IP TM. After 24 hours, the liver tissues were analysed. ( C ) TG levels were examined in liver tissues. ( D ) The hydroxyproline content was measured. ( E,F ) Liver mRNA ( E ) and protein ( F ) expression of ER stress and lipogenesis-related genes. β-actin was used as internal control. The data are presented as the mean ± SEM values, which represent three independent experiments in triplicate. *P

Techniques Used: Blocking Assay, Activity Assay, In Vivo, Mouse Assay, Expressing

Molecular changes in liver tissue of MCD diet-fed mice treated with or without BBR. ( A,B ) Liver mRNA ( A ) and protein ( B ) expression of and ER stress-related genes. ( C ) QPCR analysis of the liver tissue samples on lipogenesis related genes. ( D–F ) Analysis of mRNA on hepatic inflammation ( D ), oxidative stress ( E ), and fibrosis- ( F ) related genes; β-actin was used as internal control. The data represent the mean ± SEM values. *P
Figure Legend Snippet: Molecular changes in liver tissue of MCD diet-fed mice treated with or without BBR. ( A,B ) Liver mRNA ( A ) and protein ( B ) expression of and ER stress-related genes. ( C ) QPCR analysis of the liver tissue samples on lipogenesis related genes. ( D–F ) Analysis of mRNA on hepatic inflammation ( D ), oxidative stress ( E ), and fibrosis- ( F ) related genes; β-actin was used as internal control. The data represent the mean ± SEM values. *P

Techniques Used: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction

67) Product Images from "Suppression of type I collagen in human scleral fibroblasts treated with extremely low-frequency electromagnetic fields"

Article Title: Suppression of type I collagen in human scleral fibroblasts treated with extremely low-frequency electromagnetic fields

Journal: Molecular Vision

doi:

Electromagnetic fields and retinal cells can affect the phosphorylation of ERK1/2, P38, Akt and JNK in human scleral fibroblast cells. Effect of exposure to ELF-EMFs on the phosphorylation of ERK1/2, P38, Akt and JNK in HFSFs treated with RPE supernatant or not. A : Western blot analysis was performed using p-ERK1/2, p-p38, ERK1/2, p38, and β-actin antibodies, respectively. B : Densitometric quantification of Phosphorylation levels were obtained by optical density (O.D.) of protein bands normalized with O.D. of β-actin band. Values (mean±SD, n=3) were expressed as relative expression levels. A quantitative analysis was performed by comparison with the untreated control. The double asterisk stands for p
Figure Legend Snippet: Electromagnetic fields and retinal cells can affect the phosphorylation of ERK1/2, P38, Akt and JNK in human scleral fibroblast cells. Effect of exposure to ELF-EMFs on the phosphorylation of ERK1/2, P38, Akt and JNK in HFSFs treated with RPE supernatant or not. A : Western blot analysis was performed using p-ERK1/2, p-p38, ERK1/2, p38, and β-actin antibodies, respectively. B : Densitometric quantification of Phosphorylation levels were obtained by optical density (O.D.) of protein bands normalized with O.D. of β-actin band. Values (mean±SD, n=3) were expressed as relative expression levels. A quantitative analysis was performed by comparison with the untreated control. The double asterisk stands for p

Techniques Used: Western Blot, Expressing

68) Product Images from "Inhibition of UII/UTR System Relieves Acute Inflammation of Liver through Preventing Activation of NF-?B Pathway in ALF Mice"

Article Title: Inhibition of UII/UTR System Relieves Acute Inflammation of Liver through Preventing Activation of NF-?B Pathway in ALF Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0064895

Effects of urantide on levels of pro-inflammatory cytokines in liver and serum. (A) TNF-α, IL-1β and IFN-γ mRNA expression in liver; Left panel shows a representative ethidium bromide-stained gel of RT-PCR products. Relative expression levels of these cytokines in liver are shown in the right panel after normalization to β-actin. (B) Serum levels of TNF-α; (C) Serum levels of IL-1β; (D) Serum levels of IFN-γ. Bars represent means ± SD (n = 6). * P
Figure Legend Snippet: Effects of urantide on levels of pro-inflammatory cytokines in liver and serum. (A) TNF-α, IL-1β and IFN-γ mRNA expression in liver; Left panel shows a representative ethidium bromide-stained gel of RT-PCR products. Relative expression levels of these cytokines in liver are shown in the right panel after normalization to β-actin. (B) Serum levels of TNF-α; (C) Serum levels of IL-1β; (D) Serum levels of IFN-γ. Bars represent means ± SD (n = 6). * P

Techniques Used: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction

Effect of urantide on NF-κB pathway activation in liver. (A) IκB and phospho- IκB in liver cytoplasmic protein extracts; Left panel shows a representative picture of Western blot, and right shows the relative levels of IκBα and phospho- IκBα protein in liver after normalization to β-actin. (B) NF-κB p65 subunit in liver nuclear protein extracts; Left panel shows a representative picture of Western blot, and right shows the relative levels of p65 protein in liver after normalization to histone. (C) DNA-binding activity of NF-κB in liver nuclear protein extracts; DNA-binding activity of NF-κB was analyzed by EMSA (top). Lane 2, 3, 5 and 6 are target reactions (liver nuclear extract+biotin-DNA probe). Lane 2: sham; lane 3: urantide; lane 5: LPS/GalN; lane 6: urantide+LPS/GalN. Lane 1 and 4 are control reactions. Lane 1: cold competitive reaction of mutation DNA probe (liver nuclear extract+biotin-DNA +200-fold molar excess of unlabeled mutation DNA); lane 4: cold competitive reaction of DNA probe (liver nuclear extract+biotin-DNA +200-fold molar excess of unlabeled DNA). The bands corresponding to NF-κB were quantitated by densitometry (lower). Bars represent means ± SD (n = 6). * P
Figure Legend Snippet: Effect of urantide on NF-κB pathway activation in liver. (A) IκB and phospho- IκB in liver cytoplasmic protein extracts; Left panel shows a representative picture of Western blot, and right shows the relative levels of IκBα and phospho- IκBα protein in liver after normalization to β-actin. (B) NF-κB p65 subunit in liver nuclear protein extracts; Left panel shows a representative picture of Western blot, and right shows the relative levels of p65 protein in liver after normalization to histone. (C) DNA-binding activity of NF-κB in liver nuclear protein extracts; DNA-binding activity of NF-κB was analyzed by EMSA (top). Lane 2, 3, 5 and 6 are target reactions (liver nuclear extract+biotin-DNA probe). Lane 2: sham; lane 3: urantide; lane 5: LPS/GalN; lane 6: urantide+LPS/GalN. Lane 1 and 4 are control reactions. Lane 1: cold competitive reaction of mutation DNA probe (liver nuclear extract+biotin-DNA +200-fold molar excess of unlabeled mutation DNA); lane 4: cold competitive reaction of DNA probe (liver nuclear extract+biotin-DNA +200-fold molar excess of unlabeled DNA). The bands corresponding to NF-κB were quantitated by densitometry (lower). Bars represent means ± SD (n = 6). * P

Techniques Used: Activation Assay, Western Blot, Binding Assay, Activity Assay, Mutagenesis

Effects of urantide on UII/UTR system expression. (A) mRNA expression of UII and UTR in liver; left panel shows a representative ethidium bromide-stained gel of RT-PCR products, and right shows relative expression levels of UII and UTR mRNA in liver after normalization to β-actin. Data represent means ± SD (n = 6 each group). (B) Immunohistochemistry of UII (left) and UTR (right) in liver; The positive cells are stained with yallow-brown color (magnification ×400). Quantification of positive signals in UII or UTR staining is shown in the bottom panel. Data represent means ± SD (n = 6 each group). (C) Levels of UII secretion in blood. The mouse serum were assayed for UII secretion via ELISA. Data represent six independent studies. Values are mean ± SD (n = 6). * P
Figure Legend Snippet: Effects of urantide on UII/UTR system expression. (A) mRNA expression of UII and UTR in liver; left panel shows a representative ethidium bromide-stained gel of RT-PCR products, and right shows relative expression levels of UII and UTR mRNA in liver after normalization to β-actin. Data represent means ± SD (n = 6 each group). (B) Immunohistochemistry of UII (left) and UTR (right) in liver; The positive cells are stained with yallow-brown color (magnification ×400). Quantification of positive signals in UII or UTR staining is shown in the bottom panel. Data represent means ± SD (n = 6 each group). (C) Levels of UII secretion in blood. The mouse serum were assayed for UII secretion via ELISA. Data represent six independent studies. Values are mean ± SD (n = 6). * P

Techniques Used: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

69) Product Images from "Rapamycin Inhibits the Growth and Collagen Production of Fibroblasts Derived from Human Urethral Scar Tissue"

Article Title: Rapamycin Inhibits the Growth and Collagen Production of Fibroblasts Derived from Human Urethral Scar Tissue

Journal: BioMed Research International

doi: 10.1155/2018/7851327

Rapamycin decreases the mRNA expressions of Smad2, eIF-4E, and the alpha-1 chains of collagen type I and collagen type III (Col1α1 and Col3α1) in FHUS . The mRNA expressions of Smad2, eIF-4E, Col1 α 1, and Col3 α 1 were determined by semiquantitative RT-PCR using agarose gel electrophoresis, with β -actin as internal control. (a) shows representative blots illustrating the effects of various rapamycin concentrations on the mRNA expressions of Smad2, eIF-4E, Col1 α 1, and Col3 α 1. (b) contains quantified data showing the concentration-dependent effects of rapamycin on the mRNA expressions of Smad2, eIF-4E, Col1 α 1, and Col3 α 1. Data shown as mean ± standard deviation ( n = 4 cell lines from four patients). ∗ P
Figure Legend Snippet: Rapamycin decreases the mRNA expressions of Smad2, eIF-4E, and the alpha-1 chains of collagen type I and collagen type III (Col1α1 and Col3α1) in FHUS . The mRNA expressions of Smad2, eIF-4E, Col1 α 1, and Col3 α 1 were determined by semiquantitative RT-PCR using agarose gel electrophoresis, with β -actin as internal control. (a) shows representative blots illustrating the effects of various rapamycin concentrations on the mRNA expressions of Smad2, eIF-4E, Col1 α 1, and Col3 α 1. (b) contains quantified data showing the concentration-dependent effects of rapamycin on the mRNA expressions of Smad2, eIF-4E, Col1 α 1, and Col3 α 1. Data shown as mean ± standard deviation ( n = 4 cell lines from four patients). ∗ P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Concentration Assay, Standard Deviation

Rapamycin decreases the protein expressions of Smad2 and eIF-4E and the levels of phosphorylated Smad2 in FHUS . The protein expressions of Smad2 and eIF-4E and the levels of phospho-Smad2 were measured using western blot, with β -actin as internal control. (a) shows representative blots illustrating the effects of various rapamycin concentrations on the levels of eIF-4E, Smad2, and phospho-Smad2 proteins. (b) quantifies the concentration-dependent effects of rapamycin on the protein expressions of Smad2 and eIF-4E. (c) shows the concentration-dependent effects of rapamycin on Smad2 phosphorylation. Data shown as mean ± standard deviation ( n = 4 cell lines from four patients). ∗∗ P
Figure Legend Snippet: Rapamycin decreases the protein expressions of Smad2 and eIF-4E and the levels of phosphorylated Smad2 in FHUS . The protein expressions of Smad2 and eIF-4E and the levels of phospho-Smad2 were measured using western blot, with β -actin as internal control. (a) shows representative blots illustrating the effects of various rapamycin concentrations on the levels of eIF-4E, Smad2, and phospho-Smad2 proteins. (b) quantifies the concentration-dependent effects of rapamycin on the protein expressions of Smad2 and eIF-4E. (c) shows the concentration-dependent effects of rapamycin on Smad2 phosphorylation. Data shown as mean ± standard deviation ( n = 4 cell lines from four patients). ∗∗ P

Techniques Used: Western Blot, Concentration Assay, Standard Deviation

70) Product Images from "Tannic Acid Accelerates Cutaneous Wound Healing in Rats Via Activation of the ERK 1/2 Signaling Pathways"

Article Title: Tannic Acid Accelerates Cutaneous Wound Healing in Rats Via Activation of the ERK 1/2 Signaling Pathways

Journal: Advances in Wound Care

doi: 10.1089/wound.2018.0853

Labels ①, ②, and ③, respectively showed the protein levels of β-actin, bFGF, Erk 1/2, and P-Erk 1/2 in skin, which were assessed by WB on 3rd day, 10th day and 21st day. Blank (A) and model (B) represent the groups that were treated as blank control or model group, and YB (C), TA (h) (D), TA (m) (E), and TA (l) (F) represent the groups that were pretreated with YB, high, middle, and low dose of TA. The values are presented as means ± standard deviation (10 rats per group). * p
Figure Legend Snippet: Labels ①, ②, and ③, respectively showed the protein levels of β-actin, bFGF, Erk 1/2, and P-Erk 1/2 in skin, which were assessed by WB on 3rd day, 10th day and 21st day. Blank (A) and model (B) represent the groups that were treated as blank control or model group, and YB (C), TA (h) (D), TA (m) (E), and TA (l) (F) represent the groups that were pretreated with YB, high, middle, and low dose of TA. The values are presented as means ± standard deviation (10 rats per group). * p

Techniques Used: Western Blot, Standard Deviation

The protein levels of β-actin, bFGF, Erk 1/2, and P-Erk 1/2 in NIH 3T3 were assessed by Western blot. A concentration of 0.1 μg/mL TA activated the Erk 1/2 pathway. The expression levels of P-Erk 1/2/Erk 1/2 were increased significantly in the 0.1 μg/mL TA group than in the control group ( p
Figure Legend Snippet: The protein levels of β-actin, bFGF, Erk 1/2, and P-Erk 1/2 in NIH 3T3 were assessed by Western blot. A concentration of 0.1 μg/mL TA activated the Erk 1/2 pathway. The expression levels of P-Erk 1/2/Erk 1/2 were increased significantly in the 0.1 μg/mL TA group than in the control group ( p

Techniques Used: Western Blot, Concentration Assay, Expressing

71) Product Images from "Akt1 regulates pulmonary fibrosis via modulating IL-13 expression in macrophages"

Article Title: Akt1 regulates pulmonary fibrosis via modulating IL-13 expression in macrophages

Journal: Innate Immunity

doi: 10.1177/1753425919861774

Akt and Akt1 were phosphorylated, and Akt1 is required for IL-13 production in macrophages by IL-33 stimulation. (a) Akt1 +/+ bone marrow–derived macrophages (BMDMs) were challenged with IL-33 (10 ng/ml) for 2 h. Phospho-Akt (Ser473), Akt, Phospho-Akt1 (Ser473) and β-actin were measured by Western blot. Quantitative analysis of the relative expression levels of phosphorylated (b) Akt and (c) phosphorylated Akt1 against total AKT using ImageJ software. (d) Akt1 +/+ and Akt1 –/– BMDMs were treated with IL-33 (10 ng/ml) for 24 and 48 h. The mRNA level of IL-13 was detected by quantitative RT-PCR. (e) The Akt1 +/+ and Akt1 –/– BMDMs were challenged with IL-33 (10 ng/ml) for 2 h. Phospho-Akt1, Phospho-p65 (Ser536), p65 and β-actin were measured by Western blot. (f) Quantitative analysis of the relative expression levels of phosphorylated p65 against total p65 using ImageJ software. Data are shown as means ± SEM. * P
Figure Legend Snippet: Akt and Akt1 were phosphorylated, and Akt1 is required for IL-13 production in macrophages by IL-33 stimulation. (a) Akt1 +/+ bone marrow–derived macrophages (BMDMs) were challenged with IL-33 (10 ng/ml) for 2 h. Phospho-Akt (Ser473), Akt, Phospho-Akt1 (Ser473) and β-actin were measured by Western blot. Quantitative analysis of the relative expression levels of phosphorylated (b) Akt and (c) phosphorylated Akt1 against total AKT using ImageJ software. (d) Akt1 +/+ and Akt1 –/– BMDMs were treated with IL-33 (10 ng/ml) for 24 and 48 h. The mRNA level of IL-13 was detected by quantitative RT-PCR. (e) The Akt1 +/+ and Akt1 –/– BMDMs were challenged with IL-33 (10 ng/ml) for 2 h. Phospho-Akt1, Phospho-p65 (Ser536), p65 and β-actin were measured by Western blot. (f) Quantitative analysis of the relative expression levels of phosphorylated p65 against total p65 using ImageJ software. Data are shown as means ± SEM. * P

Techniques Used: Derivative Assay, Western Blot, Expressing, Software, Quantitative RT-PCR

72) Product Images from "Carboxyamidotriazole-Orotate Inhibits the Growth of Imatinib-Resistant Chronic Myeloid Leukaemia Cells and Modulates Exosomes-Stimulated Angiogenesis"

Article Title: Carboxyamidotriazole-Orotate Inhibits the Growth of Imatinib-Resistant Chronic Myeloid Leukaemia Cells and Modulates Exosomes-Stimulated Angiogenesis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0042310

CTO inhibits cell proliferation of LAMA84R and K562R cell lines. using anti-phospho-Abl, anti-cAbl, anti-phospho-CrkL and anti-CrkL antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading.
Figure Legend Snippet: CTO inhibits cell proliferation of LAMA84R and K562R cell lines. using anti-phospho-Abl, anti-cAbl, anti-phospho-CrkL and anti-CrkL antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading.

Techniques Used:

CTO inhibits Akt and Erk 1/2 phosphorylation in exosomes-stimulated HUVEC. using anti-phospho-Akt, anti- Akt, anti-phospho-Erk, anti-Erk antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading.
Figure Legend Snippet: CTO inhibits Akt and Erk 1/2 phosphorylation in exosomes-stimulated HUVEC. using anti-phospho-Akt, anti- Akt, anti-phospho-Erk, anti-Erk antibodies. Blots were then stripped and subsequently reprobed with antibody against β-actin to ensure equal loading.

Techniques Used:

73) Product Images from "?Np63? Mediated Activation of Bone Morphogenetic Protein Signaling Governs Stem Cell Activity and Plasticity in Normal and Malignant Mammary Epithelial Cells"

Article Title: ?Np63? Mediated Activation of Bone Morphogenetic Protein Signaling Governs Stem Cell Activity and Plasticity in Normal and Malignant Mammary Epithelial Cells

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-12-2862

LDN193189 exhibits pharmacologic activity in vivo and causes reversion of EMT One thousand primary MMTV-Myc tumor cells were embedded in matrigel and transplanted into the dorsal right flanks of female syngeneic (FVB/N) wt recipients; N=40. At two days post transplant, mice were injected intraperitoneally with 2.5 mg/kg LDN193189 or vehicle control daily for 16 days and then sacrificed two hours after the last injection. A. Western blot analysis of 3representative tumor lysates from vehicle and LDN193189 treatment groups probing for phospho-SMAD1/5/8 and p63. β-Actin is used as a loading control. B. Representative images of 2-color Immunofluorescent analysis of mouse tumor tissue probing for P-SMAD1/5/8 (green) and Vimentin (red), a marker of EMT. Nuclei are stained with DAPI.
Figure Legend Snippet: LDN193189 exhibits pharmacologic activity in vivo and causes reversion of EMT One thousand primary MMTV-Myc tumor cells were embedded in matrigel and transplanted into the dorsal right flanks of female syngeneic (FVB/N) wt recipients; N=40. At two days post transplant, mice were injected intraperitoneally with 2.5 mg/kg LDN193189 or vehicle control daily for 16 days and then sacrificed two hours after the last injection. A. Western blot analysis of 3representative tumor lysates from vehicle and LDN193189 treatment groups probing for phospho-SMAD1/5/8 and p63. β-Actin is used as a loading control. B. Representative images of 2-color Immunofluorescent analysis of mouse tumor tissue probing for P-SMAD1/5/8 (green) and Vimentin (red), a marker of EMT. Nuclei are stained with DAPI.

Techniques Used: Activity Assay, In Vivo, Mouse Assay, Injection, Western Blot, Marker, Staining

ΔNp63α induces canonical BMP signaling in mammary epithelial cells via induction of the BMP7 ligand A. Quantitative PCR analysis of ΔNp63 and BMP7 expression in human breast cancer cell lines. Expression levels are normalized to normal hTERT-immortalized human mammary epithelial cells (IMECs) and to GAPDH. B. Ectopic ΔNp63α induces the expression of BMP7 mRNA in IMECs and HC11 cells. C. Western blots were prepared with extracts from IMECs infected with adenoviral ΔNp63α or GFP as a control, and treated with either vehicle, rhNoggin (250 ng/ml), or BMP7-neutralizing antibodies. Blots were probed with antibodies againstP-SMAD1/5/8 (identifying active BMP signaling), and total SMAD1/5/8 as a loading control. D. Western blot analysis of IMECs pre-treated with vehicle or LDN193189 for 10 minutes, followed by stimulation with vehicle orrhBMP7 (50ng/ml) for 45 minutes. Blots were probed with antibodies directed against P-SMAD1/5/8 and P-SMAD2. Total SMAD1/5/8 is used as a loading control. E. IMECs were infected with adenoviruses programmed to express GFP (control) or ΔNp63α. Following infection, cells were treated with vehicle (control) or the indicated doses of LDN193189. Blots were probed with antibodies directed against P-SMAD1/5/8. β-Actin is used as a loading control. Data is presented as mean values of triplicate points ± S.E.
Figure Legend Snippet: ΔNp63α induces canonical BMP signaling in mammary epithelial cells via induction of the BMP7 ligand A. Quantitative PCR analysis of ΔNp63 and BMP7 expression in human breast cancer cell lines. Expression levels are normalized to normal hTERT-immortalized human mammary epithelial cells (IMECs) and to GAPDH. B. Ectopic ΔNp63α induces the expression of BMP7 mRNA in IMECs and HC11 cells. C. Western blots were prepared with extracts from IMECs infected with adenoviral ΔNp63α or GFP as a control, and treated with either vehicle, rhNoggin (250 ng/ml), or BMP7-neutralizing antibodies. Blots were probed with antibodies againstP-SMAD1/5/8 (identifying active BMP signaling), and total SMAD1/5/8 as a loading control. D. Western blot analysis of IMECs pre-treated with vehicle or LDN193189 for 10 minutes, followed by stimulation with vehicle orrhBMP7 (50ng/ml) for 45 minutes. Blots were probed with antibodies directed against P-SMAD1/5/8 and P-SMAD2. Total SMAD1/5/8 is used as a loading control. E. IMECs were infected with adenoviruses programmed to express GFP (control) or ΔNp63α. Following infection, cells were treated with vehicle (control) or the indicated doses of LDN193189. Blots were probed with antibodies directed against P-SMAD1/5/8. β-Actin is used as a loading control. Data is presented as mean values of triplicate points ± S.E.

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Infection

74) Product Images from "Cdx2 regulates endo-lysosomal function and epithelial cell polarity"

Article Title: Cdx2 regulates endo-lysosomal function and epithelial cell polarity

Journal: Genes & Development

doi: 10.1101/gad.1921510

Blockage of endo-lysosomal maturation perturbs apical–basal cell polarity. ( A–C ) Caco-2 3D cyst formation assays were performed with 3-d incubation with normal growth medium (control), or medium containing 50 nM Baf.A1 or 25 μM CHL. F-actin and E-cadherin were stained in red and green, respectively. ( D , E ) Control and Baf.A1-treated cysts were stained for Prkcz (red) and E-cadherin (green). ( F , G ) Control and Baf.A1-treated cysts were stained for EEA1 (red) and E-cadherin (green). ( H ) Western blots for Lamp1, Prkcz, and Par3 using lysates from Caco-2 cells treated with inhibitors overnight at indicated concentrations. Leupeptin serves as a nonspecific protease inhibitor. β-Actin serves as a loading control. ( I , J ) Early cysts from control and CDX2 knockdown cells were stained for EEA1 (red) and E-cadherin (green). Note that control endosomes are clustered apically into the center where the prospective lumen will emerge. ( K ) Three-day-old CDX2 knockdown cysts were stained with EEA1 (red) and E-cadherin (green). Note that the knockdown cells are able to accumulate endosomes around several ectopic lumens (asterisks). Bars: A–G , K , 20 μm; I , J , 9 μm.
Figure Legend Snippet: Blockage of endo-lysosomal maturation perturbs apical–basal cell polarity. ( A–C ) Caco-2 3D cyst formation assays were performed with 3-d incubation with normal growth medium (control), or medium containing 50 nM Baf.A1 or 25 μM CHL. F-actin and E-cadherin were stained in red and green, respectively. ( D , E ) Control and Baf.A1-treated cysts were stained for Prkcz (red) and E-cadherin (green). ( F , G ) Control and Baf.A1-treated cysts were stained for EEA1 (red) and E-cadherin (green). ( H ) Western blots for Lamp1, Prkcz, and Par3 using lysates from Caco-2 cells treated with inhibitors overnight at indicated concentrations. Leupeptin serves as a nonspecific protease inhibitor. β-Actin serves as a loading control. ( I , J ) Early cysts from control and CDX2 knockdown cells were stained for EEA1 (red) and E-cadherin (green). Note that control endosomes are clustered apically into the center where the prospective lumen will emerge. ( K ) Three-day-old CDX2 knockdown cysts were stained with EEA1 (red) and E-cadherin (green). Note that the knockdown cells are able to accumulate endosomes around several ectopic lumens (asterisks). Bars: A–G , K , 20 μm; I , J , 9 μm.

Techniques Used: Incubation, Staining, Western Blot, Protease Inhibitor

75) Product Images from "Exercise and Omentin: Their Role in the Crosstalk Between Muscle and Adipose Tissues in Type 2 Diabetes Mellitus Rat Models"

Article Title: Exercise and Omentin: Their Role in the Crosstalk Between Muscle and Adipose Tissues in Type 2 Diabetes Mellitus Rat Models

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.01881

Western blot representative of omentin in diabetic animals. (A) in tissues: Molecular Weight Marker (M); Mesenteric Adipose Tissue (MES), Retroperitoneal (RET), Epididimal (EPI), Brown Adipose Tissue (BAT), Liver, and Serum. For this result, 3 independent replicates were performed. (B) Representative membrane with 4 sample of omentin in muscle (one per group) compared to MES, showed that omentin was not found in muscle (I = 6 sample per group). (C) Serum Omentin Values (ELISA) ( n = 10 sample per group). (D) Omentin (40 kDa) in MES in the experimental protocols. C, control group; RT, resistance training; AT, aerobic training; CT, combined training. Results are presented as the relative density after normalizing with β-actin protein. Data are expressed as means ± SD ( n = 6 sample per group). P
Figure Legend Snippet: Western blot representative of omentin in diabetic animals. (A) in tissues: Molecular Weight Marker (M); Mesenteric Adipose Tissue (MES), Retroperitoneal (RET), Epididimal (EPI), Brown Adipose Tissue (BAT), Liver, and Serum. For this result, 3 independent replicates were performed. (B) Representative membrane with 4 sample of omentin in muscle (one per group) compared to MES, showed that omentin was not found in muscle (I = 6 sample per group). (C) Serum Omentin Values (ELISA) ( n = 10 sample per group). (D) Omentin (40 kDa) in MES in the experimental protocols. C, control group; RT, resistance training; AT, aerobic training; CT, combined training. Results are presented as the relative density after normalizing with β-actin protein. Data are expressed as means ± SD ( n = 6 sample per group). P

Techniques Used: Western Blot, Molecular Weight, Marker, Enzyme-linked Immunosorbent Assay

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Article Snippet: .. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Bio-Rad), and immunoblotted with anti–pAurora-A kinase (Thr288; Millennium Pharmaceuticals), Aurora-A kinase, pHisH3 (Ser10), HisH3, p53, p21, p27, caspase-3, caspase-8, caspase-9, poly (ADP ribose) polymerase (PARP; Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin antibodies (Santa Cruz Biotechnology). .. Immunoreactive bands were detected by Western Blot chemiluminescence reagents (NEN Life Sciences) and exposed on Kodak-XAR film.

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Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1
Article Snippet: Western Blot Total proteins extracted from harvested tissues and cells were prepared in mammalian lysis buffer containing phosphatase and protease inhibitors, and the protein concentration was determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). .. The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Western Blot:

Article Title: Staurosporine induces apoptosis in pancreatic carcinoma cells PaTu 8988t and Panc-1 via the intrinsic signaling pathway
Article Snippet: .. Western blot analysis after time-dependent incubation with 1 µM staurosporine and endogenic expression of Bcl2, BAX, Bad, caspase-8, and caspase-9 in pancreatic and colorectal carcinoma cells The colorectal cancer cell line SW 480 did not show any time-dependent changes in the expression of the proteins BAX, caspase-8, and caspase-9 (Fig. a). .. The pancreatic cancer cell line PaTu 8988t (Fig. b) showed a time-dependent decrease in the signal strength of Bcl2 after incubation with staurosporine up to the complete absence of proteins after 24 h of incubation (column 1).

Article Title: A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
Article Snippet: Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Bio-Rad), and immunoblotted with anti–pAurora-A kinase (Thr288; Millennium Pharmaceuticals), Aurora-A kinase, pHisH3 (Ser10), HisH3, p53, p21, p27, caspase-3, caspase-8, caspase-9, poly (ADP ribose) polymerase (PARP; Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin antibodies (Santa Cruz Biotechnology). .. Immunoreactive bands were detected by Western Blot chemiluminescence reagents (NEN Life Sciences) and exposed on Kodak-XAR film.

Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1
Article Snippet: Paragraph title: 2.11. Western Blot ... The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Mouse Assay:

Article Title: Citral Is Renoprotective for Focal Segmental Glomerulosclerosis by Inhibiting Oxidative Stress and Apoptosis and Activating Nrf2 Pathway in Mice
Article Snippet: .. Since an increased Bax/Bcl-2 ratio is associated with caspase-9 and caspase-3 activation [ ], we then measured renal levels of Bax and Bcl-2 and found that the Bax/Bcl-2 ratio was increased in FSGS+vehicle mice compared to normal control mice at days 14 and 28 (both p < 0.01) and this effect was significantly decreased in FSGS+Citral mice at days 14 and 28 (both p < 0.01) ( ). .. These findings suggest that the reduction in renal apoptosis seen in FSGS+Citral mice is due to suppression of the intrinsic pathway of apoptosis.

Article Title: Oral treatment with the herbal formula B307 alleviates cardiac toxicity in doxorubicin-treated mice via suppressing oxidative stress, inflammation, and apoptosis
Article Snippet: .. Also, we observed that cardiac expressions of Cyto-C, Bax, calpain, caspase 12, caspase 9, and caspase 3 were significantly suppressed in DOX-treated mice under oral B307 treatment. ..

Article Title: Oral treatment with the herbal formula B307 alleviates cardiac toxicity in doxorubicin-treated mice via suppressing oxidative stress, inflammation, and apoptosis
Article Snippet: .. As to the DOX-treated mice, cardiac expressions of caspase 9 and caspase 3 were visibly reduced under oral B307 treatment. shows that only quantified caspase 3 levels in the heart tissue of the mice were significantly reduced under oral B307 treatment (sham group vs B307 group, P < 0.05) but quantified pro-caspase 9, c-caspase 9, pro-caspase 3, and c-caspase 3 levels were significantly enhanced under DOX treatment (sham group vs DOX group, P < 0.01). .. As to the DOX-treated mice, quantified pro-caspase 9, c-caspase 9, pro-caspase 3, and c-caspase 3 levels in the heart tissue were significantly reduced under oral B307 treatment (DOX group vs B307+DOX group, P < 0.01).

Activation Assay:

Article Title: Citral Is Renoprotective for Focal Segmental Glomerulosclerosis by Inhibiting Oxidative Stress and Apoptosis and Activating Nrf2 Pathway in Mice
Article Snippet: .. Since an increased Bax/Bcl-2 ratio is associated with caspase-9 and caspase-3 activation [ ], we then measured renal levels of Bax and Bcl-2 and found that the Bax/Bcl-2 ratio was increased in FSGS+vehicle mice compared to normal control mice at days 14 and 28 (both p < 0.01) and this effect was significantly decreased in FSGS+Citral mice at days 14 and 28 (both p < 0.01) ( ). .. These findings suggest that the reduction in renal apoptosis seen in FSGS+Citral mice is due to suppression of the intrinsic pathway of apoptosis.

Incubation:

Article Title: Staurosporine induces apoptosis in pancreatic carcinoma cells PaTu 8988t and Panc-1 via the intrinsic signaling pathway
Article Snippet: .. Western blot analysis after time-dependent incubation with 1 µM staurosporine and endogenic expression of Bcl2, BAX, Bad, caspase-8, and caspase-9 in pancreatic and colorectal carcinoma cells The colorectal cancer cell line SW 480 did not show any time-dependent changes in the expression of the proteins BAX, caspase-8, and caspase-9 (Fig. a). .. The pancreatic cancer cell line PaTu 8988t (Fig. b) showed a time-dependent decrease in the signal strength of Bcl2 after incubation with staurosporine up to the complete absence of proteins after 24 h of incubation (column 1).

Article Title: A novel Aurora-A kinase inhibitor MLN8237 induces cytotoxicity and cell-cycle arrest in multiple myeloma
Article Snippet: To synchronize mitotic cells, MM cells were incubated in the absence or presence of nocodazole (400 ng/mL) for 16 hours; and both unsynchronized and synchronized cells were then exposed to MLN8237 (0.5-1μM) for 1 to 72 hours. .. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes (Bio-Rad), and immunoblotted with anti–pAurora-A kinase (Thr288; Millennium Pharmaceuticals), Aurora-A kinase, pHisH3 (Ser10), HisH3, p53, p21, p27, caspase-3, caspase-8, caspase-9, poly (ADP ribose) polymerase (PARP; Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin antibodies (Santa Cruz Biotechnology).

Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1
Article Snippet: .. The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody. .. Antibody binding was visualized with an ECL chemiluminescence system (GE Healthcare Bio-Sciences, Little Chalfont, UK).

other:

Article Title: Pelvic and hypogastric nerves are injured in a rat prostatectomy model, contributing to development of stress urinary incontinence
Article Snippet: Apoptotic mechanism We examined the pathway by which apoptosis takes place in the rat MPG after injury, by examining if caspase 9 and/or caspase 8 proteins were increased in the CN, PN, HYG and ANC nerves from 1–7 days after CN crush.

Article Title: Citral Is Renoprotective for Focal Segmental Glomerulosclerosis by Inhibiting Oxidative Stress and Apoptosis and Activating Nrf2 Pathway in Mice
Article Snippet: Furthermore, Citral administration resulted in decreased renal levels of activated caspase-3 and caspase-9 (but not of activated caspase-8) as well as Bax/Bcl-2 ratio ( ).

Article Title: Dietary Restriction Promotes Vessel Maturation in a Mouse Astrocytoma
Article Snippet: Antibodies and Reagents Antibodies were obtained against α -SMA (Sigma), β -Actin (Cell Signaling), Factor VIII (Dako), PDGF-Rβ (Santa Cruz), VEGF-R2 (Santa Cruz), and VEGF (Santa Cruz).

Article Title: TUCAN/CARDINAL/CARD8 and apoptosis resistance in non-small cell lung cancer cells
Article Snippet: Next, we examined the processing of caspase-9 in two representative lung cancer cell lines, H460 (NSCLC) and GLC4 (SCLC), after treatment for 24 and 48 h with IC80 concentrations of cisplatin.

Article Title: Oral treatment with the herbal formula B307 alleviates cardiac toxicity in doxorubicin-treated mice via suppressing oxidative stress, inflammation, and apoptosis
Article Snippet: Similarly, we found that DOX treatment induced marked apoptosis of cardiomyocytes via increasing expressions of caspase 9 and caspase 3 ( ).

Expressing:

Article Title: Staurosporine induces apoptosis in pancreatic carcinoma cells PaTu 8988t and Panc-1 via the intrinsic signaling pathway
Article Snippet: .. Western blot analysis after time-dependent incubation with 1 µM staurosporine and endogenic expression of Bcl2, BAX, Bad, caspase-8, and caspase-9 in pancreatic and colorectal carcinoma cells The colorectal cancer cell line SW 480 did not show any time-dependent changes in the expression of the proteins BAX, caspase-8, and caspase-9 (Fig. a). .. The pancreatic cancer cell line PaTu 8988t (Fig. b) showed a time-dependent decrease in the signal strength of Bcl2 after incubation with staurosporine up to the complete absence of proteins after 24 h of incubation (column 1).

Article Title: Staurosporine induces apoptosis in pancreatic carcinoma cells PaTu 8988t and Panc-1 via the intrinsic signaling pathway
Article Snippet: .. Endogenic expression of Bcl2, Bad, BAX, caspase-8, and caspase-9 in pancreatic and colorectal carcinoma cells The first aim was to obtain evidence for the actual expression of Bcl2, Bad, BAX, caspase-8, and caspase-9 in pancreatic and colorectal carcinoma cells (Fig. ). .. The pancreatic cancer cell line PaTu 8988t (column 2) showed strong expression of each of the proteins investigated, whereas the cell lines SW 480 and Panc-1 showed only expression of BAX, caspase-8, and caspase-9.

Protein Concentration:

Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1
Article Snippet: Western Blot Total proteins extracted from harvested tissues and cells were prepared in mammalian lysis buffer containing phosphatase and protease inhibitors, and the protein concentration was determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). .. The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Lysis:

Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1
Article Snippet: Western Blot Total proteins extracted from harvested tissues and cells were prepared in mammalian lysis buffer containing phosphatase and protease inhibitors, and the protein concentration was determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). .. The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

Binding Assay:

Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1
Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody. .. Antibody binding was visualized with an ECL chemiluminescence system (GE Healthcare Bio-Sciences, Little Chalfont, UK).

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    Cell Signaling Technology Inc pdgfr β erk1 2 cell signaling cascade pdgf bb activates pdgfr β
    Goniolactone C inhibits the <t>PDGFR-β/ERK1/2</t> cell signaling cascade. ( A ) A PDGFRβ Kinase Enzyme System assay (PDGFRβ Kinase Enzyme System V3731) was used to measure the in vitro activity of PDGFR-β. ( B , C ) Confluent VSMCs that had been starved for 24 h in FCS-free DMEM were treated with goniolactone C at different concentrations (0–4 μM) for 2 h and were then incubated in the presence of PDGF-BB for another 30 min. The cells were then lysed, and protein expression was analyzed using 12% SDS-PAGE. Western blot analyses were performed to detect the phosphorylation of PDGFR-β (B) and ERK1/2 (C).
    Pdgfr β Erk1 2 Cell Signaling Cascade Pdgf Bb Activates Pdgfr β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β actin
    4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and <t>β</t> -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p
    β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4722 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc insr
    UBQLN1 interacts with IGF1R, IGF2R and <t>INSR</t> (A) Schematic of <t>Ubiquilin1</t> protein. UBQLN1 is a 590 amino acid protein with an N-terminal UBL (ubiquitin-like) domain, four STI chaperone like domains in the middle and a C-terminal UBA (ubiquitin-associated) domain. (B) HEK293T cells were transfected with FLAG-tagged UBQLN1 followed by co-immunoprecipitation (IP) by anti-FLAG antibody and mass spectrometry (MS) analysis. IGF1R, IGF2R and INSR were identified as some of the top interacting partners of FLAG-UBQLN1. (C) HEK293T cells were transiently transfected with siRNA for UBQLN1 knock down (siUBQLN1) followed by co-immunoprecipitation by anti-UBQLN1 antibody and Western Blot analysis. (D) Confocal microscopy images of indirect immunofluorescence staining for FLAG-UBQLN1 (red) and IGF1R (green) in HeLa cells. Co-localization was determined for using JACop plugin of ImageJ software. Automatic threshold for images were determined by the Costes method and overlap coefficients (Mander’s Correlation Coefficients) were calculated. For the 2 chosen fields, 15.8–33.5% of FLAG-UBQLN1 overlaps with IGF1R.
    Insr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti β catenin polyclonal antibody
    SUMOylation inhibition represses the <t>Wnt/β-catenin</t> pathway. A. For myeloma cells NCI-H929, the silencing efficiency with SUMO-1 siRNA was tested 48 hours after transfection. Total RNA was then extracted, and SUMO-1 mRNA level was determined by real time RT-PCR. Whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 antibody. SUMO-1 was indicated by arrow and was about 15 kDa in size, while SUMOylation pattern was detected as conjugated proteins with SUMO-1 modification. B. Effects of SUMO-1 inhibition on TCF/β-catenin reporter activity. Myeloma cells were transfected with control siRNA or siRNA targeting SUMO-1. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after re-transfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. Data represent mean ± S.D.; *p
    Anti β Catenin Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Goniolactone C inhibits the PDGFR-β/ERK1/2 cell signaling cascade. ( A ) A PDGFRβ Kinase Enzyme System assay (PDGFRβ Kinase Enzyme System V3731) was used to measure the in vitro activity of PDGFR-β. ( B , C ) Confluent VSMCs that had been starved for 24 h in FCS-free DMEM were treated with goniolactone C at different concentrations (0–4 μM) for 2 h and were then incubated in the presence of PDGF-BB for another 30 min. The cells were then lysed, and protein expression was analyzed using 12% SDS-PAGE. Western blot analyses were performed to detect the phosphorylation of PDGFR-β (B) and ERK1/2 (C).

    Journal: Molecules

    Article Title: Goniolactone C, a Styryl Lactone Derivative, Inhibits PDGF-BB-Induced Vascular Smooth Muscle Cell Migration and Proliferation via PDGFR/ERK Signaling

    doi: 10.3390/molecules191219501

    Figure Lengend Snippet: Goniolactone C inhibits the PDGFR-β/ERK1/2 cell signaling cascade. ( A ) A PDGFRβ Kinase Enzyme System assay (PDGFRβ Kinase Enzyme System V3731) was used to measure the in vitro activity of PDGFR-β. ( B , C ) Confluent VSMCs that had been starved for 24 h in FCS-free DMEM were treated with goniolactone C at different concentrations (0–4 μM) for 2 h and were then incubated in the presence of PDGF-BB for another 30 min. The cells were then lysed, and protein expression was analyzed using 12% SDS-PAGE. Western blot analyses were performed to detect the phosphorylation of PDGFR-β (B) and ERK1/2 (C).

    Article Snippet: Goniolactone C Inhibits the PDGFR-β/ERK1/2 Cell Signaling Cascade PDGF-BB activates PDGFR-β on VSMCs, is one of the most potent mitogens and chemoattractants for vascular smooth muscle cells (VSMCs), and plays a central role in the onset and development of various vascular disorders [ , , ].

    Techniques: In Vitro, Activity Assay, Incubation, Expressing, SDS Page, Western Blot

    4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and β -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

    doi: 10.1155/2018/2747018

    Figure Lengend Snippet: 4-PG induces HO-1 mRNA and protein expression in macrophages and lung epithelial cells. (a) RAW 264.7 cells were treated with 4-PG at various concentrations (0, 5, 10, 20, and 40 μ M) for 8 h, and cell viability was determined by MTT assay. To evaluate the beneficial effect of 4-PG on HO-1 induction, cells were treated with 4-PG (0, 1, 5, and 10 μ M) at the indicated concentrations for 8 h. The mRNA and protein levels of HO-1 were measured by RT-PCR (b) and Western blotting (c). RAW 264.7 cells were treated with 4-PG (10 μ M) at the indicated time points (0, 2, 4, and 8 h). The mRNA and protein levels of HO-1 were determined by RT-PCR (d) and Western blotting (e). (f and g) PBMC and U937 cells were treated with 4-PG at the indicated concentrations (0, 1, 5, and 10 μ M) for 8 h. The mRNA and protein levels of HO-1 were determined by RT-PCR (top) and Western blotting (bottom). GAPDH and β -actin were used as internal controls. (h) RAW 264.7 cells were cotransduced with a pCignal Lenti-ARE reporter and pCignal Lenti-TK-Renilla. After treatment with 4-PG, luciferase activity was analyzed. The expression levels obtained from pCignal Lenti-ARE reporter-transduced cells without 4-PG treatment were normalized to 1. (i) RAW 264.7 cells were treated with 4-PG (10 μ M) for 8 h. Several antioxidative genes including TRX1, GCLC, and NQO1 were measured by RT-qPCR. (j and k) PBMC and U937 cells were pretreated with 4-PG (10 μ M) for 6 h followed by the stimulation of LPS (100 ng/ml) for another 4 h. (l) A549 cells pretreated with 4-PG (10 μ M) for 4 h and then stimulated with LPS (10 μ g/ml) for 6 h. The mRNA levels of HO-1, TNF- α , and IL-6 were determined by RT-PCR. Data were expressed as mean ± SD ( n = 5 determined in five independent experiments). One-way ANOVA with Turkey post hoc tests were performed; ∗ p

    Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

    Techniques: Expressing, MTT Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Quantitative RT-PCR

    4-PG prevents LPS-induced acute lung injury and upregulates HO-1 expression. (a) The scheme depicts the experimental protocol used to assess the protective effect of pterostilbene 4′-glucoside (4-PG) and pterostilbene (PTER) on LPS-induced ALI. 10-week-old mice were injected with 4-PG (10 mg/kg, i.p.) and PTER (10 mg/kg, i.p.) for 4 days prior to intranasal administration of LPS (2.5 mg/kg) for 24 h. (b) Chemical structures of 4-PG and PTER. (c) Lung sections were stained with hematoxylin and eosin (H E) for morphological evaluation, and the representative lung sections of each group are shown. Scale bar = 100 μ m. (left). Quantitative analysis of histologic lung section by lung injury score for six experimental groups. The score generates the average of two independent investigators (right). (d, e) The mRNA expression of proinflammatory cytokines and chemokines (TNF- α , IL-6, IL-1 β , CXCL1, and CXCL2) in lung tissues was detected by RT-PCR. Furthermore, mRNA and protein levels of HO-1 were assessed by RT-PCR (f, left: HO-1mRNA levels, right: quantification of the relative band density) and Western blotting (g, left: HO-1 protein levels, right: quantification of the relative band density) from lung tissues, respectively. 18S and β -actin were used as internal controls. Data were expressed as mean ± SD ( n = 5 per group); ∗∗ p

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Pterostilbene 4′-β-Glucoside Attenuates LPS-Induced Acute Lung Injury via Induction of Heme Oxygenase-1

    doi: 10.1155/2018/2747018

    Figure Lengend Snippet: 4-PG prevents LPS-induced acute lung injury and upregulates HO-1 expression. (a) The scheme depicts the experimental protocol used to assess the protective effect of pterostilbene 4′-glucoside (4-PG) and pterostilbene (PTER) on LPS-induced ALI. 10-week-old mice were injected with 4-PG (10 mg/kg, i.p.) and PTER (10 mg/kg, i.p.) for 4 days prior to intranasal administration of LPS (2.5 mg/kg) for 24 h. (b) Chemical structures of 4-PG and PTER. (c) Lung sections were stained with hematoxylin and eosin (H E) for morphological evaluation, and the representative lung sections of each group are shown. Scale bar = 100 μ m. (left). Quantitative analysis of histologic lung section by lung injury score for six experimental groups. The score generates the average of two independent investigators (right). (d, e) The mRNA expression of proinflammatory cytokines and chemokines (TNF- α , IL-6, IL-1 β , CXCL1, and CXCL2) in lung tissues was detected by RT-PCR. Furthermore, mRNA and protein levels of HO-1 were assessed by RT-PCR (f, left: HO-1mRNA levels, right: quantification of the relative band density) and Western blotting (g, left: HO-1 protein levels, right: quantification of the relative band density) from lung tissues, respectively. 18S and β -actin were used as internal controls. Data were expressed as mean ± SD ( n = 5 per group); ∗∗ p

    Article Snippet: The membrane was blocked with 5% nonfat milk in phosphate-buffered saline Tween 20 (PBS-T) and then incubated with a primary antibody against HO-1 (1 : 2000 v /v in PBS-T, Enzo Life Sciences, USA) or β -actin (1 : 2500 v /v in PBS-T, Cell Signaling Technology, MA) followed by incubation with a secondary antibody.

    Techniques: Expressing, Mouse Assay, Injection, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot

    UBQLN1 interacts with IGF1R, IGF2R and INSR (A) Schematic of Ubiquilin1 protein. UBQLN1 is a 590 amino acid protein with an N-terminal UBL (ubiquitin-like) domain, four STI chaperone like domains in the middle and a C-terminal UBA (ubiquitin-associated) domain. (B) HEK293T cells were transfected with FLAG-tagged UBQLN1 followed by co-immunoprecipitation (IP) by anti-FLAG antibody and mass spectrometry (MS) analysis. IGF1R, IGF2R and INSR were identified as some of the top interacting partners of FLAG-UBQLN1. (C) HEK293T cells were transiently transfected with siRNA for UBQLN1 knock down (siUBQLN1) followed by co-immunoprecipitation by anti-UBQLN1 antibody and Western Blot analysis. (D) Confocal microscopy images of indirect immunofluorescence staining for FLAG-UBQLN1 (red) and IGF1R (green) in HeLa cells. Co-localization was determined for using JACop plugin of ImageJ software. Automatic threshold for images were determined by the Costes method and overlap coefficients (Mander’s Correlation Coefficients) were calculated. For the 2 chosen fields, 15.8–33.5% of FLAG-UBQLN1 overlaps with IGF1R.

    Journal: The Biochemical journal

    Article Title: Regulation of Insulin-like Growth Factor Receptors by Ubiquilin1

    doi: 10.1042/BCJ20170620

    Figure Lengend Snippet: UBQLN1 interacts with IGF1R, IGF2R and INSR (A) Schematic of Ubiquilin1 protein. UBQLN1 is a 590 amino acid protein with an N-terminal UBL (ubiquitin-like) domain, four STI chaperone like domains in the middle and a C-terminal UBA (ubiquitin-associated) domain. (B) HEK293T cells were transfected with FLAG-tagged UBQLN1 followed by co-immunoprecipitation (IP) by anti-FLAG antibody and mass spectrometry (MS) analysis. IGF1R, IGF2R and INSR were identified as some of the top interacting partners of FLAG-UBQLN1. (C) HEK293T cells were transiently transfected with siRNA for UBQLN1 knock down (siUBQLN1) followed by co-immunoprecipitation by anti-UBQLN1 antibody and Western Blot analysis. (D) Confocal microscopy images of indirect immunofluorescence staining for FLAG-UBQLN1 (red) and IGF1R (green) in HeLa cells. Co-localization was determined for using JACop plugin of ImageJ software. Automatic threshold for images were determined by the Costes method and overlap coefficients (Mander’s Correlation Coefficients) were calculated. For the 2 chosen fields, 15.8–33.5% of FLAG-UBQLN1 overlaps with IGF1R.

    Article Snippet: IGF1R-beta (CST#3027), IGF1R Beta XP (CST#9750), p-IGF1R beta (CST#3918), INSR (CST#3025), IGF2R (CST#14364), AKT (CST#9272), p-AKT (CST#9271); Tubulin #B512 (Sigma); GAPDH (SantaCruz#FL335); Actin (Sigma#A5316), Ubiquilin1 (CST#14526); Anti-FLAG Affinity Gel (Sigma#A2220), INSR (CST#3025), FLAG polyclonal (Sigma# F7425).

    Techniques: Transfection, Immunoprecipitation, Mass Spectrometry, Western Blot, Confocal Microscopy, Immunofluorescence, Staining, Software

    UBQLN1 regulates expression and activity of IGF1R (A) Expression and activity of IGF1R were tested in A549 lung cancer cells following downregulation of UBQLN1 with two different siRNA (U1 KD#1 and U1 KD#2). Cells were serum starved (SF) overnight (12 hours), incubated with protein synthesis inhibitor Cycloheximide one hour prior to supplementing serum-free media with IGF1. 6 hours later, cells were harvested analyzed by Western Blot. (B) Data are normalized to Actin in non-targeting siRNA control in unstimulated cells and represented as mean+/−SEM from 2 experiments. *p ≤ 0.05. Expression of IGF2R (C) and INSR (D) were tested in A549 lung cancer cells following downregulation of UBQLN1 with two different siRNA (U1 KD#1 and U1 KD#2). Cells were cultured as in (A). IGF2R and INSR expression decreases post IGF1 stimulation in UBQLN1 deficient cells. Expression of P-AKT (D) is increased in UBQLN1 deficient cells under serum-free and stimulated conditions while T-AKT expression remains unchanged compared to control. Data are normalized to Actin in non-targeting siRNA control in unstimulated cells. (E) There is a 2-fold decrease in IGF1R mRNA expression in A549 cells that have siRNA mediated loss of UBQLN1 (p=0.0015, SEM=0.04 for U1 KD#1, p=0.0094, SEM=0.06 for U1 KD#2). Data are represented as represented as mean+/−SEM from 3 independent quantitative real-time PCR experiments done in triplicates.

    Journal: The Biochemical journal

    Article Title: Regulation of Insulin-like Growth Factor Receptors by Ubiquilin1

    doi: 10.1042/BCJ20170620

    Figure Lengend Snippet: UBQLN1 regulates expression and activity of IGF1R (A) Expression and activity of IGF1R were tested in A549 lung cancer cells following downregulation of UBQLN1 with two different siRNA (U1 KD#1 and U1 KD#2). Cells were serum starved (SF) overnight (12 hours), incubated with protein synthesis inhibitor Cycloheximide one hour prior to supplementing serum-free media with IGF1. 6 hours later, cells were harvested analyzed by Western Blot. (B) Data are normalized to Actin in non-targeting siRNA control in unstimulated cells and represented as mean+/−SEM from 2 experiments. *p ≤ 0.05. Expression of IGF2R (C) and INSR (D) were tested in A549 lung cancer cells following downregulation of UBQLN1 with two different siRNA (U1 KD#1 and U1 KD#2). Cells were cultured as in (A). IGF2R and INSR expression decreases post IGF1 stimulation in UBQLN1 deficient cells. Expression of P-AKT (D) is increased in UBQLN1 deficient cells under serum-free and stimulated conditions while T-AKT expression remains unchanged compared to control. Data are normalized to Actin in non-targeting siRNA control in unstimulated cells. (E) There is a 2-fold decrease in IGF1R mRNA expression in A549 cells that have siRNA mediated loss of UBQLN1 (p=0.0015, SEM=0.04 for U1 KD#1, p=0.0094, SEM=0.06 for U1 KD#2). Data are represented as represented as mean+/−SEM from 3 independent quantitative real-time PCR experiments done in triplicates.

    Article Snippet: IGF1R-beta (CST#3027), IGF1R Beta XP (CST#9750), p-IGF1R beta (CST#3918), INSR (CST#3025), IGF2R (CST#14364), AKT (CST#9272), p-AKT (CST#9271); Tubulin #B512 (Sigma); GAPDH (SantaCruz#FL335); Actin (Sigma#A5316), Ubiquilin1 (CST#14526); Anti-FLAG Affinity Gel (Sigma#A2220), INSR (CST#3025), FLAG polyclonal (Sigma# F7425).

    Techniques: Expressing, Activity Assay, Incubation, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction

    SUMOylation inhibition represses the Wnt/β-catenin pathway. A. For myeloma cells NCI-H929, the silencing efficiency with SUMO-1 siRNA was tested 48 hours after transfection. Total RNA was then extracted, and SUMO-1 mRNA level was determined by real time RT-PCR. Whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 antibody. SUMO-1 was indicated by arrow and was about 15 kDa in size, while SUMOylation pattern was detected as conjugated proteins with SUMO-1 modification. B. Effects of SUMO-1 inhibition on TCF/β-catenin reporter activity. Myeloma cells were transfected with control siRNA or siRNA targeting SUMO-1. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after re-transfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. Data represent mean ± S.D.; *p

    Journal: American Journal of Cancer Research

    Article Title: β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

    doi:

    Figure Lengend Snippet: SUMOylation inhibition represses the Wnt/β-catenin pathway. A. For myeloma cells NCI-H929, the silencing efficiency with SUMO-1 siRNA was tested 48 hours after transfection. Total RNA was then extracted, and SUMO-1 mRNA level was determined by real time RT-PCR. Whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 antibody. SUMO-1 was indicated by arrow and was about 15 kDa in size, while SUMOylation pattern was detected as conjugated proteins with SUMO-1 modification. B. Effects of SUMO-1 inhibition on TCF/β-catenin reporter activity. Myeloma cells were transfected with control siRNA or siRNA targeting SUMO-1. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after re-transfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. Data represent mean ± S.D.; *p

    Article Snippet: Then 20 μL was loaded onto Tricine-SDS-PAGE gels, and probed with anti-β-catenin polyclonal antibody (rabbit, CST).

    Techniques: Inhibition, Transfection, Quantitative RT-PCR, Modification, Activity Assay, Incubation, Plasmid Preparation, Negative Control, Luciferase

    Overexpression of β-catenin rescued Wnt/β-catenin pathway activity and partially prevented increased apoptosis and growth inhibition induced by SUMOylation inhibition. A. Myeloma cells NCI-H929 were treated with SUMO-1 siRNA and/or β-catenin (S33Y) plasmid for 48 hours. Protein samples were subjected to western blot analysis for the Wnt/β-catenin downstream effector proteins (c-myc, cyclinD1, survivin) with indicated antibodies. B. The distribution of apoptotic cells following treatment of myeloma cells with SUMO-1 siRNA, β-catenin (S33Y) expression vector, or both were evaluated using annexin V and propidium iodide (PI) double staining. Early apoptotic cells (Annexin-V + and PI - ) were displayed in the lower right quadrant and late apoptotic cells (Annexin-V + and PI + ) were shown in the upper right quadrant. C. The percentage of all the apoptotic cells (including Annexin-V + PI - and Annexin-V + PI + cells) were indicated by Annexin-V + cells shown as mean ± SD from three independent experiments. D. The growth inhibition of myeloma cells following different treatments was shown by the percentage of relative survival rate (OD value of sample/OD value of control in each group×100%) (*p

    Journal: American Journal of Cancer Research

    Article Title: β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

    doi:

    Figure Lengend Snippet: Overexpression of β-catenin rescued Wnt/β-catenin pathway activity and partially prevented increased apoptosis and growth inhibition induced by SUMOylation inhibition. A. Myeloma cells NCI-H929 were treated with SUMO-1 siRNA and/or β-catenin (S33Y) plasmid for 48 hours. Protein samples were subjected to western blot analysis for the Wnt/β-catenin downstream effector proteins (c-myc, cyclinD1, survivin) with indicated antibodies. B. The distribution of apoptotic cells following treatment of myeloma cells with SUMO-1 siRNA, β-catenin (S33Y) expression vector, or both were evaluated using annexin V and propidium iodide (PI) double staining. Early apoptotic cells (Annexin-V + and PI - ) were displayed in the lower right quadrant and late apoptotic cells (Annexin-V + and PI + ) were shown in the upper right quadrant. C. The percentage of all the apoptotic cells (including Annexin-V + PI - and Annexin-V + PI + cells) were indicated by Annexin-V + cells shown as mean ± SD from three independent experiments. D. The growth inhibition of myeloma cells following different treatments was shown by the percentage of relative survival rate (OD value of sample/OD value of control in each group×100%) (*p

    Article Snippet: Then 20 μL was loaded onto Tricine-SDS-PAGE gels, and probed with anti-β-catenin polyclonal antibody (rabbit, CST).

    Techniques: Over Expression, Activity Assay, Inhibition, Plasmid Preparation, Western Blot, Expressing, Double Staining

    SUMOylation inhibition promotes the degradation of β-catenin via the ubiquitin-proteasomal pathway. A. Myeloma cells were treated with SUMO-1 siRNA or negative control for 48 hours. Total RNA was then extracted, and β-catenin mRNA level was determined by real time RT-PCR. B. Myeloma cells NCI-H929 and RPMI-8226 were transfected with control or siRNA targeting SUMO-1. After 48 h incubation, whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 or anti-β-catenin antibody respectively. C. Myeloma cells NCI-H929 were treated with mounting doses of SUMO-1 siRNA (30 nM, 50 nM, 100 nM) for 48 h. Then whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 or anti-β-catenin antibody respectively. D. Myeloma cells were transfected with control or siRNA targeting SUMO-1. After 48 hours, cells were treated with CHX (100 μg/ml) for indicated time periods (0, 0.5, 1, 2 hours). Cell lysate was subjected to western blot with anti-β-catenin antibody. The relative intensity of β-catenin proteins at the indicated time points was quantified using SmartViewsoftware. In the negative control group, the intensity was standardized to 100% of the control sample (CHX 0 h); while in the SUMO-1 siRNA group, it was standardized to 100% of the control sample in the SUMO-1 siRNA group (CHX 0 h). This result is a representative of three independent experiments. E. Myeloma cells NCI-H929 were transfected with SUMO-1 siRNA or negative control. After 48 hours, the cells were harvested under denaturing conditions and immunoprecipitated with anti-β-catenin antibody. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-ubiquitin and anti-β-catenin antibodies respectively. F. Myeloma cells NCI-H929 were transfected with SUMO-1 siRNA or negative control. 48 hours after transfection, cells were treated with MG132 (10 μM) for 5 hours. The protein extracts were then analyzed by western blot using antibodies against β-catenin, SUMO-1 or actin as loading control. G. Myeloma cells were transfected with SUMO-1 siRNA together with β-catenin (WT) or β-catenin (S33Y) plasmids. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after retransfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. (Data represent mean ± S.D.; **p

    Journal: American Journal of Cancer Research

    Article Title: β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

    doi:

    Figure Lengend Snippet: SUMOylation inhibition promotes the degradation of β-catenin via the ubiquitin-proteasomal pathway. A. Myeloma cells were treated with SUMO-1 siRNA or negative control for 48 hours. Total RNA was then extracted, and β-catenin mRNA level was determined by real time RT-PCR. B. Myeloma cells NCI-H929 and RPMI-8226 were transfected with control or siRNA targeting SUMO-1. After 48 h incubation, whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 or anti-β-catenin antibody respectively. C. Myeloma cells NCI-H929 were treated with mounting doses of SUMO-1 siRNA (30 nM, 50 nM, 100 nM) for 48 h. Then whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 or anti-β-catenin antibody respectively. D. Myeloma cells were transfected with control or siRNA targeting SUMO-1. After 48 hours, cells were treated with CHX (100 μg/ml) for indicated time periods (0, 0.5, 1, 2 hours). Cell lysate was subjected to western blot with anti-β-catenin antibody. The relative intensity of β-catenin proteins at the indicated time points was quantified using SmartViewsoftware. In the negative control group, the intensity was standardized to 100% of the control sample (CHX 0 h); while in the SUMO-1 siRNA group, it was standardized to 100% of the control sample in the SUMO-1 siRNA group (CHX 0 h). This result is a representative of three independent experiments. E. Myeloma cells NCI-H929 were transfected with SUMO-1 siRNA or negative control. After 48 hours, the cells were harvested under denaturing conditions and immunoprecipitated with anti-β-catenin antibody. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-ubiquitin and anti-β-catenin antibodies respectively. F. Myeloma cells NCI-H929 were transfected with SUMO-1 siRNA or negative control. 48 hours after transfection, cells were treated with MG132 (10 μM) for 5 hours. The protein extracts were then analyzed by western blot using antibodies against β-catenin, SUMO-1 or actin as loading control. G. Myeloma cells were transfected with SUMO-1 siRNA together with β-catenin (WT) or β-catenin (S33Y) plasmids. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after retransfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. (Data represent mean ± S.D.; **p

    Article Snippet: Then 20 μL was loaded onto Tricine-SDS-PAGE gels, and probed with anti-β-catenin polyclonal antibody (rabbit, CST).

    Techniques: Inhibition, Negative Control, Quantitative RT-PCR, Transfection, Incubation, Western Blot, Immunoprecipitation, SDS Page, Plasmid Preparation, Luciferase, Activity Assay

    Endogenous β-catenin is modified by SUMO-1 in myeloma cells. A. Myeloma cells RPMI-8226 and NCI-H929 were harvested under denaturing conditions as described under “Experimental Procedures” and immunoprecipitated with anti-β–catenin rabbit antibody or IgG as a control. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 antibodies respectively. SUMOylated β-catenin and non-SUMOylated β-catenin were indicated by arrow head and arrow, respectively. B. NCI-H929 was transfected with Flag-tagged β-catenin or together with His-tagged SUMO-1 or Myc-tagged SENP1, after 48 hours, cells were harvested and then subjected to western blot with anti-Flag antibody. C. Endogenous β-catenin co-localized with SUMO-1. Myeloma cells NCI-H929 were fixed and stained with anti-β-catenin mouse antibody (green) and anti-SUMO-1 rabbit antibody (red). DNA was stained with DAPI. The images were taken by confocal microscopy as described under “Experimental Procedures”. D. After transfection of negative control and/or SUMO-1 siRNA for 48 hours, whole cell lysates were immunoprecipitated with anti-β-catenin antibody. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 respectively. SUMOylated β-catenin was indicated by arrow head. E. Bone marrow samples were isolated from myeloma patients. Total lysate was prepared from 5 × 10 6 cells in each sample and immunoprecipitated with anti-β–catenin rabbit antibody as mentioned above. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 antibodies respectively. SUMOylated β-catenin and non-SUMOylated β-catenin were indicated by arrow head and arrow, respectively.

    Journal: American Journal of Cancer Research

    Article Title: β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

    doi:

    Figure Lengend Snippet: Endogenous β-catenin is modified by SUMO-1 in myeloma cells. A. Myeloma cells RPMI-8226 and NCI-H929 were harvested under denaturing conditions as described under “Experimental Procedures” and immunoprecipitated with anti-β–catenin rabbit antibody or IgG as a control. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 antibodies respectively. SUMOylated β-catenin and non-SUMOylated β-catenin were indicated by arrow head and arrow, respectively. B. NCI-H929 was transfected with Flag-tagged β-catenin or together with His-tagged SUMO-1 or Myc-tagged SENP1, after 48 hours, cells were harvested and then subjected to western blot with anti-Flag antibody. C. Endogenous β-catenin co-localized with SUMO-1. Myeloma cells NCI-H929 were fixed and stained with anti-β-catenin mouse antibody (green) and anti-SUMO-1 rabbit antibody (red). DNA was stained with DAPI. The images were taken by confocal microscopy as described under “Experimental Procedures”. D. After transfection of negative control and/or SUMO-1 siRNA for 48 hours, whole cell lysates were immunoprecipitated with anti-β-catenin antibody. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 respectively. SUMOylated β-catenin was indicated by arrow head. E. Bone marrow samples were isolated from myeloma patients. Total lysate was prepared from 5 × 10 6 cells in each sample and immunoprecipitated with anti-β–catenin rabbit antibody as mentioned above. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 antibodies respectively. SUMOylated β-catenin and non-SUMOylated β-catenin were indicated by arrow head and arrow, respectively.

    Article Snippet: Then 20 μL was loaded onto Tricine-SDS-PAGE gels, and probed with anti-β-catenin polyclonal antibody (rabbit, CST).

    Techniques: Modification, Immunoprecipitation, SDS Page, Transfection, Western Blot, Staining, Confocal Microscopy, Negative Control, Isolation