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Active-site mTOR inhibitors (asTORi) augment HSV1 infection specifically in transformed cells. (A) Primary mouse embryonic fibroblasts (MEFs), human foreskin fibroblasts (HFF), and (B) human glioblastoma cell lines U251N and HTB-14 were pretreated with DMSO, rapamycin (RAP 100nM), or the asTORi PP242 (2μM) for 30min followed by infection with wild type HSV1 at a MOI of 0.1 for 48 hours in presence of the inhibitors. Viral infection was monitored by Western blot using antibodies against HSV1 antigens (top panel - 4E-BP1 and β-actin expression were used to monitor drug efficacy and loading, respectively), and plaque titration (bottom panel—results are presented as titers normalized to DMSO control set at 100% ± SD (n = 3)). (C) Non-transformed MEFs and HFF, as well as different transformed human cell lines, were infected with GFP-expressing HSV1-dICP0 for 48 hours at a MOI of 0.1. Resulting infection was assessed by fluorescence microscopy. (D) Transformed (4T1 and NT2196) and non-transformed (NMuMG) mouse mammary cell lines were pretreated with DMSO, rapamycin (100nM), PP242 (2μM) or INK1341 (100nM) for 30 min followed by infection with a GFP-expressing HSV1-dICP0 (0.1 MOI for 48 hours in presence of the inhibitors). Viral protein synthesis was monitored by Western blot against HSV1. Drug efficacy was monitored by phosphorylation of <t>rpS6</t> and 4E-BP1. Total rpS6 and β-actin expression were used as loading controls.
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1) Product Images from "Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis"

Article Title: Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1007264

Active-site mTOR inhibitors (asTORi) augment HSV1 infection specifically in transformed cells. (A) Primary mouse embryonic fibroblasts (MEFs), human foreskin fibroblasts (HFF), and (B) human glioblastoma cell lines U251N and HTB-14 were pretreated with DMSO, rapamycin (RAP 100nM), or the asTORi PP242 (2μM) for 30min followed by infection with wild type HSV1 at a MOI of 0.1 for 48 hours in presence of the inhibitors. Viral infection was monitored by Western blot using antibodies against HSV1 antigens (top panel - 4E-BP1 and β-actin expression were used to monitor drug efficacy and loading, respectively), and plaque titration (bottom panel—results are presented as titers normalized to DMSO control set at 100% ± SD (n = 3)). (C) Non-transformed MEFs and HFF, as well as different transformed human cell lines, were infected with GFP-expressing HSV1-dICP0 for 48 hours at a MOI of 0.1. Resulting infection was assessed by fluorescence microscopy. (D) Transformed (4T1 and NT2196) and non-transformed (NMuMG) mouse mammary cell lines were pretreated with DMSO, rapamycin (100nM), PP242 (2μM) or INK1341 (100nM) for 30 min followed by infection with a GFP-expressing HSV1-dICP0 (0.1 MOI for 48 hours in presence of the inhibitors). Viral protein synthesis was monitored by Western blot against HSV1. Drug efficacy was monitored by phosphorylation of rpS6 and 4E-BP1. Total rpS6 and β-actin expression were used as loading controls.
Figure Legend Snippet: Active-site mTOR inhibitors (asTORi) augment HSV1 infection specifically in transformed cells. (A) Primary mouse embryonic fibroblasts (MEFs), human foreskin fibroblasts (HFF), and (B) human glioblastoma cell lines U251N and HTB-14 were pretreated with DMSO, rapamycin (RAP 100nM), or the asTORi PP242 (2μM) for 30min followed by infection with wild type HSV1 at a MOI of 0.1 for 48 hours in presence of the inhibitors. Viral infection was monitored by Western blot using antibodies against HSV1 antigens (top panel - 4E-BP1 and β-actin expression were used to monitor drug efficacy and loading, respectively), and plaque titration (bottom panel—results are presented as titers normalized to DMSO control set at 100% ± SD (n = 3)). (C) Non-transformed MEFs and HFF, as well as different transformed human cell lines, were infected with GFP-expressing HSV1-dICP0 for 48 hours at a MOI of 0.1. Resulting infection was assessed by fluorescence microscopy. (D) Transformed (4T1 and NT2196) and non-transformed (NMuMG) mouse mammary cell lines were pretreated with DMSO, rapamycin (100nM), PP242 (2μM) or INK1341 (100nM) for 30 min followed by infection with a GFP-expressing HSV1-dICP0 (0.1 MOI for 48 hours in presence of the inhibitors). Viral protein synthesis was monitored by Western blot against HSV1. Drug efficacy was monitored by phosphorylation of rpS6 and 4E-BP1. Total rpS6 and β-actin expression were used as loading controls.

Techniques Used: Infection, Transformation Assay, Western Blot, Expressing, Titration, Fluorescence, Microscopy

2) Product Images from "ATR/Chk1 signaling induces autophagy through sumoylated RhoB-mediated lysosomal translocation of TSC2 after DNA damage"

Article Title: ATR/Chk1 signaling induces autophagy through sumoylated RhoB-mediated lysosomal translocation of TSC2 after DNA damage

Journal: Nature Communications

doi: 10.1038/s41467-018-06556-9

Chk1 phosphorylates RhoB to promote its sumoylation and translocation to lysosomes. a Chk1 activity is required for translocation of RhoB to lysosomes. HeLa cells pretreated 0.5 h with or without Chk1 inhibitor (0.2 μM) were subjected to immunofluorescence assay 2 h after UV (80 Jm −2 ) or 4 h after MMS (0.5 mM) treatment to examine the localization of endogenous RhoB and LAMP1. Scale bar, 10 μm. b Chk1 activity is essential for UV or MMS-induced sumoylation of endogenous RhoB. HeLa cells with expression of His-SUMO2 were treated as in panel a and then subjected to sumoylation assay. SUMO2 conjugation of RhoB was detected by immunoblotting with RhoB antibody. c Chk1 interacts with endogenous RhoB. HeLa cells transduced with Flag-tagged Chk1-D130A mutant (F-Chk1-D130A) were subjected to anti-RhoB IP followed by immunoblotting with anti-Chk1 to detect associated F-Chk1-D130A. d Chk1 phosphorylates RhoB at its threonine residues. In vitro kinase assay was carried out as described in Methods . Phosphorylated RhoB was detected by immunoblotting using phospho-threonine or phospho-serine antibodies. e Chk1 phosphorylates RhoB at Thr173 and Thr175. RhoB WT or T173,175A mutant (2A) purified from bacteria were used to perform in vitro kinase assay. f Phosphorylation of RhoB by Chk1 promotes its binding to PIAS1. HEK293T cells with expression of indicated combination of Flag-tagged PIAS1 (F-PIAS1) and HA-tagged wild-type (WT), T173,175E (2E), or T173,175 A (2A) RhoB were subjected to coimmunoprecipitation assay 1 h after treated with or without UV (80 Jm −2 ) to detect associated RhoB. g Phosphorylation of RhoB by Chk1 is required for UV or MMS-induced sumoylation. HEK293T cells with expression of indicated combination of HA-tagged SUMO2 (HA-SUMO2) and His-tagged RhoB (WT, 2E, or 2A) were subjected to sumoylation assay 1 h after treated with UV (80 Jm −2 ) or 2 h after treated with MMS (0.5 mM) to detect the SUMO2 conjugation of RhoB. h Chk1-mediated phosphorylation is essential for UV or MMS-induced lysosomal translocation of RhoB. U2OS cells with expression of HA-tagged RhoB (WT, 2E, or 2A) were subjected to immunofluorescence assay 4 h after treated with UV (80 Jm −2 ) or MMS (0.5 mM). Scale bar, 10 μm
Figure Legend Snippet: Chk1 phosphorylates RhoB to promote its sumoylation and translocation to lysosomes. a Chk1 activity is required for translocation of RhoB to lysosomes. HeLa cells pretreated 0.5 h with or without Chk1 inhibitor (0.2 μM) were subjected to immunofluorescence assay 2 h after UV (80 Jm −2 ) or 4 h after MMS (0.5 mM) treatment to examine the localization of endogenous RhoB and LAMP1. Scale bar, 10 μm. b Chk1 activity is essential for UV or MMS-induced sumoylation of endogenous RhoB. HeLa cells with expression of His-SUMO2 were treated as in panel a and then subjected to sumoylation assay. SUMO2 conjugation of RhoB was detected by immunoblotting with RhoB antibody. c Chk1 interacts with endogenous RhoB. HeLa cells transduced with Flag-tagged Chk1-D130A mutant (F-Chk1-D130A) were subjected to anti-RhoB IP followed by immunoblotting with anti-Chk1 to detect associated F-Chk1-D130A. d Chk1 phosphorylates RhoB at its threonine residues. In vitro kinase assay was carried out as described in Methods . Phosphorylated RhoB was detected by immunoblotting using phospho-threonine or phospho-serine antibodies. e Chk1 phosphorylates RhoB at Thr173 and Thr175. RhoB WT or T173,175A mutant (2A) purified from bacteria were used to perform in vitro kinase assay. f Phosphorylation of RhoB by Chk1 promotes its binding to PIAS1. HEK293T cells with expression of indicated combination of Flag-tagged PIAS1 (F-PIAS1) and HA-tagged wild-type (WT), T173,175E (2E), or T173,175 A (2A) RhoB were subjected to coimmunoprecipitation assay 1 h after treated with or without UV (80 Jm −2 ) to detect associated RhoB. g Phosphorylation of RhoB by Chk1 is required for UV or MMS-induced sumoylation. HEK293T cells with expression of indicated combination of HA-tagged SUMO2 (HA-SUMO2) and His-tagged RhoB (WT, 2E, or 2A) were subjected to sumoylation assay 1 h after treated with UV (80 Jm −2 ) or 2 h after treated with MMS (0.5 mM) to detect the SUMO2 conjugation of RhoB. h Chk1-mediated phosphorylation is essential for UV or MMS-induced lysosomal translocation of RhoB. U2OS cells with expression of HA-tagged RhoB (WT, 2E, or 2A) were subjected to immunofluorescence assay 4 h after treated with UV (80 Jm −2 ) or MMS (0.5 mM). Scale bar, 10 μm

Techniques Used: Translocation Assay, Activity Assay, Immunofluorescence, Expressing, Conjugation Assay, Transduction, Mutagenesis, In Vitro, Kinase Assay, Purification, Binding Assay, Co-Immunoprecipitation Assay

Phosphorylated and sumoylated RhoB recruits TSC2 to lysosomes to inhibit mTORC1 activity. a Knockout of RhoB prevents UV or MMS-induced downregulation of phosphorylated ULK1 and S6K. RhoB +/+ or RhoB − / − cells were subjected to immunoblotting assay 2 h after UV (80 Jm −2 ) or 4 h after MMS (0.5 mM). b Sumoylation of RhoB is required for UV or MMS-induced downregulation of phosphorylated ULK1 and S6K. RhoB − / − cells transduced with HA-tagged RhoB (WT or 4KR) were treated and subjected to immunoblotting assay as in panel a . c UV or MMS treatment induces colocalization of endogenous RhoB and TSC2. HeLa cells were treated as in panel a and subjected to immunofluorescence assay to examine the localization of endogenous RhoB and TSC2. Scale bar, 10 μm. d Knockout of RhoB attenuates translocation of TSC2 to lysosomes after UV or MMS treatment. RhoB +/+ or RhoB − / − cells were treated as in panel a and subjected to immunofluorescence to examine the colocalization of endogenous TSC2 and LAMP1. e Sumoylation of RhoB is critical for UV or MMS-induced lysosomal translocation of TSC2. RhoB − / − cells transduced with HA-tagged WT or 4KR RhoB were treated as in panel a and subjected to immunofluorescence assay. The dot lines outline cells with expression of RhoB WT or 4KR. Scale bar, 10 μm. f UV or MMS treatment enhances interaction between RhoB and endogenous TSC2. HeLa cells were treated as in panel a and subjected to anti-TSC2 immunoprecipitation followed by immunoblotting to detect associated RhoB. g Phosphorylation of RhoB is required for its interaction with TSC2 induced by UV or MMS treatment. HeLa cells transduced with HA-tagged RhoB (WT, 2E, or 2A) were treated, subjected to anti-TSC2 immunoprecipitation and immunoblotted as in panel g . h Sumoylation of RhoB is not responsible for its binding to TSC2. HeLa cells transduced with HA-tagged RhoB (WT or 4KR) were treated, subjected to anti-TSC2 immunoprecipitation and immunoblotted as in panel g
Figure Legend Snippet: Phosphorylated and sumoylated RhoB recruits TSC2 to lysosomes to inhibit mTORC1 activity. a Knockout of RhoB prevents UV or MMS-induced downregulation of phosphorylated ULK1 and S6K. RhoB +/+ or RhoB − / − cells were subjected to immunoblotting assay 2 h after UV (80 Jm −2 ) or 4 h after MMS (0.5 mM). b Sumoylation of RhoB is required for UV or MMS-induced downregulation of phosphorylated ULK1 and S6K. RhoB − / − cells transduced with HA-tagged RhoB (WT or 4KR) were treated and subjected to immunoblotting assay as in panel a . c UV or MMS treatment induces colocalization of endogenous RhoB and TSC2. HeLa cells were treated as in panel a and subjected to immunofluorescence assay to examine the localization of endogenous RhoB and TSC2. Scale bar, 10 μm. d Knockout of RhoB attenuates translocation of TSC2 to lysosomes after UV or MMS treatment. RhoB +/+ or RhoB − / − cells were treated as in panel a and subjected to immunofluorescence to examine the colocalization of endogenous TSC2 and LAMP1. e Sumoylation of RhoB is critical for UV or MMS-induced lysosomal translocation of TSC2. RhoB − / − cells transduced with HA-tagged WT or 4KR RhoB were treated as in panel a and subjected to immunofluorescence assay. The dot lines outline cells with expression of RhoB WT or 4KR. Scale bar, 10 μm. f UV or MMS treatment enhances interaction between RhoB and endogenous TSC2. HeLa cells were treated as in panel a and subjected to anti-TSC2 immunoprecipitation followed by immunoblotting to detect associated RhoB. g Phosphorylation of RhoB is required for its interaction with TSC2 induced by UV or MMS treatment. HeLa cells transduced with HA-tagged RhoB (WT, 2E, or 2A) were treated, subjected to anti-TSC2 immunoprecipitation and immunoblotted as in panel g . h Sumoylation of RhoB is not responsible for its binding to TSC2. HeLa cells transduced with HA-tagged RhoB (WT or 4KR) were treated, subjected to anti-TSC2 immunoprecipitation and immunoblotted as in panel g

Techniques Used: Activity Assay, Knock-Out, Transduction, Immunofluorescence, Translocation Assay, Expressing, Immunoprecipitation, Binding Assay

Sumoylated RhoB is translocated to lysosomes. a Sumoylation of RhoB is specific to ultraviolet (UV) or methyl methanesulphonate (MMS) treatment. One hour after treated with UV (80 Jm −2 ) or 2 h after treated with MMS (0.5 mM), camptothecin (CPT) (10 μM), or doxorubicin (DOX) (0.5 μM), HEK293T cells expressing HA-SUMO2 and His-RhoB were subjected to sumoylation assay to detect the SUMO2 conjugation of RhoB. b UV or MMS treatment enhances sumoylation of endogenous RhoB. HeLa cells with expression of His-SUMO2 were subjected to sumoylation assay 2 h after treated with UV (80 Jm −2 ) or 4 h after treated with MMS (0.5 mM). SUMO2 conjugation of RhoB was detected by immunoblotting with RhoB antibody. c Endogenous RhoB colocalizes with LAMP1 after UV or MMS but not CPT or DOX treatment. HeLa cells were subjected to immunofluorescence assay 2 h after treated with or without UV (80 Jm −2 ), MMS (0.5 mM), CPT (10 μM), or DOX (0.5 μM). Scale bar, 10 μm. d RhoB WT but not RhoB-4KR colocalizes with LAMP1 after UV or MMS treatment. U2OS cells expressing HA-RhoB (WT or 4KR) were subjected to immunofluorescence assay 4 h after UV (80 Jm −2 ) or MMS (0.5 mM) treatment to examine the localization of HA-RhoB and endogenous LAMP1. Scale bar, 10 μm
Figure Legend Snippet: Sumoylated RhoB is translocated to lysosomes. a Sumoylation of RhoB is specific to ultraviolet (UV) or methyl methanesulphonate (MMS) treatment. One hour after treated with UV (80 Jm −2 ) or 2 h after treated with MMS (0.5 mM), camptothecin (CPT) (10 μM), or doxorubicin (DOX) (0.5 μM), HEK293T cells expressing HA-SUMO2 and His-RhoB were subjected to sumoylation assay to detect the SUMO2 conjugation of RhoB. b UV or MMS treatment enhances sumoylation of endogenous RhoB. HeLa cells with expression of His-SUMO2 were subjected to sumoylation assay 2 h after treated with UV (80 Jm −2 ) or 4 h after treated with MMS (0.5 mM). SUMO2 conjugation of RhoB was detected by immunoblotting with RhoB antibody. c Endogenous RhoB colocalizes with LAMP1 after UV or MMS but not CPT or DOX treatment. HeLa cells were subjected to immunofluorescence assay 2 h after treated with or without UV (80 Jm −2 ), MMS (0.5 mM), CPT (10 μM), or DOX (0.5 μM). Scale bar, 10 μm. d RhoB WT but not RhoB-4KR colocalizes with LAMP1 after UV or MMS treatment. U2OS cells expressing HA-RhoB (WT or 4KR) were subjected to immunofluorescence assay 4 h after UV (80 Jm −2 ) or MMS (0.5 mM) treatment to examine the localization of HA-RhoB and endogenous LAMP1. Scale bar, 10 μm

Techniques Used: Cycling Probe Technology, Expressing, Conjugation Assay, Immunofluorescence

3) Product Images from "Immunoregulatory Protein B7-H3 Regulates Cancer Stem Cell Enrichment and Drug Resistance through MVP-mediated MEK Activation"

Article Title: Immunoregulatory Protein B7-H3 Regulates Cancer Stem Cell Enrichment and Drug Resistance through MVP-mediated MEK Activation

Journal: Oncogene

doi: 10.1038/s41388-018-0407-9

MEK activation is required for B7-H3 induced breast stem cells and acinar disruption A-C. HMLECD24 low CD44 high and MCF-10A-B7H3 were treated U0126 and DMSO(control) for 24 hours. Cells were incubated with CD24 and CD44 antibodies for 45 min, washed, and then analyzed by flow cytometry. D-E. HMLE-B7H3 and MCF-10A-B7H3 cells were transfected with scramble and MEK1/2 siRNA respectively. 48 hours later after transfection, cells were incubated with CD24 and CD44 antibodies for 45 min, washed, and then analyzed by flow cytometry. G. Cells were seeded in 3D Matrigel culture and treated with U0126 or DMSO as control. F. Phase-contrast images showing acinar morphology ofHMLECD24 high CD44 low with U0126 and DMSO.
Figure Legend Snippet: MEK activation is required for B7-H3 induced breast stem cells and acinar disruption A-C. HMLECD24 low CD44 high and MCF-10A-B7H3 were treated U0126 and DMSO(control) for 24 hours. Cells were incubated with CD24 and CD44 antibodies for 45 min, washed, and then analyzed by flow cytometry. D-E. HMLE-B7H3 and MCF-10A-B7H3 cells were transfected with scramble and MEK1/2 siRNA respectively. 48 hours later after transfection, cells were incubated with CD24 and CD44 antibodies for 45 min, washed, and then analyzed by flow cytometry. G. Cells were seeded in 3D Matrigel culture and treated with U0126 or DMSO as control. F. Phase-contrast images showing acinar morphology ofHMLECD24 high CD44 low with U0126 and DMSO.

Techniques Used: Activation Assay, Incubation, Flow Cytometry, Cytometry, Transfection

B7-H3 increases breast stem cells A. HMLE-CD24 low CD44 high and HMLE-CD24 high CD44 low cells were sorted form parental HMLE cells. Cell lysates were prepared for Western blotting with an antibody against B7-H3, and β-actin was used as a loading control. B-D. 1 × 10 6 HMLE-vector, HMLE-B7H3, MCF-10A-vector, MCF-10A-B7H3, MCF-10AT-Scr, MCF-10AT-sh1, MCF-10AT-sh2, HMLE-CD24 low CD44 high vector, HMLE-CD24 low CD44 high crispr B7H3-1, and HMLE- CD24 low CD44 high crispr B7H3-2 stable cells were incubated with CD24 and CD44 antibodies for 45 min, washed, and then analyzed by flow cytometry. E. SKBR3-vector, SKBR3-B7H3, HMLE-vector, HMLE-B7H3, MCF-10A-vector, and MCF-10A-B7H3 cells were seeded in ultralow attachment 96-well plates at different cell numbers with conditioned medium. After 5–10 days incubation, the mammosphere (diameter > 75 μm) number was counted. X-axis represents the number of seeded cells per well. Columns represent the mean of three independent experiments; Bars represent S.E. **, p
Figure Legend Snippet: B7-H3 increases breast stem cells A. HMLE-CD24 low CD44 high and HMLE-CD24 high CD44 low cells were sorted form parental HMLE cells. Cell lysates were prepared for Western blotting with an antibody against B7-H3, and β-actin was used as a loading control. B-D. 1 × 10 6 HMLE-vector, HMLE-B7H3, MCF-10A-vector, MCF-10A-B7H3, MCF-10AT-Scr, MCF-10AT-sh1, MCF-10AT-sh2, HMLE-CD24 low CD44 high vector, HMLE-CD24 low CD44 high crispr B7H3-1, and HMLE- CD24 low CD44 high crispr B7H3-2 stable cells were incubated with CD24 and CD44 antibodies for 45 min, washed, and then analyzed by flow cytometry. E. SKBR3-vector, SKBR3-B7H3, HMLE-vector, HMLE-B7H3, MCF-10A-vector, and MCF-10A-B7H3 cells were seeded in ultralow attachment 96-well plates at different cell numbers with conditioned medium. After 5–10 days incubation, the mammosphere (diameter > 75 μm) number was counted. X-axis represents the number of seeded cells per well. Columns represent the mean of three independent experiments; Bars represent S.E. **, p

Techniques Used: Western Blot, Plasmid Preparation, CRISPR, Incubation, Flow Cytometry, Cytometry

B7-H3 overexpression disrupts polarity and acinar structure with increased invasion potential A-C. Cells were seeded in 3D Matrigel culture and grown for 8 days. Phase-contrast images show acinar morphology ofHMLECD24 high CD44 low , HMLECD24 low CD44 high, HMLE-vector, HMLE-B7H3, MDA468-vector, MDA468-B7H3, MCF-12A-vector, MCF-12A-B7H3, HMLECD24 low CD44 high crispr vector, and HMLECD24 low CD44 high B7H3crispr cells. D. Cells were seeded in 3D Matrigel culture and grown for 8 days. Cells were stained with epithelial marker (E-cadherin, red) and DAPI (blue).
Figure Legend Snippet: B7-H3 overexpression disrupts polarity and acinar structure with increased invasion potential A-C. Cells were seeded in 3D Matrigel culture and grown for 8 days. Phase-contrast images show acinar morphology ofHMLECD24 high CD44 low , HMLECD24 low CD44 high, HMLE-vector, HMLE-B7H3, MDA468-vector, MDA468-B7H3, MCF-12A-vector, MCF-12A-B7H3, HMLECD24 low CD44 high crispr vector, and HMLECD24 low CD44 high B7H3crispr cells. D. Cells were seeded in 3D Matrigel culture and grown for 8 days. Cells were stained with epithelial marker (E-cadherin, red) and DAPI (blue).

Techniques Used: Over Expression, Plasmid Preparation, CRISPR, Staining, Marker

B7-H3 regulates MEK activation in vitro and in vivo A-B. MDA468-vector, MDA468-B7H3, MCF-10A-vector, MCF-10-B7H3, HMLE-vector, HMLE-B7H3, SKB3-vector, SKBR3-B7H3, MCF-10AT-Scr, MCF-10AT-sh1(B7H3), and MCF-10AT-sh2 (B7H3) cells were collected. Cell lysates were prepared for Western blotting with an antibody against B7-H3, MVP, total MEK, MEK-p, and β-actin(or tubulin) was used as a loading control. C-D. Tissue microarrays were stained with B7-H3, MVP, and MEK-P antibodies. The intensity of the positive staining (scores: negative=0, weak=1, moderate=2, strong=3) was scored. Then, the number of positive-stained cells (scores:
Figure Legend Snippet: B7-H3 regulates MEK activation in vitro and in vivo A-B. MDA468-vector, MDA468-B7H3, MCF-10A-vector, MCF-10-B7H3, HMLE-vector, HMLE-B7H3, SKB3-vector, SKBR3-B7H3, MCF-10AT-Scr, MCF-10AT-sh1(B7H3), and MCF-10AT-sh2 (B7H3) cells were collected. Cell lysates were prepared for Western blotting with an antibody against B7-H3, MVP, total MEK, MEK-p, and β-actin(or tubulin) was used as a loading control. C-D. Tissue microarrays were stained with B7-H3, MVP, and MEK-P antibodies. The intensity of the positive staining (scores: negative=0, weak=1, moderate=2, strong=3) was scored. Then, the number of positive-stained cells (scores:

Techniques Used: Activation Assay, In Vitro, In Vivo, Plasmid Preparation, Western Blot, Staining

MEK inhibitor resensitized B7-H3 overexpressing breast cancer cells to Taxol MDA468-vector and MDA468-B7H3 cells were seeded in 96 well plate at 8×10 3 cells/well. Cells were treated with medium, U0126, Taxol, and Taxol plus U0126 respectively for 48 hours. Cell images were taken by microscope.
Figure Legend Snippet: MEK inhibitor resensitized B7-H3 overexpressing breast cancer cells to Taxol MDA468-vector and MDA468-B7H3 cells were seeded in 96 well plate at 8×10 3 cells/well. Cells were treated with medium, U0126, Taxol, and Taxol plus U0126 respectively for 48 hours. Cell images were taken by microscope.

Techniques Used: Plasmid Preparation, Microscopy

B7-H3 regulates MEK activation through MVP A-B. IP of B7-H3 (or MVP) and control mouse IgG followed by immunoblot analysis of MDA468-B7H3 with whole cell lysates as positive control. C-D. Immunoblot analysis of WCL or IP of MVP and control mouse IgG from lysates from MCF-12A-wt-B7H3, MCF-12A-B7H3-D489(cytosolic domain deletion), MDA468-wt-B7H3, MDA468-B7H3-D489(cytosolic domain deletion). E-F. Cells were transfected with scramble and MVP siRNAs respectively. 48 hours later after transfection, cell lysates were prepared for Western blotting with an antibody against B7-H3, MVP, total MEK, MEK-p, and β-actin(or tubulin) was used as a loading control. G. Immunoblot analysis of IP of MEK and control mouse IgG of lysates from MDA468-vector and MDA468-B7H3. H. Immunoblot analysis of IP of MVP (or B7-H3) and control mouse IgG of lysates from MDA468-vector and MDA468-B7H3. I. Immunoblot analysis of IP of MEK and control mouse IgG of lysates from MDA468-B7H3(scramble) and MDA468-B7H3(MVP shRNA).
Figure Legend Snippet: B7-H3 regulates MEK activation through MVP A-B. IP of B7-H3 (or MVP) and control mouse IgG followed by immunoblot analysis of MDA468-B7H3 with whole cell lysates as positive control. C-D. Immunoblot analysis of WCL or IP of MVP and control mouse IgG from lysates from MCF-12A-wt-B7H3, MCF-12A-B7H3-D489(cytosolic domain deletion), MDA468-wt-B7H3, MDA468-B7H3-D489(cytosolic domain deletion). E-F. Cells were transfected with scramble and MVP siRNAs respectively. 48 hours later after transfection, cell lysates were prepared for Western blotting with an antibody against B7-H3, MVP, total MEK, MEK-p, and β-actin(or tubulin) was used as a loading control. G. Immunoblot analysis of IP of MEK and control mouse IgG of lysates from MDA468-vector and MDA468-B7H3. H. Immunoblot analysis of IP of MVP (or B7-H3) and control mouse IgG of lysates from MDA468-vector and MDA468-B7H3. I. Immunoblot analysis of IP of MEK and control mouse IgG of lysates from MDA468-B7H3(scramble) and MDA468-B7H3(MVP shRNA).

Techniques Used: Activation Assay, Positive Control, Transfection, Western Blot, Plasmid Preparation, shRNA

4) Product Images from "The pro-metastasis effect of circANKS1B in breast cancer"

Article Title: The pro-metastasis effect of circANKS1B in breast cancer

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0914-x

USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the pGL3-basic vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p
Figure Legend Snippet: USF1 transcriptionally elevates TGF-β1 expression in breast cancer. a The relative luciferase activities were analyzed after co-transfection with various TGF-β1 promoter reporters or the pGL3-basic vector and USF1 or control vector. b Schematic of the location of E-box motif bound by USF1 in TGF-β1 promoter region. c Wild-type (WT) or mutant (Mut) TGF-β1 luciferase reporter vector was co-transfected with USF1 or control vector into MCF-7 and MDA-MB-231 cells, after 48 h of co-transfection, the luciferase activities were tested. d ChIP-qPCR analysis of USF1 binding to the TGF-β1 promoter region in MCF-7 and MDA-MB-231 cells. RNA polymerase II (RNAPII) was used as a positive control. e qRT-PCR (left) and immunoblot (right) analysis of TGF-β1 mRNA expression in USF1-overexpressing MCF-7 cells and USF1 knockdown MDA-MB-231 cells. β-actin was used as a loading control. f The correlation between USF1 and TGF-β1 expression in breast cancer tissues from TCGA database was analyzed by Spearman correlation coefficients ( r = 0.223, n = 1109, p

Techniques Used: Expressing, Luciferase, Cotransfection, Plasmid Preparation, Mutagenesis, Transfection, Multiple Displacement Amplification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Positive Control, Quantitative RT-PCR

ESRP1 promotes circANKS1B formation and it is also a target of USF1 in breast cancer. a qRT-PCR analysis of circANKS1B and linear ANKS1B in MCF-7 and MDA-MB-231 cells with ESRP1 overexpression. b Schematic of circANKS1B minigene with four wild-type (WT) or mutant (Mut) ESRP1 binding sites. c RIP analysis of ESRP1-binding to wild-type (WT) or mutant (Mut) circANKS1B minigene using an antibody against ESRP1. d qRT-PCR analysis of circANKS1B after co-transfection with si-ESRP1 or si-NC and wild-type (WT) or various mutant (Mut) circANKS1B minigenes. e Representative IHC images of low (patient # 56) and high (patient # 28) ESRP1 expression in breast cancer tissues. Scale bar = 20 μm. f A strong correlation between ESRP1 and circANKS1B expression in breast cancer tissues assessed by Spearman correlation coefficients ( r = 0.626, n = 165, p
Figure Legend Snippet: ESRP1 promotes circANKS1B formation and it is also a target of USF1 in breast cancer. a qRT-PCR analysis of circANKS1B and linear ANKS1B in MCF-7 and MDA-MB-231 cells with ESRP1 overexpression. b Schematic of circANKS1B minigene with four wild-type (WT) or mutant (Mut) ESRP1 binding sites. c RIP analysis of ESRP1-binding to wild-type (WT) or mutant (Mut) circANKS1B minigene using an antibody against ESRP1. d qRT-PCR analysis of circANKS1B after co-transfection with si-ESRP1 or si-NC and wild-type (WT) or various mutant (Mut) circANKS1B minigenes. e Representative IHC images of low (patient # 56) and high (patient # 28) ESRP1 expression in breast cancer tissues. Scale bar = 20 μm. f A strong correlation between ESRP1 and circANKS1B expression in breast cancer tissues assessed by Spearman correlation coefficients ( r = 0.626, n = 165, p

Techniques Used: Quantitative RT-PCR, Multiple Displacement Amplification, Over Expression, Mutagenesis, Binding Assay, Cotransfection, Immunohistochemistry, Expressing

The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. ** p
Figure Legend Snippet: The ESRP1/circANKS1B/miR-148a/152-3p/USF1 feedback loop promotes breast cancer invasion and metastasis via inducing TGF-β1/Smad-mediated EMT. a-b Wound healing, transwell migration and invasion assays for circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μm. Representative images are shown at 0 and 24 h after gap creation. Scale bar = 20 μm. c Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with ESRP1, USF1 or TGF-β1 vector, or miR-148a/152-3p inhibitors. d Immunoblot analysis of p-Smad2, p-Smad3, Smad2/3, E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells transfected with si-ESRP1, si-USF1 or miR-148a/152-3p mimics or treated with LY2109761 at a final concentration of 10 μM. GAPDH was used as a loading control. e The illustration summarizes our findings. CircANKS1B, as miR-148a-3p and miR-152-3p sponge, increases USF1 expression by eliminating miR-148a/152-3p-mediated repression of USF1, and then, USF1 can respectively transcriptionally up-regulate ESRP1 and TGF-β1 expression via directly binding to the E-box motifs in their promoter regions. Subsequently, ESRP1 promotes circANKS1B generation, and TGF-β1 activates its downstream Smad signaling to induce EMT, thereby enhancing breast cancer invasion and metastasis. Data were represented as means ± S.D. of at least three independent experiments. ** p

Techniques Used: Migration, Transfection, Concentration Assay, Multiple Displacement Amplification, Plasmid Preparation, Expressing, Binding Assay

CircANKS1B promotes breast cancer invasion and metastasis by sponge activity of miR-148a-3p and miR-152-3p and up-regulation of USF1. a-b qRT-PCR analysis of ARF4, FGF7, USF1, FZD6, NFAT5 and SOX5 in circANKS1B-overexpressing MCF-7 cells and circANKS1B knockdown MDA-MB-231 cells. c Schematic of luciferase reporter vectors containing wild-type (WT) or mutant (Mut) putative miR-148a/152-3p binding sites of USF1 3′ UTR. d Wild-type or mutant USF1 3′ UTR luciferase reporter vector was co-transfected with miR-148a/152-3p mimics or control mimics into MCF-7 and MDA-MB-231 cells, after 48 h, the luciferase activities were measured. e The relative luciferase activities were analyzed after co-transfection with circANKS1B or control vector, miR-148a/152-3p mimics or control mimics and wild-type (WT) USF1 3′ UTR luciferase reporter vectors. f Immunoblot analysis of USF1 protein expression after co-transfection with circANKS1B or control vector and miR-148a/152-3p mimics or control mimics. GAPDH was used as a loading control. g Transwell invasion assay was performed after co-transfection with circANKS1B or control vector and miR-148a/152-3p mimics or control mimics. h Analysis of USF1 expression in breast cancer patients with or without lymph node metastasis. Using median USF1 H-score value as cutoff. i Representative IHC images of low (patient # 10) and high (patient # 89) USF1 expression in breast cancer tissues. Scale bar = 20 μm. j A strong correlation between circANKS1B and USF1 expression in breast cancer tissues analyzed by Spearman correlation coefficients ( r = 0.572, n = 165, p
Figure Legend Snippet: CircANKS1B promotes breast cancer invasion and metastasis by sponge activity of miR-148a-3p and miR-152-3p and up-regulation of USF1. a-b qRT-PCR analysis of ARF4, FGF7, USF1, FZD6, NFAT5 and SOX5 in circANKS1B-overexpressing MCF-7 cells and circANKS1B knockdown MDA-MB-231 cells. c Schematic of luciferase reporter vectors containing wild-type (WT) or mutant (Mut) putative miR-148a/152-3p binding sites of USF1 3′ UTR. d Wild-type or mutant USF1 3′ UTR luciferase reporter vector was co-transfected with miR-148a/152-3p mimics or control mimics into MCF-7 and MDA-MB-231 cells, after 48 h, the luciferase activities were measured. e The relative luciferase activities were analyzed after co-transfection with circANKS1B or control vector, miR-148a/152-3p mimics or control mimics and wild-type (WT) USF1 3′ UTR luciferase reporter vectors. f Immunoblot analysis of USF1 protein expression after co-transfection with circANKS1B or control vector and miR-148a/152-3p mimics or control mimics. GAPDH was used as a loading control. g Transwell invasion assay was performed after co-transfection with circANKS1B or control vector and miR-148a/152-3p mimics or control mimics. h Analysis of USF1 expression in breast cancer patients with or without lymph node metastasis. Using median USF1 H-score value as cutoff. i Representative IHC images of low (patient # 10) and high (patient # 89) USF1 expression in breast cancer tissues. Scale bar = 20 μm. j A strong correlation between circANKS1B and USF1 expression in breast cancer tissues analyzed by Spearman correlation coefficients ( r = 0.572, n = 165, p

Techniques Used: Activity Assay, Quantitative RT-PCR, Multiple Displacement Amplification, Luciferase, Mutagenesis, Binding Assay, Plasmid Preparation, Transfection, Cotransfection, Expressing, Transwell Invasion Assay, Immunohistochemistry

5) Product Images from "An Aqueous Extract of Herbal Medicine ALWPs Enhances Cognitive Performance and Inhibits LPS-Induced Neuroinflammation via FAK/NF-κB Signaling Pathways"

Article Title: An Aqueous Extract of Herbal Medicine ALWPs Enhances Cognitive Performance and Inhibits LPS-Induced Neuroinflammation via FAK/NF-κB Signaling Pathways

Journal: Frontiers in Aging Neuroscience

doi: 10.3389/fnagi.2018.00269

ALWPs decreased LPS-induced nuclear NF-κB (Ser536) levels. (A) BV2 microglial cells were pretreated with ALWPs (500 μg/ml) or PBS for 5 h, followed by treatment with LPS (1 μg/ml) or PBS for 45 min and subcellular fractionation (nucleus vs. cytosol). Western blotting was conducted on the cytosolic fraction using antibodies against p-IκBα, IκBα, NF-κB, and β-actin (as a cytosolic marker). (B–D) Quantification of the data from (A) (p-IκBα: con, n = 12; LPS, n = 12; ALWPs + LPS, n = 12; IκBα: con, n = 12; LPS, n = 12; ALWPs + LPS, n = 12; NF-κB: con, n = 12; LPS, n = 12; ALWPs + LPS, n = 12). (E) Western blotting was performed on the nuclear fraction using antibodies against NF-κB and PCNA (as a nuclear marker). (F) Quantification of the data from (E) (con, n = 12; LPS, n = 12; ALWPs + LPS, n = 12). (G) BV2 microglial cells were pretreated with ALWPs (500 μg/ml) or PBS for 5 h and then treated with LPS (1 μg/ml) or PBS for 45 min. Cells were fixed and immunostained with anti-CD11b and anti-p-NF-κB (Ser536) antibodies (40× confocal images). (H) Quantification of the data from (G) (con, n = 122 cells; LPS, n = 129 cells; LPS + ALWPs, n = 111 cells). (I) BV2 microglial cells were pretreated with PF-573228 (a FAK inhibitor, 5 μM) or vehicle (1% DMSO) for 30 min, followed by treatment with ALWPs (500 μg/ml) or PBS for 30 min and finally LPS (1 μg/ml) or PBS for 5 h. Cells were fixed and immunostained with anti-CD11b and anti-p-NF-κB (Ser536) antibodies. (J) Quantification of the data from (I) (con, n = 260 cells; LPS, n = 331 cells; LPS + ALWPs, n = 265 cells; LPS + FAK inhibitor, n = 188 cells; LPS + FAK inhibitor + ALWPs, n = 199 cells). Scale bar 20 μm (40× confocal images). ∗ p
Figure Legend Snippet: ALWPs decreased LPS-induced nuclear NF-κB (Ser536) levels. (A) BV2 microglial cells were pretreated with ALWPs (500 μg/ml) or PBS for 5 h, followed by treatment with LPS (1 μg/ml) or PBS for 45 min and subcellular fractionation (nucleus vs. cytosol). Western blotting was conducted on the cytosolic fraction using antibodies against p-IκBα, IκBα, NF-κB, and β-actin (as a cytosolic marker). (B–D) Quantification of the data from (A) (p-IκBα: con, n = 12; LPS, n = 12; ALWPs + LPS, n = 12; IκBα: con, n = 12; LPS, n = 12; ALWPs + LPS, n = 12; NF-κB: con, n = 12; LPS, n = 12; ALWPs + LPS, n = 12). (E) Western blotting was performed on the nuclear fraction using antibodies against NF-κB and PCNA (as a nuclear marker). (F) Quantification of the data from (E) (con, n = 12; LPS, n = 12; ALWPs + LPS, n = 12). (G) BV2 microglial cells were pretreated with ALWPs (500 μg/ml) or PBS for 5 h and then treated with LPS (1 μg/ml) or PBS for 45 min. Cells were fixed and immunostained with anti-CD11b and anti-p-NF-κB (Ser536) antibodies (40× confocal images). (H) Quantification of the data from (G) (con, n = 122 cells; LPS, n = 129 cells; LPS + ALWPs, n = 111 cells). (I) BV2 microglial cells were pretreated with PF-573228 (a FAK inhibitor, 5 μM) or vehicle (1% DMSO) for 30 min, followed by treatment with ALWPs (500 μg/ml) or PBS for 30 min and finally LPS (1 μg/ml) or PBS for 5 h. Cells were fixed and immunostained with anti-CD11b and anti-p-NF-κB (Ser536) antibodies. (J) Quantification of the data from (I) (con, n = 260 cells; LPS, n = 331 cells; LPS + ALWPs, n = 265 cells; LPS + FAK inhibitor, n = 188 cells; LPS + FAK inhibitor + ALWPs, n = 199 cells). Scale bar 20 μm (40× confocal images). ∗ p

Techniques Used: Fractionation, Western Blot, Marker

6) Product Images from "Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway"

Article Title: Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0787-2

ERK/p38 is downstream of RhoA/ROCK in supervillin-mediated cell metastasis during hypoxia. a. MHCC-97H cells that had been transfected with SV1, SV4, or SV5 plasmids were treated with PBS or the MEK inhibitor PD0325901 (10 μM) for 1 h under hypoxic conditions before assaying for phosphorylated p38, phosphorylated ERK, Snail1, and Vimentin by immunoblotting. β-tubulin was used as the loading control. b, c. MHCC-97H cells that had been transfected with SV1, SV4, or SV5 plasmids were treated with PBS or the MEK inhibitor PD0325901 (10 μM) for 1 h under hypoxic conditions before assay for cell migration ( b ) and invasion ( c ). The number of migrated SV1, SV4, or SV5 cells treated with PD0325901 was compared to those control cells treated with PBS. D. MHCC-97H and Huh-7 cells co-transfected with control or supervillin-specific siRNA and a RhoA(WT), RhoA(V14), or RhoA(N19) plasmids for 48 h were treated with PBS or the ROCK inhibitor Y27632 2HCl (10 μM) for 16 h during hypoxia, and then assayed for phosphorylated p38 and ERK by immunoblotting. β-actin was used as the loading control
Figure Legend Snippet: ERK/p38 is downstream of RhoA/ROCK in supervillin-mediated cell metastasis during hypoxia. a. MHCC-97H cells that had been transfected with SV1, SV4, or SV5 plasmids were treated with PBS or the MEK inhibitor PD0325901 (10 μM) for 1 h under hypoxic conditions before assaying for phosphorylated p38, phosphorylated ERK, Snail1, and Vimentin by immunoblotting. β-tubulin was used as the loading control. b, c. MHCC-97H cells that had been transfected with SV1, SV4, or SV5 plasmids were treated with PBS or the MEK inhibitor PD0325901 (10 μM) for 1 h under hypoxic conditions before assay for cell migration ( b ) and invasion ( c ). The number of migrated SV1, SV4, or SV5 cells treated with PD0325901 was compared to those control cells treated with PBS. D. MHCC-97H and Huh-7 cells co-transfected with control or supervillin-specific siRNA and a RhoA(WT), RhoA(V14), or RhoA(N19) plasmids for 48 h were treated with PBS or the ROCK inhibitor Y27632 2HCl (10 μM) for 16 h during hypoxia, and then assayed for phosphorylated p38 and ERK by immunoblotting. β-actin was used as the loading control

Techniques Used: Transfection, Migration

Supervillin regulates hypoxia-induced epithelial-mesenchymal transition (EMT) of HCC. a. Bright-field microscopy showing the morphological changes that occur when HepG2 and Huh-7 cells are cultured for 16 h in hypoxia. b. Immunoblots showing the changes in E-cadherin, Vimentin, Snail1, and supervillin isoforms during hypoxia after treatment with supervillin-specific siRNA. HepG2 and Huh-7 cells were transfected with a control or supervillin-specific siRNA and incubated under normoxia for 48 h followed by being scratched and exposed to normoxia or hypoxia for 16 h; β-tubulin was used as the loading control. c. Immunofluorescence staining showing that the relative amounts and localization of E-cadherin, Snail1, and Vimentin under normal and hypoxic conditions in Huh-7 cells treated with control or supervillin-specific siRNA. Scale bar = 50 μm
Figure Legend Snippet: Supervillin regulates hypoxia-induced epithelial-mesenchymal transition (EMT) of HCC. a. Bright-field microscopy showing the morphological changes that occur when HepG2 and Huh-7 cells are cultured for 16 h in hypoxia. b. Immunoblots showing the changes in E-cadherin, Vimentin, Snail1, and supervillin isoforms during hypoxia after treatment with supervillin-specific siRNA. HepG2 and Huh-7 cells were transfected with a control or supervillin-specific siRNA and incubated under normoxia for 48 h followed by being scratched and exposed to normoxia or hypoxia for 16 h; β-tubulin was used as the loading control. c. Immunofluorescence staining showing that the relative amounts and localization of E-cadherin, Snail1, and Vimentin under normal and hypoxic conditions in Huh-7 cells treated with control or supervillin-specific siRNA. Scale bar = 50 μm

Techniques Used: Microscopy, Cell Culture, Western Blot, Transfection, Incubation, Immunofluorescence, Staining

7) Product Images from "An allelic variant in the intergenic region between ERAP1 and ERAP2 correlates with an inverse expression of the two genes"

Article Title: An allelic variant in the intergenic region between ERAP1 and ERAP2 correlates with an inverse expression of the two genes

Journal: Scientific Reports

doi: 10.1038/s41598-018-28799-8

ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 β−Actin copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value
Figure Legend Snippet: ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 β−Actin copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value

Techniques Used: Inhibition

8) Product Images from "p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity"

Article Title: p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity

Journal: Scientific Reports

doi: 10.1038/s41598-019-40238-w

PCAF-mediated δ-catenin acetylation promotes autophagic degradation of δ-catenin. ( A , B ) The acetyltransferase activity of PCAF is required for the downregulation of δ-catenin. However, proteasome inhibition does not suppress the effect of PCAF on downregulating δ -catenin. HEK293Tcells transfected with GFP-δ-catenin and Flag-PCAF ( A ) and Rv/δ cells transfected with Flag-PCAF ( B ) were treated with the proteasome inhibitor MG132 (10 μM) or the histone acetyltransferase inhibitor Garcinol (5 µM) or Baf A1 (100 nM) or transfected with shPCAF and incubated for 12 h, and then cell lysates were subjected to immunoblotting. ( C ) Autophagy inhibitors attenuate PCAF-mediated δ-catenin degradation. HEK293T cells were transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with the autophagy inhibitors chloroquine (CQ, 100 μM), bafilomycin A1 (BafA1, 100 nM), or 3-methyladenine (3-MA, 5 mM), and cell lysates were subjected to immunoblotting. ( D ) Autophagy inhibitors increase the stability of δ-catenin. HEK293T cells transfected with δ-catenin and treated with 0.2 µM TSA were treated with MG132 (10 μM) or Baf A1 (100 nM) or 3-MA (5 mM) and incubated for 12 h, and then treated with cycloheximide (CHX, 20 ng/ml) for indicated time (h), and then cell lysates were subjected to immunoblotting. α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. **p
Figure Legend Snippet: PCAF-mediated δ-catenin acetylation promotes autophagic degradation of δ-catenin. ( A , B ) The acetyltransferase activity of PCAF is required for the downregulation of δ-catenin. However, proteasome inhibition does not suppress the effect of PCAF on downregulating δ -catenin. HEK293Tcells transfected with GFP-δ-catenin and Flag-PCAF ( A ) and Rv/δ cells transfected with Flag-PCAF ( B ) were treated with the proteasome inhibitor MG132 (10 μM) or the histone acetyltransferase inhibitor Garcinol (5 µM) or Baf A1 (100 nM) or transfected with shPCAF and incubated for 12 h, and then cell lysates were subjected to immunoblotting. ( C ) Autophagy inhibitors attenuate PCAF-mediated δ-catenin degradation. HEK293T cells were transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with the autophagy inhibitors chloroquine (CQ, 100 μM), bafilomycin A1 (BafA1, 100 nM), or 3-methyladenine (3-MA, 5 mM), and cell lysates were subjected to immunoblotting. ( D ) Autophagy inhibitors increase the stability of δ-catenin. HEK293T cells transfected with δ-catenin and treated with 0.2 µM TSA were treated with MG132 (10 μM) or Baf A1 (100 nM) or 3-MA (5 mM) and incubated for 12 h, and then treated with cycloheximide (CHX, 20 ng/ml) for indicated time (h), and then cell lysates were subjected to immunoblotting. α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. **p

Techniques Used: Activity Assay, Inhibition, Transfection, Incubation

Multiple lysine residues in the N-terminus are responsible for PCAF-mediated δ-catenin downregulation. ( A ) Schematic representation of the triple arginine mutation at Lys360, Lys371, and Lys428 (FL KR), and the deletion/arginine mutation constructs of δ-catenin 1–499, 1–499 KR, 85–499, 85–499 KR, 1–499∆N KR, and 325–499 KR. ( B ) Deletion mutants 85–499 KR and 325–499 KR of δ-catenin were not affected by PCAF. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibody. ( C ) 3-MA restored the downregulated FL KR, 85–499, and 1–499∆N KR mutants of δ-catenin except the 85–499 KR mutation. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs and incubated with 3-MA (1 mM) for 24 h, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibodies. ( D ) PCAF did not acetylate δ-catenin 85–499 KR mutation. HEK293T cells were transfected with full length GFP-δ-catenin or 85–499 KR mutant together with or without Flag-PCAF, and each cell lysates were subjected to immunoprecipitation with anti-acetylated-lysine, followed by immunoblotting of precipitated proteins. α-Tubulin or ß-actin was used as a loading control. Relative values of δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. *p
Figure Legend Snippet: Multiple lysine residues in the N-terminus are responsible for PCAF-mediated δ-catenin downregulation. ( A ) Schematic representation of the triple arginine mutation at Lys360, Lys371, and Lys428 (FL KR), and the deletion/arginine mutation constructs of δ-catenin 1–499, 1–499 KR, 85–499, 85–499 KR, 1–499∆N KR, and 325–499 KR. ( B ) Deletion mutants 85–499 KR and 325–499 KR of δ-catenin were not affected by PCAF. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibody. ( C ) 3-MA restored the downregulated FL KR, 85–499, and 1–499∆N KR mutants of δ-catenin except the 85–499 KR mutation. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs and incubated with 3-MA (1 mM) for 24 h, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibodies. ( D ) PCAF did not acetylate δ-catenin 85–499 KR mutation. HEK293T cells were transfected with full length GFP-δ-catenin or 85–499 KR mutant together with or without Flag-PCAF, and each cell lysates were subjected to immunoprecipitation with anti-acetylated-lysine, followed by immunoblotting of precipitated proteins. α-Tubulin or ß-actin was used as a loading control. Relative values of δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. *p

Techniques Used: Mutagenesis, Construct, Transfection, Expressing, Incubation, Immunoprecipitation

PCAF and HDACs regulate the acetylation status and levels of δ-catenin. ( A , B ) PCAF decreases δ-catenin levels. HEK293T ( A ) and δ-catenin-overexpressing CWR22Rv-1 (Rv/δ) ( B ) cells were transfected as indicated, and cell lysates were subjected to immunoblotting. ( C – F ) PCAF interacts with and acetylates δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoprecipitation with anti-δ-catenin ( C ), anti-Flag ( D ), or anti-acetylated-lysine ( E , F ), followed by immunoblotting of precipitated proteins. ( G – I ) HDACs deacetylate and upregulate δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoblotting ( G ) or immunoprecipitation with anti-acetylated-lysine ( H ). At 12 h post-transfection of HEK293T cells with GFP-δ-catenin and HDAC1, cells were treated with 0.2 µM trichostatin A (TSA) or transfected with Flag-PCAF, and incubated for 24 h, followed by immunoblotting with anti-δ-catenin and anti-Flag antibodies ( I ). α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin/HDACs ratios from three different experimental results are shown as a bar graph ( G ). Values are presented as the mean ± SEM. “−”, Mock transfection or vehicle treatment.
Figure Legend Snippet: PCAF and HDACs regulate the acetylation status and levels of δ-catenin. ( A , B ) PCAF decreases δ-catenin levels. HEK293T ( A ) and δ-catenin-overexpressing CWR22Rv-1 (Rv/δ) ( B ) cells were transfected as indicated, and cell lysates were subjected to immunoblotting. ( C – F ) PCAF interacts with and acetylates δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoprecipitation with anti-δ-catenin ( C ), anti-Flag ( D ), or anti-acetylated-lysine ( E , F ), followed by immunoblotting of precipitated proteins. ( G – I ) HDACs deacetylate and upregulate δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoblotting ( G ) or immunoprecipitation with anti-acetylated-lysine ( H ). At 12 h post-transfection of HEK293T cells with GFP-δ-catenin and HDAC1, cells were treated with 0.2 µM trichostatin A (TSA) or transfected with Flag-PCAF, and incubated for 24 h, followed by immunoblotting with anti-δ-catenin and anti-Flag antibodies ( I ). α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin/HDACs ratios from three different experimental results are shown as a bar graph ( G ). Values are presented as the mean ± SEM. “−”, Mock transfection or vehicle treatment.

Techniques Used: Transfection, Immunoprecipitation, Incubation

9) Product Images from "SUMOylation of G9a regulates its function as an activator of myoblast proliferation"

Article Title: SUMOylation of G9a regulates its function as an activator of myoblast proliferation

Journal: Cell Death & Disease

doi: 10.1038/s41419-019-1465-9

G9a SUMOylation regulates E2F1/PCAF (P300/CBP-associated factor) association. a HEK293T cells were co-transfected with E2F1 and Flag-G9a or Flag-G9a4KR. Lysates were immunoprecipitated with Flag-agarose beads and immunoblotted with anti-E2F1, anti-G9a, and anti-β-actin antibodies. Numbers at the bottom of the panel indicate the relative interaction of E2F1 with G9a and G9a4KR, which was quantified by calculating the ratio of E2F1 to Flag band intensities in the immunoprecipitated material. The interaction with G9a was given a value of 1. b HEK293T cells were co-transfected with PCAF and Flag-G9a or Flag-G9a4KR. Lysates were immunoprecipitated with anti-PCAF antibody and immunoblotted with anti-PCAF, anti-G9a, and anti-β-actin antibodies. The relative interaction of PCAF with G9a and G9a4KR was quantified by calculating the ratio of G9a to PCAF band intensities in the immunoprecipitated material. The interaction with G9a was given a value of 1. c C2C12 cells were transfected with Flag-G9a or Flag-G9a4KR. Lysates were immunoprecipitated with anti-E2F1 antibody and immunoblotted with anti-PCAF and anti-FLAG antibodies. The ratio of PCAF to E2F1 band intensities was quantified in the immunoprecipitated material. Numbers at the bottom of the panel show interaction relative to control cells which was given a value of 1. d Flag-G9a was transfected with or without SENP1 as indicated. Lysates were immunoprecipitated with anti-E2F1 antibody and immunoblotted with anti-PCAF, anti-E2F1, and anti-β-actin antibodies. The band intensities of PCAF with E2F1 were quantified in the immunoprecipitated material. The interaction in the presence of G9a was given a value of 1. e Cells stably expressing pBABE-G9a or pBABE-G9a4KR were transfected with 200 ng of Cyclin D1 promoter (pD1luc) reporter. After 48 h, the luciferase activity was measured using dual luciferase reporter assays. f pBABE and pBABE-G9a cells were transfected with 200 ng of Cyclin D1 promoter (pD1luc) with or without SENP1. Luciferase activity was measured using dual luciferase reporter assays. g HEK293T cells were co-transfected with Flag-G9a and PCAF or PCAF-SUMO interaction motif (SIM) mutant (PCAF*). Lysates were immunoprecipitated with anti-PCAF antibody, followed by immunoblotting with anti-PCAF, anti-G9a, and anti- β-actin antibodies. The interaction of PCAF and PCAF* with G9a was quantified by calculating the ratio of G9a to PCAF band intensities in the immunoprecipitated material. Numbers at the bottom of the panel indicate interaction relative to PCAF that was given a value of 1. h pBABE and pBABE-G9a cells were co-transfected with 200 ng of Cyclin D1 reporter along with PCAF or PCAF SIM mutant (PCAF*). Luciferase activity was measured using dual luciferase reporter assays. i Chromatin immunoprecipitation (ChIP) assays were performed with anti-E2F1, anti-PCAF, and anti-H3K9ac antibodies at Cyclin D1 and dihydrofolate reductase (DHFR) promoters in proliferating pBABE, pBABE-G9a, or pBABE-G9a4KR cells. As a negative control, G9a ChIP was performed at the β-actin promoter in pBABE, pBABE-G9a, and pBABE-G9a4KR cells. j No change in G9a occupancy was seen in Flag-G9a and Flag-G9a4KR cells using anti-Flag antibody. .Significance was determined using Student’s t-test (*p
Figure Legend Snippet: G9a SUMOylation regulates E2F1/PCAF (P300/CBP-associated factor) association. a HEK293T cells were co-transfected with E2F1 and Flag-G9a or Flag-G9a4KR. Lysates were immunoprecipitated with Flag-agarose beads and immunoblotted with anti-E2F1, anti-G9a, and anti-β-actin antibodies. Numbers at the bottom of the panel indicate the relative interaction of E2F1 with G9a and G9a4KR, which was quantified by calculating the ratio of E2F1 to Flag band intensities in the immunoprecipitated material. The interaction with G9a was given a value of 1. b HEK293T cells were co-transfected with PCAF and Flag-G9a or Flag-G9a4KR. Lysates were immunoprecipitated with anti-PCAF antibody and immunoblotted with anti-PCAF, anti-G9a, and anti-β-actin antibodies. The relative interaction of PCAF with G9a and G9a4KR was quantified by calculating the ratio of G9a to PCAF band intensities in the immunoprecipitated material. The interaction with G9a was given a value of 1. c C2C12 cells were transfected with Flag-G9a or Flag-G9a4KR. Lysates were immunoprecipitated with anti-E2F1 antibody and immunoblotted with anti-PCAF and anti-FLAG antibodies. The ratio of PCAF to E2F1 band intensities was quantified in the immunoprecipitated material. Numbers at the bottom of the panel show interaction relative to control cells which was given a value of 1. d Flag-G9a was transfected with or without SENP1 as indicated. Lysates were immunoprecipitated with anti-E2F1 antibody and immunoblotted with anti-PCAF, anti-E2F1, and anti-β-actin antibodies. The band intensities of PCAF with E2F1 were quantified in the immunoprecipitated material. The interaction in the presence of G9a was given a value of 1. e Cells stably expressing pBABE-G9a or pBABE-G9a4KR were transfected with 200 ng of Cyclin D1 promoter (pD1luc) reporter. After 48 h, the luciferase activity was measured using dual luciferase reporter assays. f pBABE and pBABE-G9a cells were transfected with 200 ng of Cyclin D1 promoter (pD1luc) with or without SENP1. Luciferase activity was measured using dual luciferase reporter assays. g HEK293T cells were co-transfected with Flag-G9a and PCAF or PCAF-SUMO interaction motif (SIM) mutant (PCAF*). Lysates were immunoprecipitated with anti-PCAF antibody, followed by immunoblotting with anti-PCAF, anti-G9a, and anti- β-actin antibodies. The interaction of PCAF and PCAF* with G9a was quantified by calculating the ratio of G9a to PCAF band intensities in the immunoprecipitated material. Numbers at the bottom of the panel indicate interaction relative to PCAF that was given a value of 1. h pBABE and pBABE-G9a cells were co-transfected with 200 ng of Cyclin D1 reporter along with PCAF or PCAF SIM mutant (PCAF*). Luciferase activity was measured using dual luciferase reporter assays. i Chromatin immunoprecipitation (ChIP) assays were performed with anti-E2F1, anti-PCAF, and anti-H3K9ac antibodies at Cyclin D1 and dihydrofolate reductase (DHFR) promoters in proliferating pBABE, pBABE-G9a, or pBABE-G9a4KR cells. As a negative control, G9a ChIP was performed at the β-actin promoter in pBABE, pBABE-G9a, and pBABE-G9a4KR cells. j No change in G9a occupancy was seen in Flag-G9a and Flag-G9a4KR cells using anti-Flag antibody. .Significance was determined using Student’s t-test (*p

Techniques Used: Transfection, Immunoprecipitation, Stable Transfection, Expressing, Luciferase, Activity Assay, Mutagenesis, Chromatin Immunoprecipitation, Negative Control

10) Product Images from "Elk1 affects katanin and spastin proteins via differential transcriptional and post-transcriptional regulations"

Article Title: Elk1 affects katanin and spastin proteins via differential transcriptional and post-transcriptional regulations

Journal: PLoS ONE

doi: 10.1371/journal.pone.0212518

Western blotting results of Elk1 and Elk1-3R effects on both endogenous katanin-p60 and spastin proteins. (A) Analysis of SH-SY5Y transfection efficacy with His-tag including pCMV6_Elk1 and pCMV6_Elk1-3R vectors. (B) Western blotting results showing katanin-p60 level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (C) Quantification of katanin-p60 level was performed by normalizing band intensities of katanin-p60 to band intensities of ß-actin. Results indicated that both Elk1 and Elk1-3R decreased the katanin-p60 level significantly and the difference between them was not significant. SEM values are 0, 0.01, and 0.02, respectively (n = 4). (D) Western blotting results showing spastin level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (E) Quantification of spastin level was done as previously described. Results indicated that there was no significant spastin level difference between Elk1 and Elk1-3R transfected cells. However, they were significant compared to untransfected cells. SEM values are 0, 0.03, and 0.03, respectively (n = 4).
Figure Legend Snippet: Western blotting results of Elk1 and Elk1-3R effects on both endogenous katanin-p60 and spastin proteins. (A) Analysis of SH-SY5Y transfection efficacy with His-tag including pCMV6_Elk1 and pCMV6_Elk1-3R vectors. (B) Western blotting results showing katanin-p60 level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (C) Quantification of katanin-p60 level was performed by normalizing band intensities of katanin-p60 to band intensities of ß-actin. Results indicated that both Elk1 and Elk1-3R decreased the katanin-p60 level significantly and the difference between them was not significant. SEM values are 0, 0.01, and 0.02, respectively (n = 4). (D) Western blotting results showing spastin level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (E) Quantification of spastin level was done as previously described. Results indicated that there was no significant spastin level difference between Elk1 and Elk1-3R transfected cells. However, they were significant compared to untransfected cells. SEM values are 0, 0.03, and 0.03, respectively (n = 4).

Techniques Used: Western Blot, Transfection

ICC results of Elk1 and Elk1-3R effects on endogenous katanin-p60 level. Asterisk symbol represents untransfected cells, whereas the cells indicated with arrows are either Elk1 or Elk1-3R overexpressing cells. The images were taken using 63X objective at zoom 2.6. (A) The level of endogenous katanin-p60 protein is reduced in Elk1 overexpressed SH-SY5Y cells compared to untransfected cells. (B) Relative fluorescence density graph shows that the expression of katanin-p60 was 5.5 fold decreased in Elk1 overexpressed cells compared to control cells. SEM values are 0 and 0.02, respectively. (C) The decrease in endogenous katanin-p60 level is observed in Elk1-3R overexpressed SH-SY5Y cells compared to untransfected cells. (D) Relative fluorescence density graph shows that the expression of katanin-p60 was 4.76 fold decreased in Elk1-3R overexpressed cells compared to control cells. SEM values are 0 and 0.03, respectively.
Figure Legend Snippet: ICC results of Elk1 and Elk1-3R effects on endogenous katanin-p60 level. Asterisk symbol represents untransfected cells, whereas the cells indicated with arrows are either Elk1 or Elk1-3R overexpressing cells. The images were taken using 63X objective at zoom 2.6. (A) The level of endogenous katanin-p60 protein is reduced in Elk1 overexpressed SH-SY5Y cells compared to untransfected cells. (B) Relative fluorescence density graph shows that the expression of katanin-p60 was 5.5 fold decreased in Elk1 overexpressed cells compared to control cells. SEM values are 0 and 0.02, respectively. (C) The decrease in endogenous katanin-p60 level is observed in Elk1-3R overexpressed SH-SY5Y cells compared to untransfected cells. (D) Relative fluorescence density graph shows that the expression of katanin-p60 was 4.76 fold decreased in Elk1-3R overexpressed cells compared to control cells. SEM values are 0 and 0.03, respectively.

Techniques Used: Immunocytochemistry, Fluorescence, Expressing

11) Product Images from "Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles"

Article Title: Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles

Journal: Scientific Reports

doi: 10.1038/s41598-019-41029-z

Several autophagic proteins are beneficial for HCMV production. Stable HFF cell lines either expressing a dominant-negative mutant of ATG4B or knockdown for the indicated autophagic proteins (LC3B, ATG5, BECN1 or ULK1) and their corresponding control cell lines (WT: wild type HFF, shSCR: scrambled shRNA, shNT: non-target shRNA) have been used. ( A ) Immunoblot analysis of p62, LC3B, ATG5, BECN1 and ULK1 expression confirms the inhibition of autophagy in the indicated cell lines. Actin was used as a loading control. Full-length blots are presented in Fig. S7 ( B ) Viral titers were determined 10 days post-infection with HCMV AD169 strain at MOI 0.01 in the different cell lines. Error bars indicate SEM from at least three independent experiments. *p
Figure Legend Snippet: Several autophagic proteins are beneficial for HCMV production. Stable HFF cell lines either expressing a dominant-negative mutant of ATG4B or knockdown for the indicated autophagic proteins (LC3B, ATG5, BECN1 or ULK1) and their corresponding control cell lines (WT: wild type HFF, shSCR: scrambled shRNA, shNT: non-target shRNA) have been used. ( A ) Immunoblot analysis of p62, LC3B, ATG5, BECN1 and ULK1 expression confirms the inhibition of autophagy in the indicated cell lines. Actin was used as a loading control. Full-length blots are presented in Fig. S7 ( B ) Viral titers were determined 10 days post-infection with HCMV AD169 strain at MOI 0.01 in the different cell lines. Error bars indicate SEM from at least three independent experiments. *p

Techniques Used: Expressing, Dominant Negative Mutation, shRNA, Inhibition, Infection

12) Product Images from "Protective Effect of Hesperidin Against Sepsis-Induced Lung Injury by Inducing the Heat-Stable Protein 70 (Hsp70)/Toll-Like Receptor 4 (TLR4)/ Myeloid Differentiation Primary Response 88 (MyD88) Pathway"

Article Title: Protective Effect of Hesperidin Against Sepsis-Induced Lung Injury by Inducing the Heat-Stable Protein 70 (Hsp70)/Toll-Like Receptor 4 (TLR4)/ Myeloid Differentiation Primary Response 88 (MyD88) Pathway

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

doi: 10.12659/MSM.912490

Effect of hesperidin on the expression of Bcl-2, caspase-3, TLR-4, and HSP70 protein in the lung tissue of CLP-induced lung injury mice. Mean ±SEM (n=6). ## p
Figure Legend Snippet: Effect of hesperidin on the expression of Bcl-2, caspase-3, TLR-4, and HSP70 protein in the lung tissue of CLP-induced lung injury mice. Mean ±SEM (n=6). ## p

Techniques Used: Expressing, Mouse Assay

13) Product Images from "Usp7 regulates Hippo pathway through deubiquitinating the transcriptional coactivator Yorkie"

Article Title: Usp7 regulates Hippo pathway through deubiquitinating the transcriptional coactivator Yorkie

Journal: Nature Communications

doi: 10.1038/s41467-019-08334-7

Usp7 deubiquitinates and stabilizes Yorkie (Yki). a Immunoblots of lysates from S2 cells transfected with Myc-Yki and treated by CHX for indicated intervals. To prevent the function of lysosome or proteasome, the cells were treated with corresponding inhibitors. Of note, Myc-Yki was unstable, whereas lysosome inhibitor could hamper its degradation. Actin acts as a loading control. Quantification analyses were shown on right. b Endogenous Yki protein was unstable and underwent lysosome-mediated degradation. Actin acts as a loading control. Quantification analyses were shown on right. c S2 cells treated by CHX for indicated intervals were analyzed by subcellular fractionation. Of note, the cytoplasmic Yki was degraded by lysosome, whereas the nuclear Yki was degraded by proteasome. Lamin C and Tubulin are used as loading controls. d Myc-Yki-NLS protein was degraded by proteasome in S2 cells. Actin acts as a loading control. Quantification analyses were shown on right. e Myc-Myr-Yki protein was degraded by lysosome in S2 cells. Actin acts as a loading control. Quantification analyses were shown on right. f Immunoblots of lysates from S2 cells expressing indicated constructs and treated with CHX for indicated intervals. Quantification analyses were shown on right. Notably, Usp7 effectively blocked Yki degradation. g Knockdown of usp7 promoted Yki degradation. h S2 cells transfected with indicated constructs were analyzed by subcellular fractionation. Of note, Usp7 increased the nuclear Yki protein levels. Quantification analyses were shown on right. Lamin C and Tubulin are used as loading controls. i Usp7 increased Myc-Yki-NLS protein level. j Knockdown of usp7 decreased nuclear Yki protein. k Immunoblots of immunoprecipitates (top) or lysates (bottom three panels) from S2 cells expressing indicated proteins and treated with MG132 plus NH 4 Cl for 4 h. Knockdown of usp7 promoted Yki ubiquitination. l Usp7 and Usp7-ΔMATH attenuated, but Usp7-CA promoted endogenous Yki ubiquitination. m Usp7 attenuated, whereas Usp7-CA promoted Yki-NLS ubiquitination. For statistical results, data are means ± SEM. n = 3 biological-independent experiments. Above all, the arrowhead indicates heavy IgG
Figure Legend Snippet: Usp7 deubiquitinates and stabilizes Yorkie (Yki). a Immunoblots of lysates from S2 cells transfected with Myc-Yki and treated by CHX for indicated intervals. To prevent the function of lysosome or proteasome, the cells were treated with corresponding inhibitors. Of note, Myc-Yki was unstable, whereas lysosome inhibitor could hamper its degradation. Actin acts as a loading control. Quantification analyses were shown on right. b Endogenous Yki protein was unstable and underwent lysosome-mediated degradation. Actin acts as a loading control. Quantification analyses were shown on right. c S2 cells treated by CHX for indicated intervals were analyzed by subcellular fractionation. Of note, the cytoplasmic Yki was degraded by lysosome, whereas the nuclear Yki was degraded by proteasome. Lamin C and Tubulin are used as loading controls. d Myc-Yki-NLS protein was degraded by proteasome in S2 cells. Actin acts as a loading control. Quantification analyses were shown on right. e Myc-Myr-Yki protein was degraded by lysosome in S2 cells. Actin acts as a loading control. Quantification analyses were shown on right. f Immunoblots of lysates from S2 cells expressing indicated constructs and treated with CHX for indicated intervals. Quantification analyses were shown on right. Notably, Usp7 effectively blocked Yki degradation. g Knockdown of usp7 promoted Yki degradation. h S2 cells transfected with indicated constructs were analyzed by subcellular fractionation. Of note, Usp7 increased the nuclear Yki protein levels. Quantification analyses were shown on right. Lamin C and Tubulin are used as loading controls. i Usp7 increased Myc-Yki-NLS protein level. j Knockdown of usp7 decreased nuclear Yki protein. k Immunoblots of immunoprecipitates (top) or lysates (bottom three panels) from S2 cells expressing indicated proteins and treated with MG132 plus NH 4 Cl for 4 h. Knockdown of usp7 promoted Yki ubiquitination. l Usp7 and Usp7-ΔMATH attenuated, but Usp7-CA promoted endogenous Yki ubiquitination. m Usp7 attenuated, whereas Usp7-CA promoted Yki-NLS ubiquitination. For statistical results, data are means ± SEM. n = 3 biological-independent experiments. Above all, the arrowhead indicates heavy IgG

Techniques Used: Western Blot, Transfection, Fractionation, Expressing, Construct

A proposed model of Usp7 deubiquitinating Yorkie (Yki). When Hippo (Hpo) pathway turns on, Hpo phosphorylates and activates Wts. In turn, Wts phosphorylates the transcriptional coactivator Yki, culminating Yki retention in the cytoplasm via binding with 14-3-3. When Hpo pathway is closed, Yki translocates into the nucleus to activate the target gene expression with the assistance of Sd. The nuclear Yki protein is destabilized by proteasome, which is attenuated by Usp7
Figure Legend Snippet: A proposed model of Usp7 deubiquitinating Yorkie (Yki). When Hippo (Hpo) pathway turns on, Hpo phosphorylates and activates Wts. In turn, Wts phosphorylates the transcriptional coactivator Yki, culminating Yki retention in the cytoplasm via binding with 14-3-3. When Hpo pathway is closed, Yki translocates into the nucleus to activate the target gene expression with the assistance of Sd. The nuclear Yki protein is destabilized by proteasome, which is attenuated by Usp7

Techniques Used: Binding Assay, Expressing

Usp7 regulates Hippo (Hpo) pathway through Yorkie (Yki). a A wing disc with hpo knockdown was stained to show GFP (green) and CycE (white). b A wing disc simultaneous expressing hpo RNA interference (RNAi) and usp7 RNAi was stained to show GFP (green) and CycE (white). Of note, knockdown of usp7 decreased CycE level in hpo RNAi background. c A wing disc with hpo knockdown was stained to show Ci (red) and diap1 -lacZ (white). d Knockdown of usp7 attenuated diap1 -lacZ expression in hpo RNAi background. e A wing disc expressing wts RNAi was stained to show GFP (green) and CycE (white). f Knockdown of usp7 decreased CycE protein level under wts RNAi background. g A wing disc expressing wts RNAi was stained to show Ci (red) and diap1 -lacZ (white). h Knockdown of usp7 decreased diap1 -lacZ expression under wts RNAi background. i Knockdown of yki decreased diap1 -lacZ expression. j A wing disc simultaneous expressing yki RNAi and UAS- usp7 was stained to show Ci (red) and diap1 -lacZ (white). Of note, overexpression of usp7 could not restore the decreased diap1 -lacZ caused by yki knockdown. k A wing disc with yki ectopic expression by En-gal4 was stained to show GFP (green) and CycE (white). Overexpression of yki resulted in CycE upregulation and overgrowth. l Knockdown of usp7 did not rescue the phenotypes induced by yki overexpression. m Knockdown of usp7 decreased Yki protein level. n A wing disc carrying usp7 KG06814 clones were stained to show the expression of GFP (green) and Yki (white). Of note, usp7 mutation resulted in decreased Yki expression (marked by arrows). o A wing disc expressing yki-1w was stained to show Ci (red) and ban -lacZ (white). p A wing disc simultaneous expressing yki-1w and Fg- usp7 was stained to show Fg (green) and ban -lacZ (white). Notably, overexpression of usp7 further elevated ban -lacZ level and resulted in serious overgrowth. Scale bars: 50 μm for all images
Figure Legend Snippet: Usp7 regulates Hippo (Hpo) pathway through Yorkie (Yki). a A wing disc with hpo knockdown was stained to show GFP (green) and CycE (white). b A wing disc simultaneous expressing hpo RNA interference (RNAi) and usp7 RNAi was stained to show GFP (green) and CycE (white). Of note, knockdown of usp7 decreased CycE level in hpo RNAi background. c A wing disc with hpo knockdown was stained to show Ci (red) and diap1 -lacZ (white). d Knockdown of usp7 attenuated diap1 -lacZ expression in hpo RNAi background. e A wing disc expressing wts RNAi was stained to show GFP (green) and CycE (white). f Knockdown of usp7 decreased CycE protein level under wts RNAi background. g A wing disc expressing wts RNAi was stained to show Ci (red) and diap1 -lacZ (white). h Knockdown of usp7 decreased diap1 -lacZ expression under wts RNAi background. i Knockdown of yki decreased diap1 -lacZ expression. j A wing disc simultaneous expressing yki RNAi and UAS- usp7 was stained to show Ci (red) and diap1 -lacZ (white). Of note, overexpression of usp7 could not restore the decreased diap1 -lacZ caused by yki knockdown. k A wing disc with yki ectopic expression by En-gal4 was stained to show GFP (green) and CycE (white). Overexpression of yki resulted in CycE upregulation and overgrowth. l Knockdown of usp7 did not rescue the phenotypes induced by yki overexpression. m Knockdown of usp7 decreased Yki protein level. n A wing disc carrying usp7 KG06814 clones were stained to show the expression of GFP (green) and Yki (white). Of note, usp7 mutation resulted in decreased Yki expression (marked by arrows). o A wing disc expressing yki-1w was stained to show Ci (red) and ban -lacZ (white). p A wing disc simultaneous expressing yki-1w and Fg- usp7 was stained to show Fg (green) and ban -lacZ (white). Notably, overexpression of usp7 further elevated ban -lacZ level and resulted in serious overgrowth. Scale bars: 50 μm for all images

Techniques Used: Staining, Expressing, Over Expression, Clone Assay, Mutagenesis

Usp7 binds Yorkie (Yki). a, b Schematic drawings show the domains or motifs in Usp7 ( a ) and Yki ( b ) and their truncated fragments used in subsequent co-IP and pull-down assays. Black and blue bars denote MATH domain and core catalytic domain of Usp7 ( a ). Black bars represent WW domains of Yki ( b ). c Immunoblots of immunoprecipitates (top two panels) or lysates (bottom two panels) from S2 cells expressing indicated constructs. Of note, Myc-Yki could pull-down Fg-Usp7 in S2 cells. d Fg-Usp7 could pull-down Myc-Yki in S2 cells. e Endogenous Yki protein pulled down endogenous Usp7 in S2 cells. f Extracts from S2 cells expressing Myc-Yki were incubated with GST or GST-Usp7. The bound proteins were analyzed by IB. Asterisks mark GST fusion proteins. g Usp7-ΔMATH could bind Yki. h Yki did not compete with Ci to bind Usp7. i - j Usp7 exclusively interacted with N-terminal region of Yki in S2 cells. Asterisks mark expressed Yki truncated fragments. k Pull-down between Fg-Usp7 and GST or GST-tagged Yki fragments. Asterisks indicate expressed GST fusion proteins. l Hippo (Hpo) decreased, whereas the kinase dead form Hpo-KD increased Usp7-Yki interaction. m Wts attenuated, whereas the kinase dead form Wts-KD promoted Usp7-Yki association. Above all, the solid arrowhead indicates heavy IgG ( c , d , g , i , j ), whereas the hollow arrowhead light IgG ( i )
Figure Legend Snippet: Usp7 binds Yorkie (Yki). a, b Schematic drawings show the domains or motifs in Usp7 ( a ) and Yki ( b ) and their truncated fragments used in subsequent co-IP and pull-down assays. Black and blue bars denote MATH domain and core catalytic domain of Usp7 ( a ). Black bars represent WW domains of Yki ( b ). c Immunoblots of immunoprecipitates (top two panels) or lysates (bottom two panels) from S2 cells expressing indicated constructs. Of note, Myc-Yki could pull-down Fg-Usp7 in S2 cells. d Fg-Usp7 could pull-down Myc-Yki in S2 cells. e Endogenous Yki protein pulled down endogenous Usp7 in S2 cells. f Extracts from S2 cells expressing Myc-Yki were incubated with GST or GST-Usp7. The bound proteins were analyzed by IB. Asterisks mark GST fusion proteins. g Usp7-ΔMATH could bind Yki. h Yki did not compete with Ci to bind Usp7. i - j Usp7 exclusively interacted with N-terminal region of Yki in S2 cells. Asterisks mark expressed Yki truncated fragments. k Pull-down between Fg-Usp7 and GST or GST-tagged Yki fragments. Asterisks indicate expressed GST fusion proteins. l Hippo (Hpo) decreased, whereas the kinase dead form Hpo-KD increased Usp7-Yki interaction. m Wts attenuated, whereas the kinase dead form Wts-KD promoted Usp7-Yki association. Above all, the solid arrowhead indicates heavy IgG ( c , d , g , i , j ), whereas the hollow arrowhead light IgG ( i )

Techniques Used: Co-Immunoprecipitation Assay, Western Blot, Expressing, Construct, Incubation

14) Product Images from "Combination of Berberine with Resveratrol Improves the Lipid-Lowering Efficacy"

Article Title: Combination of Berberine with Resveratrol Improves the Lipid-Lowering Efficacy

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123903

The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p
Figure Legend Snippet: The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

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

15) Product Images from "A Prenylated Xanthone, Cudratricusxanthone A, Isolated from Cudrania tricuspidata Inhibits Lipopolysaccharide-Induced Neuroinflammation through Inhibition of NF-κB and p38 MAPK Pathways in BV2 Microglia"

Article Title: A Prenylated Xanthone, Cudratricusxanthone A, Isolated from Cudrania tricuspidata Inhibits Lipopolysaccharide-Induced Neuroinflammation through Inhibition of NF-κB and p38 MAPK Pathways in BV2 Microglia

Journal: Molecules

doi: 10.3390/molecules21091240

The effects of cudratricusxanthone A ( 1 ) on IκB-α phosphorylation and degradation ( A ); NF-κB translocation ( B , C ); NF-κB localization as determined by immunofluorescence analysis ( D ); and NF-κB DNA binding activity ( E ) in BV2 microglia. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A ( 1 ), and stimulated for 1 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. Western blot analyses of IκB-α and p -IκB-α in the cytoplasm ( A ) and NF-κB in the cytoplasm ( B ) and nucleus ( C ) and immunofluorescence analyses ( E ) were carried out as described in the Experimental Section. The band intensity was quantified by densitometry and normalized to β-actin and PCNA, and the values are presented at the bottom of the each band. Relative data represent the means ± SDs of three experiments. * p
Figure Legend Snippet: The effects of cudratricusxanthone A ( 1 ) on IκB-α phosphorylation and degradation ( A ); NF-κB translocation ( B , C ); NF-κB localization as determined by immunofluorescence analysis ( D ); and NF-κB DNA binding activity ( E ) in BV2 microglia. Cells were pretreated for 3 h with the indicated concentrations of cudratricusxanthone A ( 1 ), and stimulated for 1 h with LPS (1 μg/mL). The LPS treatment was performed in the presence of compound. Western blot analyses of IκB-α and p -IκB-α in the cytoplasm ( A ) and NF-κB in the cytoplasm ( B ) and nucleus ( C ) and immunofluorescence analyses ( E ) were carried out as described in the Experimental Section. The band intensity was quantified by densitometry and normalized to β-actin and PCNA, and the values are presented at the bottom of the each band. Relative data represent the means ± SDs of three experiments. * p

Techniques Used: Translocation Assay, Immunofluorescence, Binding Assay, Activity Assay, Western Blot

16) Product Images from "MicroRNA 214 inhibits adipocyte enhancer-binding protein 1 activity and increases the sensitivity of chemotherapy in colorectal cancer"

Article Title: MicroRNA 214 inhibits adipocyte enhancer-binding protein 1 activity and increases the sensitivity of chemotherapy in colorectal cancer

Journal: Oncology Letters

doi: 10.3892/ol.2018.9623

Kaplan-Meier survival analysis of primary colorectal cancer patients (n=62) following surgical resection with high AEBP1 expression (n=39) and low AEBP1 expression (n=23). (A) OS following surgery and (B) DFS following surgery. AEBP1, adipocyte enhancer-binding protein 1; OS, overall survival; DFS, disease-free survival.
Figure Legend Snippet: Kaplan-Meier survival analysis of primary colorectal cancer patients (n=62) following surgical resection with high AEBP1 expression (n=39) and low AEBP1 expression (n=23). (A) OS following surgery and (B) DFS following surgery. AEBP1, adipocyte enhancer-binding protein 1; OS, overall survival; DFS, disease-free survival.

Techniques Used: Expressing, Binding Assay

17) Product Images from "Membrane metallo-endopeptidase (Neprilysin) regulates inflammatory response and insulin signaling in white preadipocytes"

Article Title: Membrane metallo-endopeptidase (Neprilysin) regulates inflammatory response and insulin signaling in white preadipocytes

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2019.01.006

Knockdown of MME perturbs insulin signaling and increases IRα but not IRβ . Immortalized human neck white subcutaneous preadipocytes were transiently transfected with siRNA targeting MME, non-targeting siRNA, or Omapatrilat for 48 h. Cells were starved for 24 h before a 20-minute insulin stimulation (100 nM) followed by protein isolation and western blot. (A) Western blot of knockdown of MME or treatment with Omapatrilat in immortalized subcutaneous preadipocytes. (B–K) Densitometric analysis of protein levels. The levels of unphosphorylated proteins were grouped by treatment (Control, MME KD, or Ompatrilat). The levels of phosphorylated proteins were grouped by treatment and insulin stimulation (black = no insulin, grey = 20 min after 100 nM insulin). Omapatrilat = dual ACE/MME pharmacological inhibitor. Asterisks indicate p ≤ 0.05 by Student's t -test. N = 3 or 6. Bars indicate mean ± s.e.m.
Figure Legend Snippet: Knockdown of MME perturbs insulin signaling and increases IRα but not IRβ . Immortalized human neck white subcutaneous preadipocytes were transiently transfected with siRNA targeting MME, non-targeting siRNA, or Omapatrilat for 48 h. Cells were starved for 24 h before a 20-minute insulin stimulation (100 nM) followed by protein isolation and western blot. (A) Western blot of knockdown of MME or treatment with Omapatrilat in immortalized subcutaneous preadipocytes. (B–K) Densitometric analysis of protein levels. The levels of unphosphorylated proteins were grouped by treatment (Control, MME KD, or Ompatrilat). The levels of phosphorylated proteins were grouped by treatment and insulin stimulation (black = no insulin, grey = 20 min after 100 nM insulin). Omapatrilat = dual ACE/MME pharmacological inhibitor. Asterisks indicate p ≤ 0.05 by Student's t -test. N = 3 or 6. Bars indicate mean ± s.e.m.

Techniques Used: Transfection, Isolation, Western Blot

18) Product Images from "IL-33 suppresses GSK-3β activation through an ST2-independent MyD88/TRAF6/RIP/PI3K/Akt pathway"

Article Title: IL-33 suppresses GSK-3β activation through an ST2-independent MyD88/TRAF6/RIP/PI3K/Akt pathway

Journal: Heliyon

doi: 10.1016/j.heliyon.2018.e00971

PDK1 is not implicated in IL-33-induced Akt activation and GSK-3β inactivation. PC-12 cells, transfected with the NC siRNA or the PDK1 siRNA, were treated with IL-33 (1 ng/mL) for 30 min, followed by Western blotting using antibodies against Akt, pT308-Akt, pS473-Akt, GSK-3β, pS9-GSK-3β, and pY216-GSK-3β. ( A ) ( D ) Western blot images. The arrow shown in ( D ) indicates the signal band at 46 kDa for pY216-GSK-3β. ( B ) ( C ) In the graphs, each column represents the mean (±SEM) signal intensity for pT308-Akt and pS473-Akt relative to the signal intensity for Akt (n = 8 Western blot results from independent experiments). ( E ) ( F ) In the graphs, each column represents the mean (±SEM) signal intensity for pS9-GSK-3β and pY216-GSK-3β relative to the signal intensity for GSK-3β (n = 8 Western blot results from independent experiments). P values shown in the graphs, ANOVA followed by a Bonferroni correction.
Figure Legend Snippet: PDK1 is not implicated in IL-33-induced Akt activation and GSK-3β inactivation. PC-12 cells, transfected with the NC siRNA or the PDK1 siRNA, were treated with IL-33 (1 ng/mL) for 30 min, followed by Western blotting using antibodies against Akt, pT308-Akt, pS473-Akt, GSK-3β, pS9-GSK-3β, and pY216-GSK-3β. ( A ) ( D ) Western blot images. The arrow shown in ( D ) indicates the signal band at 46 kDa for pY216-GSK-3β. ( B ) ( C ) In the graphs, each column represents the mean (±SEM) signal intensity for pT308-Akt and pS473-Akt relative to the signal intensity for Akt (n = 8 Western blot results from independent experiments). ( E ) ( F ) In the graphs, each column represents the mean (±SEM) signal intensity for pS9-GSK-3β and pY216-GSK-3β relative to the signal intensity for GSK-3β (n = 8 Western blot results from independent experiments). P values shown in the graphs, ANOVA followed by a Bonferroni correction.

Techniques Used: Activation Assay, Transfection, Western Blot

PI3K is implicated in IL-33-induced Akt activation and GSK-3β inactivation. PC-12 cells, transfected with the NC siRNA or the PI3K siRNA, were treated with IL-33 (1 ng/mL) for 30 min, followed by Western blotting using antibodies against Akt, pT308-Akt, pS473-Akt, GSK-3β, pS9-GSK-3β, and pY216-GSK-3β. ( A ) ( D ) Western blot images. The arrow shown in ( D ) indicates the signal band at 46 kDa for pY216-GSK-3β. ( B ) ( C ) In the graphs, each column represents the mean (±SEM) signal intensity for pT308-Akt and pS473-Akt relative to the signal intensity for Akt (n = 8 Western blot results from independent experiments). ( E ) ( F ) In the graphs, each column represents the mean (±SEM) signal intensity for pS9-GSK-3β and pY216-GSK-3β relative to the signal intensity for GSK-3β (n = 8 Western blot results from independent experiments). P values shown in the graphs, ANOVA followed by a Bonferroni correction.
Figure Legend Snippet: PI3K is implicated in IL-33-induced Akt activation and GSK-3β inactivation. PC-12 cells, transfected with the NC siRNA or the PI3K siRNA, were treated with IL-33 (1 ng/mL) for 30 min, followed by Western blotting using antibodies against Akt, pT308-Akt, pS473-Akt, GSK-3β, pS9-GSK-3β, and pY216-GSK-3β. ( A ) ( D ) Western blot images. The arrow shown in ( D ) indicates the signal band at 46 kDa for pY216-GSK-3β. ( B ) ( C ) In the graphs, each column represents the mean (±SEM) signal intensity for pT308-Akt and pS473-Akt relative to the signal intensity for Akt (n = 8 Western blot results from independent experiments). ( E ) ( F ) In the graphs, each column represents the mean (±SEM) signal intensity for pS9-GSK-3β and pY216-GSK-3β relative to the signal intensity for GSK-3β (n = 8 Western blot results from independent experiments). P values shown in the graphs, ANOVA followed by a Bonferroni correction.

Techniques Used: Activation Assay, Transfection, Western Blot

RIP is implicated in IL-33-induced Akt activation and GSK-3β inactivation. PC-12 cells, transfected with the NC siRNA or the RIP siRNA, were treated with IL-33 (1 ng/mL) for 30 min, followed by Western blotting using antibodies against Akt, pT308-Akt, pS473-Akt, GSK-3β, pS9-GSK-3β, and pY216-GSK-3β. ( A ) ( D ) Western blot images. The arrow shown in ( D ) indicates the signal band at 46 kDa for pY216-GSK-3β. ( B ) ( C ) In the graphs, each column represents the mean (±SEM) signal intensity for pT308-Akt and pS473-Akt relative to the signal intensity for Akt (n = 8 Western blot results from independent experiments). ( E ) ( F ) In the graphs, each column represents the mean (±SEM) signal intensity for pS9-GSK-3β and pY216-GSK-3β relative to the signal intensity for GSK-3β (n = 8 Western blot results from independent experiments). P values shown in the graphs, ANOVA followed by a Bonferroni correction.
Figure Legend Snippet: RIP is implicated in IL-33-induced Akt activation and GSK-3β inactivation. PC-12 cells, transfected with the NC siRNA or the RIP siRNA, were treated with IL-33 (1 ng/mL) for 30 min, followed by Western blotting using antibodies against Akt, pT308-Akt, pS473-Akt, GSK-3β, pS9-GSK-3β, and pY216-GSK-3β. ( A ) ( D ) Western blot images. The arrow shown in ( D ) indicates the signal band at 46 kDa for pY216-GSK-3β. ( B ) ( C ) In the graphs, each column represents the mean (±SEM) signal intensity for pT308-Akt and pS473-Akt relative to the signal intensity for Akt (n = 8 Western blot results from independent experiments). ( E ) ( F ) In the graphs, each column represents the mean (±SEM) signal intensity for pS9-GSK-3β and pY216-GSK-3β relative to the signal intensity for GSK-3β (n = 8 Western blot results from independent experiments). P values shown in the graphs, ANOVA followed by a Bonferroni correction.

Techniques Used: Activation Assay, Transfection, Western Blot

TAK1 is not implicated in IL-33-induced Akt activation and GSK-3β inactivation. PC-12 cells, transfected with the NC siRNA or the TAK1 siRNA, were treated with IL-33 (1 ng/mL) for 30 min, followed by Western blotting using antibodies against Akt, pT308-Akt, pS473-Akt, GSK-3β, pS9-GSK-3β, and pY216-GSK-3β. ( A ) ( D ) Western blot images. The arrow shown in ( D ) indicates the signal band at 46 kDa for pY216-GSK-3β. ( B ) ( C ) In the graphs, each column represents the mean (±SEM) signal intensity for pT308-Akt and pS473-Akt relative to the signal intensity for Akt (n = 8 Western blot results from independent experiments). ( E ) ( F ) In the graphs, each column represents the mean (±SEM) signal intensity for pS9-GSK-3β and pY216-GSK-3β relative to the signal intensity for GSK-3β (n = 8 Western blot results from independent experiments. P values shown in the graphs, ANOVA followed by a Bonferroni correction.
Figure Legend Snippet: TAK1 is not implicated in IL-33-induced Akt activation and GSK-3β inactivation. PC-12 cells, transfected with the NC siRNA or the TAK1 siRNA, were treated with IL-33 (1 ng/mL) for 30 min, followed by Western blotting using antibodies against Akt, pT308-Akt, pS473-Akt, GSK-3β, pS9-GSK-3β, and pY216-GSK-3β. ( A ) ( D ) Western blot images. The arrow shown in ( D ) indicates the signal band at 46 kDa for pY216-GSK-3β. ( B ) ( C ) In the graphs, each column represents the mean (±SEM) signal intensity for pT308-Akt and pS473-Akt relative to the signal intensity for Akt (n = 8 Western blot results from independent experiments). ( E ) ( F ) In the graphs, each column represents the mean (±SEM) signal intensity for pS9-GSK-3β and pY216-GSK-3β relative to the signal intensity for GSK-3β (n = 8 Western blot results from independent experiments. P values shown in the graphs, ANOVA followed by a Bonferroni correction.

Techniques Used: Activation Assay, Transfection, Western Blot

19) Product Images from "Spicatoside A in red Liriope platyphylla displays a laxative effect in a constipation rat model via regulating mAChRs and ER stress signaling"

Article Title: Spicatoside A in red Liriope platyphylla displays a laxative effect in a constipation rat model via regulating mAChRs and ER stress signaling

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2018.3960

Detection of the ER stress response. (A) Expression levels of ER stress-related proteins IRE1α, IRE1β, p-IRE1, JNK, p-JNK, eIF2α and p-eIF2α were measured by western blot analysis. Band intensities were evaluated relative to the intensity of the actin bands. Data are presented as mean ± standard deviation from three replicates (N=5-6 rats per treatment group). * P
Figure Legend Snippet: Detection of the ER stress response. (A) Expression levels of ER stress-related proteins IRE1α, IRE1β, p-IRE1, JNK, p-JNK, eIF2α and p-eIF2α were measured by western blot analysis. Band intensities were evaluated relative to the intensity of the actin bands. Data are presented as mean ± standard deviation from three replicates (N=5-6 rats per treatment group). * P

Techniques Used: Expressing, Western Blot, Standard Deviation

20) Product Images from "All-trans retinoic acid alters the expression of the tight junction proteins Claudin-1 and -4 and epidermal barrier function-associated genes in the epidermis"

Article Title: All-trans retinoic acid alters the expression of the tight junction proteins Claudin-1 and -4 and epidermal barrier function-associated genes in the epidermis

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2019.4098

mRNA and protein expression levels of CLDN1 and CLDN4 in immortalized keratinocyte HaCaT cells treated with ATRA. HaCaT cells were incubated with or without 1 µ M ATRA for 36 h. (A) In the cells, CLDN-4, TJP3 and JGB4 were upregulated, while CLDN1 was downregulated. (B) In mice, CLDN1 and FLG were downregulated, while CLDN4 and CLDN2 were upregulated. (C) CLDN1 and CLDN4 protein expression levels were determined by western blotting. β-actin was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments performed in triplicate (n=3). * P
Figure Legend Snippet: mRNA and protein expression levels of CLDN1 and CLDN4 in immortalized keratinocyte HaCaT cells treated with ATRA. HaCaT cells were incubated with or without 1 µ M ATRA for 36 h. (A) In the cells, CLDN-4, TJP3 and JGB4 were upregulated, while CLDN1 was downregulated. (B) In mice, CLDN1 and FLG were downregulated, while CLDN4 and CLDN2 were upregulated. (C) CLDN1 and CLDN4 protein expression levels were determined by western blotting. β-actin was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments performed in triplicate (n=3). * P

Techniques Used: Expressing, Incubation, Mouse Assay, Western Blot, Standard Deviation

21) Product Images from "Cofilin-Mediated Actin Stress Response Is Maladaptive in Heat-Stressed Embryos"

Article Title: Cofilin-Mediated Actin Stress Response Is Maladaptive in Heat-Stressed Embryos

Journal: Cell reports

doi: 10.1016/j.celrep.2019.02.092

Cofilin Mediates an ASR that Changes Actin Organization in Nuclei and Cytoplasm (A) Surface views show G-actin Red in furrow tips in cofilin +/− embryos at the indicated temperatures. Intra-nuclear rods (yellow arrows) are reduced in cofilin +/− embryos at 32°C. (B) Percentage of embryos with rods in wild-type (WT) and cofilin +/− embryos at the indicated temperatures (n ≥ 14 embryos per condition, with ~500 nuclei analyzed per embryo; mean ± SE). (C) Representative western blots for Dm -Cofilin, Pi-cofilin from WT embryos at the indicated temperatures. β-actin is used as a loading control. Antibody validation in Figure S2 . (D) Ratio phosphorylated to total Cofilin in WT embryos at the indicated temperatures (n = 4 independent experiments; mean ± SE). (E) F-actin levels in furrow tips in WT embryos at the indicated temperatures (n ≥ 29 embryos per temperature, with 15 furrows analyzed per embryo; mean ± SE). Normalization to Histone-GFP embryos at 25°C, according to Figure S3 . (F) Cross sections show FRAP of furrow tip F-actin (G-actin Red ) in WT embryos at the indicated temperatures. Yellow boxes show bleached furrow tips. Pre, immediate pre-bleach time point; bleach, immediate post-bleach time point; sec, seconds after bleach. (G) FRAP kinetics for furrow tip F-actin in WT and cofilin +/− embryos at indicated temperatures. Each point represents one embryo (n ≥ 14 embryos per temperature, with 1–3 furrows analyzed per embryo; horizontal lines represent means ± SE). (H) F-actin levels in furrow tips in WT and cofilin +/− embryos at 32°C (n R 48 embryos per condition, with 15 furrows analyzed per embryo; mean ± SE). Normalization to Histone-GFP embryos at 32°C. (E) and (H) correspond to scatterplots in Figures S4C and S4D . Student’s t test used to calculate p values in (B), (E), (G), and (H).
Figure Legend Snippet: Cofilin Mediates an ASR that Changes Actin Organization in Nuclei and Cytoplasm (A) Surface views show G-actin Red in furrow tips in cofilin +/− embryos at the indicated temperatures. Intra-nuclear rods (yellow arrows) are reduced in cofilin +/− embryos at 32°C. (B) Percentage of embryos with rods in wild-type (WT) and cofilin +/− embryos at the indicated temperatures (n ≥ 14 embryos per condition, with ~500 nuclei analyzed per embryo; mean ± SE). (C) Representative western blots for Dm -Cofilin, Pi-cofilin from WT embryos at the indicated temperatures. β-actin is used as a loading control. Antibody validation in Figure S2 . (D) Ratio phosphorylated to total Cofilin in WT embryos at the indicated temperatures (n = 4 independent experiments; mean ± SE). (E) F-actin levels in furrow tips in WT embryos at the indicated temperatures (n ≥ 29 embryos per temperature, with 15 furrows analyzed per embryo; mean ± SE). Normalization to Histone-GFP embryos at 25°C, according to Figure S3 . (F) Cross sections show FRAP of furrow tip F-actin (G-actin Red ) in WT embryos at the indicated temperatures. Yellow boxes show bleached furrow tips. Pre, immediate pre-bleach time point; bleach, immediate post-bleach time point; sec, seconds after bleach. (G) FRAP kinetics for furrow tip F-actin in WT and cofilin +/− embryos at indicated temperatures. Each point represents one embryo (n ≥ 14 embryos per temperature, with 1–3 furrows analyzed per embryo; horizontal lines represent means ± SE). (H) F-actin levels in furrow tips in WT and cofilin +/− embryos at 32°C (n R 48 embryos per condition, with 15 furrows analyzed per embryo; mean ± SE). Normalization to Histone-GFP embryos at 32°C. (E) and (H) correspond to scatterplots in Figures S4C and S4D . Student’s t test used to calculate p values in (B), (E), (G), and (H).

Techniques Used: Western Blot, Size-exclusion Chromatography

ASR Is Maladaptive for Cellularization and Embryo Development (A) FRAP kinetics for furrow tip F-actin in embryos of indicated genotypes at 32°C. Each point represents one embryo (n ≥ 14 embryos per genotype, with 1–3 furrows analyzed per embryo). (B) Surface views show furrow tip F-actin (phalloidin, green) and nuclei (Hoechst, purple) in embryos of indicated genotypes at 32°C. Multinucleate cells highlighted by orange nuclei in corresponding segmented images. (C) Severity of multinucleation in embryos of indicated genotypes at 32°C. Each point represents one embryo (n ≥ 11 embryos per genotype, with ~150 nuclei analyzed per embryo). (D) Data from (4C) on shortened y axis to show no difference between wild-type (WT) and cofilin +/− embryos. (E) Larval hatching rates for indicated conditions (n ≥ 5 independent experiments, with ≥ 9 embryos per experiment; mean ± SE). Corresponding scatterplot in Figure S4F . Student’s t test used to calculate p values in (A), (C), (D), and (E). Horizontal lines represent means ± SE in (A), (C), and (D).
Figure Legend Snippet: ASR Is Maladaptive for Cellularization and Embryo Development (A) FRAP kinetics for furrow tip F-actin in embryos of indicated genotypes at 32°C. Each point represents one embryo (n ≥ 14 embryos per genotype, with 1–3 furrows analyzed per embryo). (B) Surface views show furrow tip F-actin (phalloidin, green) and nuclei (Hoechst, purple) in embryos of indicated genotypes at 32°C. Multinucleate cells highlighted by orange nuclei in corresponding segmented images. (C) Severity of multinucleation in embryos of indicated genotypes at 32°C. Each point represents one embryo (n ≥ 11 embryos per genotype, with ~150 nuclei analyzed per embryo). (D) Data from (4C) on shortened y axis to show no difference between wild-type (WT) and cofilin +/− embryos. (E) Larval hatching rates for indicated conditions (n ≥ 5 independent experiments, with ≥ 9 embryos per experiment; mean ± SE). Corresponding scatterplot in Figure S4F . Student’s t test used to calculate p values in (A), (C), (D), and (E). Horizontal lines represent means ± SE in (A), (C), and (D).

Techniques Used:

22) Product Images from "Pioglitazone increases VEGFR3 expression and promotes activation of M2 macrophages via the peroxisome proliferator-activated receptor γ"

Article Title: Pioglitazone increases VEGFR3 expression and promotes activation of M2 macrophages via the peroxisome proliferator-activated receptor γ

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2019.9945

Effect of pioglitazone on the expression of F4/80 and VEGFR3  in vivo . Immunofluorescence analysis of F4/80 and VEGFR3 co-localization (white arrows) in kidney sections from mice in the sham, UUO and UUO + pioglitazone groups. Pio, pioglitazone; UUO, unilateral ureteral obstruction; VEGFR3, vascular endothelial growth factor receptor 3.
Figure Legend Snippet: Effect of pioglitazone on the expression of F4/80 and VEGFR3 in vivo . Immunofluorescence analysis of F4/80 and VEGFR3 co-localization (white arrows) in kidney sections from mice in the sham, UUO and UUO + pioglitazone groups. Pio, pioglitazone; UUO, unilateral ureteral obstruction; VEGFR3, vascular endothelial growth factor receptor 3.

Techniques Used: Expressing, In Vivo, Immunofluorescence, Mouse Assay

23) Product Images from "Clostridium perfringens α-toxin impairs granulocyte colony-stimulating factor receptor-mediated granulocyte production while triggering septic shock"

Article Title: Clostridium perfringens α-toxin impairs granulocyte colony-stimulating factor receptor-mediated granulocyte production while triggering septic shock

Journal: Communications Biology

doi: 10.1038/s42003-019-0280-2

α-Toxin makes neutrophils insensitive to granulocyte colony-stimulating factor (G-CSF). a Bone marrow cells derived from wild-type (WT), Tlr2 − / − (TLR2 −/− ), Tlr4 − / − (TLR4 −/− ), and Myd88 − / − (MYD88 −/− ) mice were cultured for 24 h in the presence or absence (Control) of 100 ng ml −1 α-toxin (α-Toxin), and flow cytometric analysis was performed. The frequency of CD11b + Ly-6G high neutrophils is shown ( n = 3 per condition). b – e Magnetically isolated Ly-6G + cells were cultured for 24 h in the presence or absence of the indicated concentrations of G-CSF and α-toxin. The viable cells were determined using a Cell Counting Kit-8 ( b , n = 3 per condition). Whole-cell extracts were analyzed by immunoblotting with specific antibodies against G-CSFR and β-actin, and the density of bands was measured ( c , d , n = 3 per condition). Representative blots are shown of three independent experiments, raw gel images are available in Supplementary Figure 9 ( c ). Localization of G-CSFR was monitored by an immunostaining assay ( e ). Scale bar, 20 µm. f Ly-6G + cells were cultured for 30 min in the presence or absence of 100 ng ml −1 α-toxin. The cells were subjected to immunofluorescence analysis of ceramide. Scale bar, 20 µm. g – i Ly-6G + cells were cultured for 24 h in the presence or absence of the indicated concentrations of G-CSF and C 2 -ceramide. The viable cells were determined using a Cell Counting Kit-8 ( g , n = 4 per condition). Whole-cell extracts were analyzed by immunoblotting with specific antibodies against G-CSFR and β-actin, and the density of bands was measured ( h , i , n = 3 per condition). Representative blots are shown of three independent experiments, raw gel images are available in Supplementary Figure 9 ( h ). One-way analysis of variance was employed to assess statistical significance. Values are mean ± standard deviation. Similar results were obtained in two independent experiments
Figure Legend Snippet: α-Toxin makes neutrophils insensitive to granulocyte colony-stimulating factor (G-CSF). a Bone marrow cells derived from wild-type (WT), Tlr2 − / − (TLR2 −/− ), Tlr4 − / − (TLR4 −/− ), and Myd88 − / − (MYD88 −/− ) mice were cultured for 24 h in the presence or absence (Control) of 100 ng ml −1 α-toxin (α-Toxin), and flow cytometric analysis was performed. The frequency of CD11b + Ly-6G high neutrophils is shown ( n = 3 per condition). b – e Magnetically isolated Ly-6G + cells were cultured for 24 h in the presence or absence of the indicated concentrations of G-CSF and α-toxin. The viable cells were determined using a Cell Counting Kit-8 ( b , n = 3 per condition). Whole-cell extracts were analyzed by immunoblotting with specific antibodies against G-CSFR and β-actin, and the density of bands was measured ( c , d , n = 3 per condition). Representative blots are shown of three independent experiments, raw gel images are available in Supplementary Figure 9 ( c ). Localization of G-CSFR was monitored by an immunostaining assay ( e ). Scale bar, 20 µm. f Ly-6G + cells were cultured for 30 min in the presence or absence of 100 ng ml −1 α-toxin. The cells were subjected to immunofluorescence analysis of ceramide. Scale bar, 20 µm. g – i Ly-6G + cells were cultured for 24 h in the presence or absence of the indicated concentrations of G-CSF and C 2 -ceramide. The viable cells were determined using a Cell Counting Kit-8 ( g , n = 4 per condition). Whole-cell extracts were analyzed by immunoblotting with specific antibodies against G-CSFR and β-actin, and the density of bands was measured ( h , i , n = 3 per condition). Representative blots are shown of three independent experiments, raw gel images are available in Supplementary Figure 9 ( h ). One-way analysis of variance was employed to assess statistical significance. Values are mean ± standard deviation. Similar results were obtained in two independent experiments

Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Flow Cytometry, Isolation, Cell Counting, Immunostaining, Immunofluorescence, Standard Deviation

24) Product Images from "Nimbolide protects against endotoxin-induced acute respiratory distress syndrome by inhibiting TNF-α mediated NF-κB and HDAC-3 nuclear translocation"

Article Title: Nimbolide protects against endotoxin-induced acute respiratory distress syndrome by inhibiting TNF-α mediated NF-κB and HDAC-3 nuclear translocation

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1247-9

Nimbolide interacts with TNF-α and inhibits TNF-α regulated inflammatory signaling. a Docking model of nimbolide in the active site of TNF-α (PDB ID: 2AZ5) and ( b ) its ligand-protein interactions in the binding site of TNF-α. The dark pink dashed lines represent hydrogen bonds. H-bond distances (in Å) between heteroatoms of ligand and amino acid residues are as follows: Ser60 (3.4Å) and Leu120 (3.2Å). The red line indicates arene-arene interaction with Tyr59. A549 cells were pre-treated with nimbolide for 24 h and stimulated by LPS (1 µg/ml) for 12 h. Animals were pre-treated with nimbolide (0.3, 1 and 3 mg/kg) for 5 days later LPS (50 μg) was administered. c TNF-α protein expression was analyzed by confocal microscope. d A 5-µm-sized sections of lung tissues were subjected to IHC to determine TNF-α expression and images were captured at ×400 magnification. Protein expressions of TNF-α, p-p38 MAPK, p-IKK-α/β, p-IκB-α, p-NF-κB, p-GSK-3β, and mTOR were determined by western blotting in ( e ) A549 cells and ( f ) lung tissues, respectively. The HDACs such as HDAC-1, 2, 3, and 4 protein expressions were studied in ( g ) A549 cells and ( h ) lung tissues. i Nimbolide (0.05, 0.1, 1, and 2.5 µM) upon HDAC levels were analyzed by HDAC fluorometric kit and HDAC inhibitory activity was compared with trichostatin A (2.5 µM). Data represented as mean ± SEM (n=3 independent experiments). * P
Figure Legend Snippet: Nimbolide interacts with TNF-α and inhibits TNF-α regulated inflammatory signaling. a Docking model of nimbolide in the active site of TNF-α (PDB ID: 2AZ5) and ( b ) its ligand-protein interactions in the binding site of TNF-α. The dark pink dashed lines represent hydrogen bonds. H-bond distances (in Å) between heteroatoms of ligand and amino acid residues are as follows: Ser60 (3.4Å) and Leu120 (3.2Å). The red line indicates arene-arene interaction with Tyr59. A549 cells were pre-treated with nimbolide for 24 h and stimulated by LPS (1 µg/ml) for 12 h. Animals were pre-treated with nimbolide (0.3, 1 and 3 mg/kg) for 5 days later LPS (50 μg) was administered. c TNF-α protein expression was analyzed by confocal microscope. d A 5-µm-sized sections of lung tissues were subjected to IHC to determine TNF-α expression and images were captured at ×400 magnification. Protein expressions of TNF-α, p-p38 MAPK, p-IKK-α/β, p-IκB-α, p-NF-κB, p-GSK-3β, and mTOR were determined by western blotting in ( e ) A549 cells and ( f ) lung tissues, respectively. The HDACs such as HDAC-1, 2, 3, and 4 protein expressions were studied in ( g ) A549 cells and ( h ) lung tissues. i Nimbolide (0.05, 0.1, 1, and 2.5 µM) upon HDAC levels were analyzed by HDAC fluorometric kit and HDAC inhibitory activity was compared with trichostatin A (2.5 µM). Data represented as mean ± SEM (n=3 independent experiments). * P

Techniques Used: Binding Assay, Expressing, Microscopy, Immunohistochemistry, Western Blot, Activity Assay

Nimbolide inhibits TNF-α regulated NF-κB and HDAC-3 protein expression and nuclear translocation. A549 cells were treated with nimbolide (2.5 µM) for 24 h and stimulated with TNF-α (10 ng/ml) for 30 min. a NF-κB and HDAC-3 protein levels were observed in both cytosolic and nuclear fraction of A549 cells by western blotting. b The confocal analysis was performed to determine the protein expressions of NF-κB and HDAC-3. All the images were captured at ×400 magnification. BEAS-2B cells were transfected with TNF-α and scrambled siRNA and incubated for 24 h. In another set of the experimental group, cells were pre-treated with nimbolide (2.5 µM) for 24 h. Then, cells were stimulated with TNF-α (10 ng/ml) for 30 min except for nimbolide alone (NIM) group (2.5 µM). The expression of TNF-α, NF-κB and HDAC-3 levels were measured by ( c ) western blotting and ( d ) confocal analysis. The images were captured at ×400 magnification
Figure Legend Snippet: Nimbolide inhibits TNF-α regulated NF-κB and HDAC-3 protein expression and nuclear translocation. A549 cells were treated with nimbolide (2.5 µM) for 24 h and stimulated with TNF-α (10 ng/ml) for 30 min. a NF-κB and HDAC-3 protein levels were observed in both cytosolic and nuclear fraction of A549 cells by western blotting. b The confocal analysis was performed to determine the protein expressions of NF-κB and HDAC-3. All the images were captured at ×400 magnification. BEAS-2B cells were transfected with TNF-α and scrambled siRNA and incubated for 24 h. In another set of the experimental group, cells were pre-treated with nimbolide (2.5 µM) for 24 h. Then, cells were stimulated with TNF-α (10 ng/ml) for 30 min except for nimbolide alone (NIM) group (2.5 µM). The expression of TNF-α, NF-κB and HDAC-3 levels were measured by ( c ) western blotting and ( d ) confocal analysis. The images were captured at ×400 magnification

Techniques Used: Expressing, Translocation Assay, Western Blot, Transfection, Incubation

Nimbolide modulates the pro- and anti-inflammatory cytokines and chemokines. The proinflammatory cytokines and chemokines (IL-6, IL-12 (p40), MIP-1α, MIP-1β, and TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) expression were determined by multiplex and ELISA. a – h Animal lung tissue lysate was prepared and evaluated for the levels of aforementioned inflammatory cytokines and chemokines by multiplex. i , j lung tissue supernatants were subjected to ELISA to determine IL-1β and TGF-β cytokines expression. k Griess assay was performed to determine the nitrite levels in cell lysate of lung tissues. l The antioxidant GSH levels were measured in lung tissue. m The MPO as well as ( n ) iNOS, nitrotyrosine, Nrf-2, HO-1, and SOD-1 protein expressions were determined by immunoblot analysis. Data presented as mean ± SEM (n=8 mice per group). * P
Figure Legend Snippet: Nimbolide modulates the pro- and anti-inflammatory cytokines and chemokines. The proinflammatory cytokines and chemokines (IL-6, IL-12 (p40), MIP-1α, MIP-1β, and TNF-α) and anti-inflammatory cytokines (IL-4, IL-10, and IL-13) expression were determined by multiplex and ELISA. a – h Animal lung tissue lysate was prepared and evaluated for the levels of aforementioned inflammatory cytokines and chemokines by multiplex. i , j lung tissue supernatants were subjected to ELISA to determine IL-1β and TGF-β cytokines expression. k Griess assay was performed to determine the nitrite levels in cell lysate of lung tissues. l The antioxidant GSH levels were measured in lung tissue. m The MPO as well as ( n ) iNOS, nitrotyrosine, Nrf-2, HO-1, and SOD-1 protein expressions were determined by immunoblot analysis. Data presented as mean ± SEM (n=8 mice per group). * P

Techniques Used: Expressing, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Griess Assay, Mouse Assay

25) Product Images from "Urolithin A gains in antiproliferative capacity by reducing the glycolytic potential via the p53/TIGAR axis in colon cancer cells"

Article Title: Urolithin A gains in antiproliferative capacity by reducing the glycolytic potential via the p53/TIGAR axis in colon cancer cells

Journal: Carcinogenesis

doi: 10.1093/carcin/bgy158

Urolithin A leads to accumulation of HCT116 cells in G 2 /M phase of the cell cycle. WT and p53−/− HCT 116 cells were treated with solvent (0.1% DMSO), 20 µM or 40 µM urolithin A for 24 and 48 h prior to PI staining of their nuclei and flow cytometric analysis of cell-cycle distribution. Bar graphs depict compiled data of three independent biological replicates. (mean ± SD, * P
Figure Legend Snippet: Urolithin A leads to accumulation of HCT116 cells in G 2 /M phase of the cell cycle. WT and p53−/− HCT 116 cells were treated with solvent (0.1% DMSO), 20 µM or 40 µM urolithin A for 24 and 48 h prior to PI staining of their nuclei and flow cytometric analysis of cell-cycle distribution. Bar graphs depict compiled data of three independent biological replicates. (mean ± SD, * P

Techniques Used: Staining, Flow Cytometry

Knockdown of TIGAR by siRNA reduces the antiproliferative properties of urolithin A. ( A ) HCT116 WT cells were left untreated (ctrl) or transfected with siRNA (200 nM final concentration, scrambled (scr) or TIGAR-specific sequences #1–3, as indicated). After recovery and 24-h treatment with solvent (0.1% DMSO) or 30 µM urolithin A cells were lysed and subjected to western blot analysis for TIGAR, p21 and actin as loading control. Representative blots and densitometric analyses of three biological replicates are depicted. ( B ) HCT116 WT cells were transfected with the siRNAs (scr/mix#1–3) as indicated. Eight hours after transfection, cells were seeded into 96-well plates and treated with different concentrations of urolithin A. After 72-h incubation, a biomass staining via crystal violet was performed. Residual biomass was plotted against the concentration of urolithin A, and IC 50 values were derived from the fitted curve ( n = 3, mean ± SD, * P
Figure Legend Snippet: Knockdown of TIGAR by siRNA reduces the antiproliferative properties of urolithin A. ( A ) HCT116 WT cells were left untreated (ctrl) or transfected with siRNA (200 nM final concentration, scrambled (scr) or TIGAR-specific sequences #1–3, as indicated). After recovery and 24-h treatment with solvent (0.1% DMSO) or 30 µM urolithin A cells were lysed and subjected to western blot analysis for TIGAR, p21 and actin as loading control. Representative blots and densitometric analyses of three biological replicates are depicted. ( B ) HCT116 WT cells were transfected with the siRNAs (scr/mix#1–3) as indicated. Eight hours after transfection, cells were seeded into 96-well plates and treated with different concentrations of urolithin A. After 72-h incubation, a biomass staining via crystal violet was performed. Residual biomass was plotted against the concentration of urolithin A, and IC 50 values were derived from the fitted curve ( n = 3, mean ± SD, * P

Techniques Used: Transfection, Concentration Assay, Western Blot, Incubation, Staining, Derivative Assay

Urolithin A leads to stabilization of p53 and expression of the p53 target genes p21 and TIGAR. WT and p53−/− HCT 116 cells were treated with 30 µM urolithin A for the indicated periods of time. Total cell lysates were then subjected to immunoblot analysis for p53, p21, TIGAR and tubulin as loading control. Representative pictures and compiled densitometric analyses from three independent experiments are depicted (mean ± SD, * P
Figure Legend Snippet: Urolithin A leads to stabilization of p53 and expression of the p53 target genes p21 and TIGAR. WT and p53−/− HCT 116 cells were treated with 30 µM urolithin A for the indicated periods of time. Total cell lysates were then subjected to immunoblot analysis for p53, p21, TIGAR and tubulin as loading control. Representative pictures and compiled densitometric analyses from three independent experiments are depicted (mean ± SD, * P

Techniques Used: Expressing

Urolithin A leads to p53/p21-dependent senescence-like growth arrest in HCT116 cells. ( A ) WT and p53−/− HCT 116 cells were subcultivated in the continuous presence of solvent (0.1% DMSO) or 20 µM urolithin A (medium exchange every other day), and CPD were plotted against time of cultivation. Representative curves of three independent experiments with consistent results are depicted. ( B ) Cells after 25 days of cultivation in DMSO or 20 µM Uro A containing growth medium were photographed. ( C ) WT, p53−/− and p21−/− were chronically treated with 20 µM urolithin A prior to fixation and staining for SA-β-gal. The percentage of blue (here: dark) stained and hence SA-Gal positive cells from at least five different samples is summarized and depicted in the bar graph. Only urolithin A-treated WT, p53−/− or p21−/− HCT116 cells are shown.
Figure Legend Snippet: Urolithin A leads to p53/p21-dependent senescence-like growth arrest in HCT116 cells. ( A ) WT and p53−/− HCT 116 cells were subcultivated in the continuous presence of solvent (0.1% DMSO) or 20 µM urolithin A (medium exchange every other day), and CPD were plotted against time of cultivation. Representative curves of three independent experiments with consistent results are depicted. ( B ) Cells after 25 days of cultivation in DMSO or 20 µM Uro A containing growth medium were photographed. ( C ) WT, p53−/− and p21−/− were chronically treated with 20 µM urolithin A prior to fixation and staining for SA-β-gal. The percentage of blue (here: dark) stained and hence SA-Gal positive cells from at least five different samples is summarized and depicted in the bar graph. Only urolithin A-treated WT, p53−/− or p21−/− HCT116 cells are shown.

Techniques Used: Staining

Urolithin A reduces the glycolytic potential in WT but not p53−/− HCT116 cells. WT and p53−/− HCT 116 cells were treated with DMSO (0.1%) or urolithin A (30 µM) for 24 h before they were subjected to extracellular flux analysis and assessment of the cell energy phenotype and metabolic potential. Data of four independent biological replicates were compiled and presented relative to the overall mean of the DMSO controls. ( A ) shows the glycolytic, ( B ) the respiratory potential. (mean ± SD,* P
Figure Legend Snippet: Urolithin A reduces the glycolytic potential in WT but not p53−/− HCT116 cells. WT and p53−/− HCT 116 cells were treated with DMSO (0.1%) or urolithin A (30 µM) for 24 h before they were subjected to extracellular flux analysis and assessment of the cell energy phenotype and metabolic potential. Data of four independent biological replicates were compiled and presented relative to the overall mean of the DMSO controls. ( A ) shows the glycolytic, ( B ) the respiratory potential. (mean ± SD,* P

Techniques Used:

26) Product Images from "Steppogenin Isolated from Cudrania tricuspidata Shows Antineuroinflammatory Effects via NF-κB and MAPK Pathways in LPS-Stimulated BV2 and Primary Rat Microglial Cells"

Article Title: Steppogenin Isolated from Cudrania tricuspidata Shows Antineuroinflammatory Effects via NF-κB and MAPK Pathways in LPS-Stimulated BV2 and Primary Rat Microglial Cells

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

doi: 10.3390/molecules22122130

The effects of steppogenin ( 1 ) on nitrite ( A ) production and iNOS and COX-2 expression ( B ) in lipopolysaccharide (LPS)-stimulated primary rat microglial cells. ( A , B ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensities were quantified by densitometry and normalized to the intensities of the β-actin band; the normalized values are presented below each band. ** p
Figure Legend Snippet: The effects of steppogenin ( 1 ) on nitrite ( A ) production and iNOS and COX-2 expression ( B ) in lipopolysaccharide (LPS)-stimulated primary rat microglial cells. ( A , B ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensities were quantified by densitometry and normalized to the intensities of the β-actin band; the normalized values are presented below each band. ** p

Techniques Used: Expressing

The effects of steppogenin ( 1 ) on nitrite ( A ) and prostaglandin E2 (PGE 2 ) ( B ) production and iNOS and COX-2 expression ( C ) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. ( A – C ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; the normalized values are presented below each band. * p
Figure Legend Snippet: The effects of steppogenin ( 1 ) on nitrite ( A ) and prostaglandin E2 (PGE 2 ) ( B ) production and iNOS and COX-2 expression ( C ) in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. ( A – C ) The cells were pretreated for 3 h with the indicated concentrations of 1 and then stimulated for 24 h with LPS (1 μg/mL). The data are presented as the mean ± SD of three experiments. The band intensity was quantified by densitometry and normalized to the intensity of the β-actin band; the normalized values are presented below each band. * p

Techniques Used: Expressing

27) Product Images from "Triptolide reduces the viability of osteosarcoma cells by reducing MKP-1 and Hsp70 expression"

Article Title: Triptolide reduces the viability of osteosarcoma cells by reducing MKP-1 and Hsp70 expression

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2016.3164

Triptolide decreases the expression of MKP-1 protein. U-2 OS cells (A and B) or MG-63 cells (C) were treated with rriptolide (0, 5, 10, 25 and 50 nM) at 37°C for 48 h. (A) Representative blot using U-2 OS cell lysates was shown. (B) Protein expression levels of MKP-1 relative to GAPDH levels in the U-2 OS cells treated with triptolide. (C) Similar experiments were performed using the MG-63 cells. Data were from at least three independent experiments.*P
Figure Legend Snippet: Triptolide decreases the expression of MKP-1 protein. U-2 OS cells (A and B) or MG-63 cells (C) were treated with rriptolide (0, 5, 10, 25 and 50 nM) at 37°C for 48 h. (A) Representative blot using U-2 OS cell lysates was shown. (B) Protein expression levels of MKP-1 relative to GAPDH levels in the U-2 OS cells treated with triptolide. (C) Similar experiments were performed using the MG-63 cells. Data were from at least three independent experiments.*P

Techniques Used: Expressing

Effects of triptolide on the mRNA expression levels of MKP-1. U-2 OS and MG-63 cells were treated with triptolide (0, 5, 10, 25 and 50 nM) at 37°C for 24 h. All of the experiments were conducted in three independent experiments. Values are presented as the mean ± standard deviation. *P
Figure Legend Snippet: Effects of triptolide on the mRNA expression levels of MKP-1. U-2 OS and MG-63 cells were treated with triptolide (0, 5, 10, 25 and 50 nM) at 37°C for 24 h. All of the experiments were conducted in three independent experiments. Values are presented as the mean ± standard deviation. *P

Techniques Used: Expressing, Standard Deviation

28) Product Images from "Expression of heteromeric amino acid transporters along the murine intestine"

Article Title: Expression of heteromeric amino acid transporters along the murine intestine

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2004.065037

Protein abundance of heteromeric amino acid transporters The segmental expression and abundance of 4F2hc (migrating at ∼80 kDa), of its associated light chains LAT1, LAT2 and y + -LAT1 (∼35–45 kDa) as well as of the rBAT-associated light chain b° ,+ AT was assessed by Western blotting (50 μg crude membrane protein per lane). Kidney and brain were loaded as control. As for mRNA, distinct patterns of protein expression were found for all subunits assayed. LAT1 was found only in brain. LAT2 showed a gradient of expression along the small intestine but was also abundant in stomach. For LAT2 a second band of higher molecular weight (approx. 120–150 kDa) was seen which was specific and may represent the heterodimer formed together with 4F2hc. y + -LAT1 expression was confined to the small intestine and kidney and increased along the small intestine. b° ,+ AT showed a similar pattern of expression to y + -LAT1. Western blots shown represent data from one representative mouse but qualitatively similar results were obtained from three different mice in separate experiments.
Figure Legend Snippet: Protein abundance of heteromeric amino acid transporters The segmental expression and abundance of 4F2hc (migrating at ∼80 kDa), of its associated light chains LAT1, LAT2 and y + -LAT1 (∼35–45 kDa) as well as of the rBAT-associated light chain b° ,+ AT was assessed by Western blotting (50 μg crude membrane protein per lane). Kidney and brain were loaded as control. As for mRNA, distinct patterns of protein expression were found for all subunits assayed. LAT1 was found only in brain. LAT2 showed a gradient of expression along the small intestine but was also abundant in stomach. For LAT2 a second band of higher molecular weight (approx. 120–150 kDa) was seen which was specific and may represent the heterodimer formed together with 4F2hc. y + -LAT1 expression was confined to the small intestine and kidney and increased along the small intestine. b° ,+ AT showed a similar pattern of expression to y + -LAT1. Western blots shown represent data from one representative mouse but qualitatively similar results were obtained from three different mice in separate experiments.

Techniques Used: Expressing, Western Blot, Molecular Weight, Mouse Assay

Localization of 4F2hc and y + -LAT1 in mouse intestine y + -LAT1 (red) colocalized with 4F2hc (green) in duodenum, jejunum and ileum to the basolateral side of the epithelial cells. A – C , only a weak signal for y + -LAT1 was seen in duodenum. D – J , similar to LAT2 a gradient of expression was seen for y + -LAT1 along the villi of the jejunum ( D – I ) and ileum ( H – J ). The red staining appearing in the lumen ( E and F ) may result from unspecific antibody binding to mucus and was also seen with pre-immune serum. Note that not all cells were stained for 4F2hc and y + -LAT1 as marked by the asterisk; these cells most likely represent goblet cells. No staining for y + -LAT1 was seen in stomach and colon. Original magnification 400×.
Figure Legend Snippet: Localization of 4F2hc and y + -LAT1 in mouse intestine y + -LAT1 (red) colocalized with 4F2hc (green) in duodenum, jejunum and ileum to the basolateral side of the epithelial cells. A – C , only a weak signal for y + -LAT1 was seen in duodenum. D – J , similar to LAT2 a gradient of expression was seen for y + -LAT1 along the villi of the jejunum ( D – I ) and ileum ( H – J ). The red staining appearing in the lumen ( E and F ) may result from unspecific antibody binding to mucus and was also seen with pre-immune serum. Note that not all cells were stained for 4F2hc and y + -LAT1 as marked by the asterisk; these cells most likely represent goblet cells. No staining for y + -LAT1 was seen in stomach and colon. Original magnification 400×.

Techniques Used: Expressing, Staining, Binding Assay

Relative mRNA abundance of 4F2hc and associated light chains The abundance of the different test mRNAs relative to that of GAPDH (2 (Ct(GAPDH)−Ct(TEST)) is indicated for each tissue tested (see Methods). 4F2hc mRNA showed a wide tissue distribution with a segmental gradient of expression in small intestine that is similar to that of LAT2 and y + -LAT1. The mean relative mRNA abundance tested independently in three mice is shown. S, stomach; D, duodenum; J, jejunum; I, ileum; C, colon; L, liver; B, brain; K, kidney.
Figure Legend Snippet: Relative mRNA abundance of 4F2hc and associated light chains The abundance of the different test mRNAs relative to that of GAPDH (2 (Ct(GAPDH)−Ct(TEST)) is indicated for each tissue tested (see Methods). 4F2hc mRNA showed a wide tissue distribution with a segmental gradient of expression in small intestine that is similar to that of LAT2 and y + -LAT1. The mean relative mRNA abundance tested independently in three mice is shown. S, stomach; D, duodenum; J, jejunum; I, ileum; C, colon; L, liver; B, brain; K, kidney.

Techniques Used: Expressing, Mouse Assay

Localization of 4F2hc and LAT2 in mouse stomach A – C , immunohistochemical labelling localized 4F2hc (green, A ) and LAT2 (red, B ) to the basolateral side of large cells along the gastric gland. D , staining of the parietal cell specific β-subunit of the gastric H + /K + -ATPase (green) identifies the cells expressing LAT2 as acid-secreting parietal cells. Original magnification 400×.
Figure Legend Snippet: Localization of 4F2hc and LAT2 in mouse stomach A – C , immunohistochemical labelling localized 4F2hc (green, A ) and LAT2 (red, B ) to the basolateral side of large cells along the gastric gland. D , staining of the parietal cell specific β-subunit of the gastric H + /K + -ATPase (green) identifies the cells expressing LAT2 as acid-secreting parietal cells. Original magnification 400×.

Techniques Used: Immunohistochemistry, Staining, Expressing

Localization of 4F2hc and LAT2 in mouse small intestine Immunohistochemistry localized 4F2hc (green) and LAT-2 (red) to the basolateral side of the epithelial cells lining the duodenum, jejunum and ileum. 4F2hc and LAT2 colocalized and showed a gradient of expression along the villi with higher expression in the tips. A and B , 4F2hc and LAT2 localization in mouse duodenum; C , overlay of 4F2hc and LAT2 (yellow). Arrows show single dispersed cells which did not show staining for 4F2 and LAT2. D , colocalization of 4F2hc and LAT2 in jejunum, overlay (yellow). E – G , localization of 4F2hc and LAT2 in ileum and overlay (yellow). Original magnifications 400×. H , higher magnification of overlay of 4F2hc and LAT2 in ileum showing colocalization of both subunits to the basolateral membrane and some vesicular intracellular structures. Note that some cells were not stained for 4F2 and LAT2 (arrows). Original magnification 600×.
Figure Legend Snippet: Localization of 4F2hc and LAT2 in mouse small intestine Immunohistochemistry localized 4F2hc (green) and LAT-2 (red) to the basolateral side of the epithelial cells lining the duodenum, jejunum and ileum. 4F2hc and LAT2 colocalized and showed a gradient of expression along the villi with higher expression in the tips. A and B , 4F2hc and LAT2 localization in mouse duodenum; C , overlay of 4F2hc and LAT2 (yellow). Arrows show single dispersed cells which did not show staining for 4F2 and LAT2. D , colocalization of 4F2hc and LAT2 in jejunum, overlay (yellow). E – G , localization of 4F2hc and LAT2 in ileum and overlay (yellow). Original magnifications 400×. H , higher magnification of overlay of 4F2hc and LAT2 in ileum showing colocalization of both subunits to the basolateral membrane and some vesicular intracellular structures. Note that some cells were not stained for 4F2 and LAT2 (arrows). Original magnification 600×.

Techniques Used: Immunohistochemistry, Expressing, Staining

29) Product Images from "Tart cherry supplementation improves working memory, hippocampal inflammation, and autophagy in aged rats"

Article Title: Tart cherry supplementation improves working memory, hippocampal inflammation, and autophagy in aged rats

Journal: Age

doi: 10.1007/s11357-016-9945-7

Tart cherry consumption reduced inflammation in the hippocampus. The results shown represent western blot ( top ) of inflammatory markers (glial fibrillary acidic protein [GFAP], NADPH oxidase-2 [NOX-2], and cyclooxygenase-2 [COX-2]) in the hippocampus of control ( opened bar ) and 2 % tart cherry-supplemented group ( hatched bar ), and densitometry analysis ( bottom ) of the immunoreactive bands normalized to β-actin, with results represented as mean ± SEM; n = 7/group. Asterisk indicates statistically significant difference between the groups ( p
Figure Legend Snippet: Tart cherry consumption reduced inflammation in the hippocampus. The results shown represent western blot ( top ) of inflammatory markers (glial fibrillary acidic protein [GFAP], NADPH oxidase-2 [NOX-2], and cyclooxygenase-2 [COX-2]) in the hippocampus of control ( opened bar ) and 2 % tart cherry-supplemented group ( hatched bar ), and densitometry analysis ( bottom ) of the immunoreactive bands normalized to β-actin, with results represented as mean ± SEM; n = 7/group. Asterisk indicates statistically significant difference between the groups ( p

Techniques Used: Western Blot

30) Product Images from "Inhibition of KPNA4 Attenuates Prostate Cancer Metastasis"

Article Title: Inhibition of KPNA4 Attenuates Prostate Cancer Metastasis

Journal: Oncogene

doi: 10.1038/onc.2016.440

Schematic model of miR-708/KPNA4/TNF-α and β signaling pathway that regulates PCa microenvironment and bone metastasis In primary prostate tumor, miR-708 decrease in the cancer cells causes abnormal high expression of KPNA4, which will subsequently increase the TNF-α and β expression. Enrichment of TNF-α and β in tumor microenvironment can promote both of cancer cell mobility and M2 polarization of TAMs. On the other hand, increased TNF-α and β can enhance the osteoclastogenesis in bone environment, and eventually accelerate prostate cancer bone metastasis.
Figure Legend Snippet: Schematic model of miR-708/KPNA4/TNF-α and β signaling pathway that regulates PCa microenvironment and bone metastasis In primary prostate tumor, miR-708 decrease in the cancer cells causes abnormal high expression of KPNA4, which will subsequently increase the TNF-α and β expression. Enrichment of TNF-α and β in tumor microenvironment can promote both of cancer cell mobility and M2 polarization of TAMs. On the other hand, increased TNF-α and β can enhance the osteoclastogenesis in bone environment, and eventually accelerate prostate cancer bone metastasis.

Techniques Used: Expressing

TNF-α and β mediates the KPNA4 induced prostate cancer migration ( a–b ) TNF-α and TNF-β expression was evaluated in the primary tumor tissue by immunofluorenscence staining. ( c ) M2-phenotype associated cytokines of primary murine macrophages that were subjected to TNF-α or TNF-β stimulation (5ng/mL) was determined by real-time PCR. ( d ) Transwell assay of PC3-shKPNA4 or scramble control cells lines in the absence or presence of U937 cells to determine the cell invasion, PC3-shKPNA4 cells were stimulated with or without recombinant TNF-α or β cytokines (5ng/mL). ( e–f ) Invasive cells were quantitated by crystal violet staining assay. * p
Figure Legend Snippet: TNF-α and β mediates the KPNA4 induced prostate cancer migration ( a–b ) TNF-α and TNF-β expression was evaluated in the primary tumor tissue by immunofluorenscence staining. ( c ) M2-phenotype associated cytokines of primary murine macrophages that were subjected to TNF-α or TNF-β stimulation (5ng/mL) was determined by real-time PCR. ( d ) Transwell assay of PC3-shKPNA4 or scramble control cells lines in the absence or presence of U937 cells to determine the cell invasion, PC3-shKPNA4 cells were stimulated with or without recombinant TNF-α or β cytokines (5ng/mL). ( e–f ) Invasive cells were quantitated by crystal violet staining assay. * p

Techniques Used: Migration, Expressing, Staining, Real-time Polymerase Chain Reaction, Transwell Assay, Recombinant

31) Product Images from "The Intracellular Interactome of Tetraspanin-enriched Microdomains Reveals Their Function as Sorting Machineries toward Exosomes *"

Article Title: The Intracellular Interactome of Tetraspanin-enriched Microdomains Reveals Their Function as Sorting Machineries toward Exosomes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.445304

Specificity of interactions between TEM receptors and intracellular proteins in cell lysates and exosomes of human primary lymphoblasts. Charts show proteomic analysis (high throughput mass spectrometry) of pulldown assays in whole cell lysates ( left ) or exosome extracts ( right ) of primary human lymphoblasts. Data correspond to interaction with α-actinin ( A ), filamin ( B ), nucleolin ( C ), Rac ( D ), elongation factor 1-α ( E ), actin ( F ), and ERMs ( G ). Western blotting of equivalent pulldowns in whole cell lysates and exosomes confirms these interactions.
Figure Legend Snippet: Specificity of interactions between TEM receptors and intracellular proteins in cell lysates and exosomes of human primary lymphoblasts. Charts show proteomic analysis (high throughput mass spectrometry) of pulldown assays in whole cell lysates ( left ) or exosome extracts ( right ) of primary human lymphoblasts. Data correspond to interaction with α-actinin ( A ), filamin ( B ), nucleolin ( C ), Rac ( D ), elongation factor 1-α ( E ), actin ( F ), and ERMs ( G ). Western blotting of equivalent pulldowns in whole cell lysates and exosomes confirms these interactions.

Techniques Used: Transmission Electron Microscopy, High Throughput Screening Assay, Mass Spectrometry, Western Blot

32) Product Images from "DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells"

Article Title: DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells

Journal: Journal of Virology

doi: 10.1128/JVI.01831-16

PLA analysis of the S-phase factors DNA repair DNA polymerases with replicating viral DNA. HEK293 cells were transfected with pIHBoV1NS1(−) or pIHBoV1. At 48 h posttransfection they were labeled with BrdU. Cells were cytospun onto slides and were costained with anti-BrdU and anti-PCNA, anti-RFC1, anti-RPA32, anti-Pol δ, anti-Pol α, or anti-Pol ε, as indicated (A), or were costained with anti-BrdU and one of the following repair proteins: Pol η, Pol ι, Pol μ, Pol κ, Pol λ, Pol β, Rev 1, or Pol ξ (B). The PLA-amplified signal is shown in red. Nuclei were stained with DAPI (blue). Confocal images were taken at a ×100 magnification.
Figure Legend Snippet: PLA analysis of the S-phase factors DNA repair DNA polymerases with replicating viral DNA. HEK293 cells were transfected with pIHBoV1NS1(−) or pIHBoV1. At 48 h posttransfection they were labeled with BrdU. Cells were cytospun onto slides and were costained with anti-BrdU and anti-PCNA, anti-RFC1, anti-RPA32, anti-Pol δ, anti-Pol α, or anti-Pol ε, as indicated (A), or were costained with anti-BrdU and one of the following repair proteins: Pol η, Pol ι, Pol μ, Pol κ, Pol λ, Pol β, Rev 1, or Pol ξ (B). The PLA-amplified signal is shown in red. Nuclei were stained with DAPI (blue). Confocal images were taken at a ×100 magnification.

Techniques Used: Proximity Ligation Assay, Transfection, Labeling, Amplification, Staining

33) Product Images from "Human Ubc9 Contributes to Production of Fully Infectious Human Immunodeficiency Virus Type 1 Virions "

Article Title: Human Ubc9 Contributes to Production of Fully Infectious Human Immunodeficiency Virus Type 1 Virions

Journal: Journal of Virology

doi: 10.1128/JVI.00237-09

Biochemical composition of viral particles. The relative levels of vRNAs and proteins packaged into equivalent numbers of defective and infectious virions (normalized to the p24 content) were analyzed. (a) VRNA packaging. RNA from pelleted virions was extracted, reverse transcribed using oligo(dT) 12-18, and quantified by real-time PCR using vRNA-specific primers. Real-time PCR data from three independent experiments is shown as the amount of vRNA packaged into virions relative to that of vRNA packaged into virions produced from cells transfected with pNL4-3 only. The error bars indicate standard deviations. (b) Packaging of cellular and HIV-1 regulatory proteins. The levels of cellular (cyclophilin A [Cyp A]) and viral (Vif, Vpr, and Vpu) regulatory proteins packaged into virions were analyzed by immunoblotting. The bands were quantified using Discovery Series Quantity One software. (c) Glycoprotein packaging into virions. Envelope packaging into virions was analyzed by immunoblotting using polyclonal anti-HIV-1 and monoclonal anti-gp41 antibodies. (d) Glycoprotein production in cell lysates. Envelope production inside transfected cells was analyzed by immunoblotting using polyclonal anti-HIV-1 and monoclonal anti-gp41 antibodies. (e) Decreases in virion infectivity are not due to defects in early postentry events. Cells were transfected with RNAs as in previous experiments, followed by DNA transfections with pNL4-3-ΔE-EGFP and pHyg-VSV-G or pNL4-3 ΔEnv alone. Media containing pseudotyped virions were harvested and clarified 24 h after DNA transfections. Virion infectivity assays were carried out as before using TZM-bl target cells. Ubc9 expression in cell lysates. No RNA (lanes 1 to 4), control RNA (lanes 5 to 7), Ubc9 RNAi (lanes 8 to 10).
Figure Legend Snippet: Biochemical composition of viral particles. The relative levels of vRNAs and proteins packaged into equivalent numbers of defective and infectious virions (normalized to the p24 content) were analyzed. (a) VRNA packaging. RNA from pelleted virions was extracted, reverse transcribed using oligo(dT) 12-18, and quantified by real-time PCR using vRNA-specific primers. Real-time PCR data from three independent experiments is shown as the amount of vRNA packaged into virions relative to that of vRNA packaged into virions produced from cells transfected with pNL4-3 only. The error bars indicate standard deviations. (b) Packaging of cellular and HIV-1 regulatory proteins. The levels of cellular (cyclophilin A [Cyp A]) and viral (Vif, Vpr, and Vpu) regulatory proteins packaged into virions were analyzed by immunoblotting. The bands were quantified using Discovery Series Quantity One software. (c) Glycoprotein packaging into virions. Envelope packaging into virions was analyzed by immunoblotting using polyclonal anti-HIV-1 and monoclonal anti-gp41 antibodies. (d) Glycoprotein production in cell lysates. Envelope production inside transfected cells was analyzed by immunoblotting using polyclonal anti-HIV-1 and monoclonal anti-gp41 antibodies. (e) Decreases in virion infectivity are not due to defects in early postentry events. Cells were transfected with RNAs as in previous experiments, followed by DNA transfections with pNL4-3-ΔE-EGFP and pHyg-VSV-G or pNL4-3 ΔEnv alone. Media containing pseudotyped virions were harvested and clarified 24 h after DNA transfections. Virion infectivity assays were carried out as before using TZM-bl target cells. Ubc9 expression in cell lysates. No RNA (lanes 1 to 4), control RNA (lanes 5 to 7), Ubc9 RNAi (lanes 8 to 10).

Techniques Used: Real-time Polymerase Chain Reaction, Produced, Transfection, Software, Infection, Expressing

34) Product Images from "Transforming growth factor β1 (TGFβ1) regulates CD44V6 expression and activity through extracellular signal-regulated kinase (ERK)-induced EGR1 in pulmonary fibrogenic fibroblasts"

Article Title: Transforming growth factor β1 (TGFβ1) regulates CD44V6 expression and activity through extracellular signal-regulated kinase (ERK)-induced EGR1 in pulmonary fibrogenic fibroblasts

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M116.752451

Increased expression of CD44v6 is associated with collagen-1 and hyaluronan synthesis in the fibrogenic fibroblasts in the bleomycin-induced pulmonary fibrosis model. A , systemic effects in response to bleomycin-induced lung fibrosis or PBS control were assessed by a time-course evaluation of body weights. Whole-lung homogenates were analyzed for acid-soluble collagen by Sircol assay ( B ) and for acid-insoluble collagen by a hydroxyproline assay ( C ) for the indicated days after bleomycin-induced lung injury. D , fibroblasts (BLMFbs) were isolated from lung tissues at days 7, 14, 21, and 45 after bleomycin-induced lung injury, and fibroblasts were isolated from PBS (MNLFBs)-treated mouse lung tissues at the same times as described under “Experimental procedures.” The effects on hyaluronan synthesis in response to bleomycin-induced lung fibrosis or to PBS control were assessed by measuring hyaluronan secreted into the medium by an ELISA-like assay. E , total RNAs were examined by real-time PCR analysis for the indicated mRNAs expressed relative to β-actin. F , lysates from BLMFbs at days 7, 14, 21, and 45 after bleomycin-induced lung injury and MNLFBs at day 0 were immunoblotted ( WB ) for CD44v6, CD44s, α-Sma, collagen-1, and β-actin (loading control) (using anti-CD44v6 (VFF-18 clone) from Chemicon), CD44s using anti-CD44 (ab157107) from Abcam, α-SMA (using anti-α-SMA (IH8) from Novus Biologicals, collagen-1 (using anti-collagen-1 (ab1209539) from Abcam), and β-actin (from EMD Millipore (RM112)). Densitometry analyses are shown below the Western blots after normalization with β-actin. The data in the experiments ( A–F ) are from three independent experiments and are expressed as means ± S.D. ( error bars ). Statistical analysis was by analysis of variance. B–E , *, p ≤ 0.001 versus PBS-treated control or day 0 control groups; F , *, p ≤ 0.001 versus day 0 control group.
Figure Legend Snippet: Increased expression of CD44v6 is associated with collagen-1 and hyaluronan synthesis in the fibrogenic fibroblasts in the bleomycin-induced pulmonary fibrosis model. A , systemic effects in response to bleomycin-induced lung fibrosis or PBS control were assessed by a time-course evaluation of body weights. Whole-lung homogenates were analyzed for acid-soluble collagen by Sircol assay ( B ) and for acid-insoluble collagen by a hydroxyproline assay ( C ) for the indicated days after bleomycin-induced lung injury. D , fibroblasts (BLMFbs) were isolated from lung tissues at days 7, 14, 21, and 45 after bleomycin-induced lung injury, and fibroblasts were isolated from PBS (MNLFBs)-treated mouse lung tissues at the same times as described under “Experimental procedures.” The effects on hyaluronan synthesis in response to bleomycin-induced lung fibrosis or to PBS control were assessed by measuring hyaluronan secreted into the medium by an ELISA-like assay. E , total RNAs were examined by real-time PCR analysis for the indicated mRNAs expressed relative to β-actin. F , lysates from BLMFbs at days 7, 14, 21, and 45 after bleomycin-induced lung injury and MNLFBs at day 0 were immunoblotted ( WB ) for CD44v6, CD44s, α-Sma, collagen-1, and β-actin (loading control) (using anti-CD44v6 (VFF-18 clone) from Chemicon), CD44s using anti-CD44 (ab157107) from Abcam, α-SMA (using anti-α-SMA (IH8) from Novus Biologicals, collagen-1 (using anti-collagen-1 (ab1209539) from Abcam), and β-actin (from EMD Millipore (RM112)). Densitometry analyses are shown below the Western blots after normalization with β-actin. The data in the experiments ( A–F ) are from three independent experiments and are expressed as means ± S.D. ( error bars ). Statistical analysis was by analysis of variance. B–E , *, p ≤ 0.001 versus PBS-treated control or day 0 control groups; F , *, p ≤ 0.001 versus day 0 control group.

Techniques Used: Expressing, Hydroxyproline Assay, Isolation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

Characterization of CD44v6 mRNA stability in 21dBLMFbs untreated or treated with TGFβ1 after the addition of ACTD. 21dBLMFbs were serum-deprived for 48 h followed by treatment with or without TGFβ1 (5 ng/ml) in the presence of ACTD (2 μg/ml) for the indicated times and analyzed for CD44v6 mRNA, CD44s mRNA, and Gapdh mRNA. As positive and negative controls, mRNA expression levels were determined from total RNAs from cells that were exposed with TGFβ1 plus ACTD with TGFβ1 antibody (5 μg/ml) or TGFβRI antibody (5 μg/ml) and with 2 μg/ml in DMSO solution plus TGFβ1 antibody or TGFβRI antibody (5 μg/ml) in serum-starved medium for the same time points (data not shown). A , real-time PCR of CD44v6 mRNA after normalization with Gapdh mRNA during the 20 h of ACTD treatment. B , representative mRNA by RT-PCR analyses. Data are representative of three independent experiments.
Figure Legend Snippet: Characterization of CD44v6 mRNA stability in 21dBLMFbs untreated or treated with TGFβ1 after the addition of ACTD. 21dBLMFbs were serum-deprived for 48 h followed by treatment with or without TGFβ1 (5 ng/ml) in the presence of ACTD (2 μg/ml) for the indicated times and analyzed for CD44v6 mRNA, CD44s mRNA, and Gapdh mRNA. As positive and negative controls, mRNA expression levels were determined from total RNAs from cells that were exposed with TGFβ1 plus ACTD with TGFβ1 antibody (5 μg/ml) or TGFβRI antibody (5 μg/ml) and with 2 μg/ml in DMSO solution plus TGFβ1 antibody or TGFβRI antibody (5 μg/ml) in serum-starved medium for the same time points (data not shown). A , real-time PCR of CD44v6 mRNA after normalization with Gapdh mRNA during the 20 h of ACTD treatment. B , representative mRNA by RT-PCR analyses. Data are representative of three independent experiments.

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

35) Product Images from "Chemoprevention of Pancreatic Cancer: Characterization of Par-4 and its Modulation by 3,3? Diindolylmethane (DIM)"

Article Title: Chemoprevention of Pancreatic Cancer: Characterization of Par-4 and its Modulation by 3,3? Diindolylmethane (DIM)

Journal: Pharmaceutical research

doi: 10.1007/s11095-008-9581-8

Western blot analysis of PAR-4 protein expression in pancreatic cancer cell lines. Panc-1 ( Lane 1 ), L3.6pl ( Lane 2 ), Colo-357 ( Lane 3 ), MiaPaCa ( Lane 4 ), BxPC-3 ( Lane 5 ) and Hs766T ( Lane 6 ). β-actin protein was used as protein loading control as shown for each blot. Cell extracts were prepared according to the procedure described under MATERIALS AND METHODS section.
Figure Legend Snippet: Western blot analysis of PAR-4 protein expression in pancreatic cancer cell lines. Panc-1 ( Lane 1 ), L3.6pl ( Lane 2 ), Colo-357 ( Lane 3 ), MiaPaCa ( Lane 4 ), BxPC-3 ( Lane 5 ) and Hs766T ( Lane 6 ). β-actin protein was used as protein loading control as shown for each blot. Cell extracts were prepared according to the procedure described under MATERIALS AND METHODS section.

Techniques Used: Western Blot, Expressing

PAR-4 expression is up-regulated by B-DIM. ( A ) Western blot analysis of lysates of L3.6pl cells treated with: DMSO (untreated; Lane 1 ); 10 μmol/L B-DIM ( Lane 2 ); 20 μmol/L ( Lane 3 ), respectively. ( B ) Western blot analysis of lysates of Colo-357 cells treated with DMSO (untreated; Lane 1 ); 10 μmol/L B-DIM ( Lane 2 ); 20 μmol/L ( Lane 3 ), respectively. β-actin protein was used as loading control as shown for each blot.
Figure Legend Snippet: PAR-4 expression is up-regulated by B-DIM. ( A ) Western blot analysis of lysates of L3.6pl cells treated with: DMSO (untreated; Lane 1 ); 10 μmol/L B-DIM ( Lane 2 ); 20 μmol/L ( Lane 3 ), respectively. ( B ) Western blot analysis of lysates of Colo-357 cells treated with DMSO (untreated; Lane 1 ); 10 μmol/L B-DIM ( Lane 2 ); 20 μmol/L ( Lane 3 ), respectively. β-actin protein was used as loading control as shown for each blot.

Techniques Used: Expressing, Western Blot

PAR-4 expression is up-regulated by B-DIM in combination with gemcitabine. ( A ) Western blot analysis of lysate extracted from Colo-357 cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+Gemcitabine 100 nmol/L, respectively. ( B ) Western blot analysis of lysate extracted from L-3.6pl cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+Gemcitabine 100 nmol/L, respectively. Western blot analysis of lysate extracted from BxPC-3 cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+ Gemcitabine 100 nmol/L, respectively. Cell lysates were prepared according to the procedure described under MATERIALS AND METHODS section.
Figure Legend Snippet: PAR-4 expression is up-regulated by B-DIM in combination with gemcitabine. ( A ) Western blot analysis of lysate extracted from Colo-357 cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+Gemcitabine 100 nmol/L, respectively. ( B ) Western blot analysis of lysate extracted from L-3.6pl cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+Gemcitabine 100 nmol/L, respectively. Western blot analysis of lysate extracted from BxPC-3 cells treated with; DMSO (untreated; Lane 1 ); B-DIM 20 μmol/L ( Lane 2 ); Gemcitabine 100 nmol/L ( Lane 3 ); B-DIM 20 μmol/L+ Gemcitabine 100 nmol/L, respectively. Cell lysates were prepared according to the procedure described under MATERIALS AND METHODS section.

Techniques Used: Expressing, Western Blot

36) Product Images from "Autoactivation of the Epstein-Barr Virus Oncogenic Protein LMP1 during Type II Latency through Opposite Roles of the NF-?B and JNK Signaling Pathways"

Article Title: Autoactivation of the Epstein-Barr Virus Oncogenic Protein LMP1 during Type II Latency through Opposite Roles of the NF-?B and JNK Signaling Pathways

Journal: Journal of Virology

doi: 10.1128/JVI.02052-05

Opposite roles of the JNK and NF-κB pathways on endogenous LMP1 expression at the mRNA and protein levels. Inhibition of (A) NF-κB activity upregulates, whereas inhibition of (B) JNK activity downregulates the endogenous LMP1 protein levels in PRI and TE1 cell lines. The cells were treated with vehicle (DMSO), sulfasalazine (1 mM and 5 mM for TE1 and PRI cells, respectively), or SP600125 (20 μM) for 16 h. Equal amounts of cells were harvested and directly lysed in 3× Laemmli buffer. Protein lysates were analyzed by Western blotting for the expression of LMP1, IκBα, and EBNA2. Equal loading of proteins in each lane was confirmed by probing the membrane with anti-β-actin antibody. (C) Sulfasalazine increases, whereas SP600125 decreases the LMP1 mRNA levels in PRI cells. PRI cells were treated with either vehicle (DMSO) or sulfasalazine (5 mM) or SP600125 (20 μM) for 16 h. Cells were then harvested, and mRNAs were extracted and subjected to real-time RT-PCR as described in Materials and Methods. LMP1 mRNA levels were normalized to the mRNA levels of three housekeeping genes (β-actin, glyceraldehyde-3-phosphate dehydrogenase, and hypoxanthine phosphoribosyltransferase), and the normalized levels in the untreated condition were arbitrarily assigned a value of 100. (D) The constitutively active form of IκBα inhibits NF-κB target genes and increases the levels of LMP1 transcripts in PRI cells. Where indicated, the PRI-pRT1-IκBm stable cell line were treated with doxycycline (2 μg/ml) (+ Dox) to induce the expression of IκBm. After induction for 24 h, the relative LMP1 mRNA levels were quantified by real-time RT-PCR as described above. After induction for 48 h, protein lysates were collected as described above and analyzed by Western blotting for the expression of EBNA2, ICAM1, and TRAF1.
Figure Legend Snippet: Opposite roles of the JNK and NF-κB pathways on endogenous LMP1 expression at the mRNA and protein levels. Inhibition of (A) NF-κB activity upregulates, whereas inhibition of (B) JNK activity downregulates the endogenous LMP1 protein levels in PRI and TE1 cell lines. The cells were treated with vehicle (DMSO), sulfasalazine (1 mM and 5 mM for TE1 and PRI cells, respectively), or SP600125 (20 μM) for 16 h. Equal amounts of cells were harvested and directly lysed in 3× Laemmli buffer. Protein lysates were analyzed by Western blotting for the expression of LMP1, IκBα, and EBNA2. Equal loading of proteins in each lane was confirmed by probing the membrane with anti-β-actin antibody. (C) Sulfasalazine increases, whereas SP600125 decreases the LMP1 mRNA levels in PRI cells. PRI cells were treated with either vehicle (DMSO) or sulfasalazine (5 mM) or SP600125 (20 μM) for 16 h. Cells were then harvested, and mRNAs were extracted and subjected to real-time RT-PCR as described in Materials and Methods. LMP1 mRNA levels were normalized to the mRNA levels of three housekeeping genes (β-actin, glyceraldehyde-3-phosphate dehydrogenase, and hypoxanthine phosphoribosyltransferase), and the normalized levels in the untreated condition were arbitrarily assigned a value of 100. (D) The constitutively active form of IκBα inhibits NF-κB target genes and increases the levels of LMP1 transcripts in PRI cells. Where indicated, the PRI-pRT1-IκBm stable cell line were treated with doxycycline (2 μg/ml) (+ Dox) to induce the expression of IκBm. After induction for 24 h, the relative LMP1 mRNA levels were quantified by real-time RT-PCR as described above. After induction for 48 h, protein lysates were collected as described above and analyzed by Western blotting for the expression of EBNA2, ICAM1, and TRAF1.

Techniques Used: Expressing, Inhibition, Activity Assay, Western Blot, Quantitative RT-PCR, Stable Transfection

37) Product Images from "Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1"

Article Title: Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1

Journal: The international journal of biochemistry & cell biology

doi: 10.1016/j.biocel.2012.10.002

Proposed mechanism of Egr-1 regulation by ethanol oxidation ADH-catalysis of ethanol oxidation produces acetaldehyde, which enhances Egr-1 gene transcription by activating Egr-1 promoter. Increased gene transcription enhances Egr-1 mRNA which precedes a rise in nuclear Egr-1 protein. CYP2E1 and ADH catalysis of ethanol oxidation generates reactive products and inhibits proteasome activity. Such inhibition stabilizes Egr-1 protein, from hydrolysis by the proteasome. Finally, Egr-1 regulates SREBP1c and TNF-α to initiate ethanol induced steatosis
Figure Legend Snippet: Proposed mechanism of Egr-1 regulation by ethanol oxidation ADH-catalysis of ethanol oxidation produces acetaldehyde, which enhances Egr-1 gene transcription by activating Egr-1 promoter. Increased gene transcription enhances Egr-1 mRNA which precedes a rise in nuclear Egr-1 protein. CYP2E1 and ADH catalysis of ethanol oxidation generates reactive products and inhibits proteasome activity. Such inhibition stabilizes Egr-1 protein, from hydrolysis by the proteasome. Finally, Egr-1 regulates SREBP1c and TNF-α to initiate ethanol induced steatosis

Techniques Used: Activity Assay, Inhibition

38) Product Images from "Tyrosine phosphatase PTPα regulates focal adhesion remodeling through Rac1 activation"

Article Title: Tyrosine phosphatase PTPα regulates focal adhesion remodeling through Rac1 activation

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00359.2007

Supermature focal adhesions (FA) in protein tyrosine phosphatase-α (PTPα)-null (PTPα −/− ) cells associated with lack of remodeling. Wild-type PTPα (PTPα wt ) and PTPα −/− cells were plated on fibronectin (10 mg/ml) for 1, 3, and 16 h and then fixed with paraformaldehyde. A : FA visualized with anti-paxillin and anti-α-actinin antibodies. Arrow denotes enlarged and elongated (supermature) FA, characterized by inclusion of α-actinin. B and C : length of peripheral FA and cell area on paxillin-stained cells after 16 h of spreading. Values are means ± SD of 40–50 measurements in 3 independent experiments. * P
Figure Legend Snippet: Supermature focal adhesions (FA) in protein tyrosine phosphatase-α (PTPα)-null (PTPα −/− ) cells associated with lack of remodeling. Wild-type PTPα (PTPα wt ) and PTPα −/− cells were plated on fibronectin (10 mg/ml) for 1, 3, and 16 h and then fixed with paraformaldehyde. A : FA visualized with anti-paxillin and anti-α-actinin antibodies. Arrow denotes enlarged and elongated (supermature) FA, characterized by inclusion of α-actinin. B and C : length of peripheral FA and cell area on paxillin-stained cells after 16 h of spreading. Values are means ± SD of 40–50 measurements in 3 independent experiments. * P

Techniques Used: Staining

Transfection of PTPα rescues FA turnover in PTPα −/− cells. A : transient transfection of PTPα −/− cells with hemagglutinin (HA)-tagged PTPα constructs. Cells were plated on fibronectin for 1 h and then stained with anti-paxillin antibodies. Transfected cells were identified by staining with anti-HA antibodies using a specific Texas Red (TR) secondary antibody (not illustrated). Arrows indicate supermature FA. D1/ΔD2, PTPα lacking D2 domain; ΔD1/D2, PTPα lacking D1 domain; ΔD1/ΔD2, PTPα lacking D1 and D2 domains. B : transient transfection of PTPα −/− cells with HA-PTPα wt . FA were visualized by cotransfection with GFP-α-actinin. Fibroblasts were allowed to spread for 1 and 3 h. FA turnover in rescued PTPα −/− cells is reflected by formation of smaller and more widely distributed FA. C : cell area after 3 h of spreading. Values are means ± SD of 50 measurements in 3 independent experiments. * P
Figure Legend Snippet: Transfection of PTPα rescues FA turnover in PTPα −/− cells. A : transient transfection of PTPα −/− cells with hemagglutinin (HA)-tagged PTPα constructs. Cells were plated on fibronectin for 1 h and then stained with anti-paxillin antibodies. Transfected cells were identified by staining with anti-HA antibodies using a specific Texas Red (TR) secondary antibody (not illustrated). Arrows indicate supermature FA. D1/ΔD2, PTPα lacking D2 domain; ΔD1/D2, PTPα lacking D1 domain; ΔD1/ΔD2, PTPα lacking D1 and D2 domains. B : transient transfection of PTPα −/− cells with HA-PTPα wt . FA were visualized by cotransfection with GFP-α-actinin. Fibroblasts were allowed to spread for 1 and 3 h. FA turnover in rescued PTPα −/− cells is reflected by formation of smaller and more widely distributed FA. C : cell area after 3 h of spreading. Values are means ± SD of 50 measurements in 3 independent experiments. * P

Techniques Used: Transfection, Construct, Staining, Cotransfection

PTPα phosphatase activity is required for FA remodeling. A : genetically modified NIH-3T3 cells lines induced to express wild-type human PTPα (PTPα wt ind ) and double-mutant Cys 433 → Ser/Cys 723 → Ser PTPα (PTPα CCSS ind ). Cells were plated on fibronectin for 3 h. FA were observed by staining for paxillin ( A ) or by transfection with GFP-α-actinin ( F ). B–E : cell area, number of FA per cell, length of peripheral FA, and shape factor (length vs. width), which indicates degree of polarization, from images in A . Values are means ± SD of 30–40 measurements in 3 independent experiments. * P
Figure Legend Snippet: PTPα phosphatase activity is required for FA remodeling. A : genetically modified NIH-3T3 cells lines induced to express wild-type human PTPα (PTPα wt ind ) and double-mutant Cys 433 → Ser/Cys 723 → Ser PTPα (PTPα CCSS ind ). Cells were plated on fibronectin for 3 h. FA were observed by staining for paxillin ( A ) or by transfection with GFP-α-actinin ( F ). B–E : cell area, number of FA per cell, length of peripheral FA, and shape factor (length vs. width), which indicates degree of polarization, from images in A . Values are means ± SD of 30–40 measurements in 3 independent experiments. * P

Techniques Used: Activity Assay, Genetically Modified, Mutagenesis, Staining, Transfection

39) Product Images from "Activation of AP-1 Transcription Factors Differentiates FGF2 and Vascular Endothelial Growth Factor Regulation of Endothelial Nitric-oxide Synthase Expression in Placental Artery Endothelial Cells *"

Article Title: Activation of AP-1 Transcription Factors Differentiates FGF2 and Vascular Endothelial Growth Factor Regulation of Endothelial Nitric-oxide Synthase Expression in Placental Artery Endothelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.092791

FGF2, but not VEGF, increases JunB/Fra1 binding to the consensus AP-1 site. A , oFPAEC were treated with or without 10 ng/ml FGF2 ( F ) or VEGF ( V ) for 3 h, and nuclear extracts were prepared. Supershift assays for AP-1 subunit members using an oligonucleotide probe containing the sheep NOS3 consensus AP-1 element were performed as described under “Experimental Procedures.” Representative “supershift” EMSA images shown depict similar results from three independent experiments. B , oFPAEC were treated with or without 10 ng/ml FGF2 ( F ) or VEGF ( V ) for 3 h and then used for ChIP assays. The real-time PCR signals obtained for IP with specific JunB/Fra1 antibodies were estimated with respect to input DNA, and results are expressed as mean ± S.E. ( n = 3) of -fold changes over controls. *, p
Figure Legend Snippet: FGF2, but not VEGF, increases JunB/Fra1 binding to the consensus AP-1 site. A , oFPAEC were treated with or without 10 ng/ml FGF2 ( F ) or VEGF ( V ) for 3 h, and nuclear extracts were prepared. Supershift assays for AP-1 subunit members using an oligonucleotide probe containing the sheep NOS3 consensus AP-1 element were performed as described under “Experimental Procedures.” Representative “supershift” EMSA images shown depict similar results from three independent experiments. B , oFPAEC were treated with or without 10 ng/ml FGF2 ( F ) or VEGF ( V ) for 3 h and then used for ChIP assays. The real-time PCR signals obtained for IP with specific JunB/Fra1 antibodies were estimated with respect to input DNA, and results are expressed as mean ± S.E. ( n = 3) of -fold changes over controls. *, p

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

FGF2, but not VEGF, increases JunB/FRA1 binding to the CRE-like site. A , oFPAEC were treated with or without 10 ng/ml FGF2 ( F ) or VEGF ( V ) for 3 h. Supershift using an oligonucleotide probe containing the sheep NOS3 promoter CRE-like element was performed as described under “Experimental Procedures.” Representative supershift EMSA images shown depict similar results from three independent experiments for binding of members of the Jun/Fos families and of CREB and phospho-CREB to the CRE-like element. B , oFPAEC were treated with or without 10 ng/ml FGF2 or VEGF for 2 h and then used for ChIP assays. The real-time PCR signals obtained for IP with specific JunB/Fra1 antibodies were estimated with respect to input DNA, and results are expressed as mean ± S.E. ( error bars ) ( n = 3) of -fold changes over controls ( Ctl ). *, p
Figure Legend Snippet: FGF2, but not VEGF, increases JunB/FRA1 binding to the CRE-like site. A , oFPAEC were treated with or without 10 ng/ml FGF2 ( F ) or VEGF ( V ) for 3 h. Supershift using an oligonucleotide probe containing the sheep NOS3 promoter CRE-like element was performed as described under “Experimental Procedures.” Representative supershift EMSA images shown depict similar results from three independent experiments for binding of members of the Jun/Fos families and of CREB and phospho-CREB to the CRE-like element. B , oFPAEC were treated with or without 10 ng/ml FGF2 or VEGF for 2 h and then used for ChIP assays. The real-time PCR signals obtained for IP with specific JunB/Fra1 antibodies were estimated with respect to input DNA, and results are expressed as mean ± S.E. ( error bars ) ( n = 3) of -fold changes over controls ( Ctl ). *, p

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, CTL Assay

FGF2-increased NOS3 up-regulation requires JunB and Fra1 but not c-Jun. A , cells were infected with or without empty, antisense ( AS ), or sense ( S ) JunB adenoviruses for 18 h. After recovery in complete culture medium, cells were starved in the presence or absence of FGF2 (10 ng/ml) for 24 h. B , cells were treated similarly to A but with sense and/or antisense c-Jun adenoviruses. C , cells were treated similarly to A but with sense and/or antisense Fra1-expressing cytomegalovirus constructs. For all treatments, total protein samples were analyzed for JunB, c-Jun, Fra1, NOS3, and β-actin levels. Representative blots shown depict a typical experiment for each panel. Bar graphs summarize data (means ± S.E. ( error bars )) of -fold control values from three independent experiments. Bars with different letters ( a versus b versus c ) differ significantly ( p
Figure Legend Snippet: FGF2-increased NOS3 up-regulation requires JunB and Fra1 but not c-Jun. A , cells were infected with or without empty, antisense ( AS ), or sense ( S ) JunB adenoviruses for 18 h. After recovery in complete culture medium, cells were starved in the presence or absence of FGF2 (10 ng/ml) for 24 h. B , cells were treated similarly to A but with sense and/or antisense c-Jun adenoviruses. C , cells were treated similarly to A but with sense and/or antisense Fra1-expressing cytomegalovirus constructs. For all treatments, total protein samples were analyzed for JunB, c-Jun, Fra1, NOS3, and β-actin levels. Representative blots shown depict a typical experiment for each panel. Bar graphs summarize data (means ± S.E. ( error bars )) of -fold control values from three independent experiments. Bars with different letters ( a versus b versus c ) differ significantly ( p

Techniques Used: Infection, Expressing, Construct

40) Product Images from "The Tyrosine 343 Residue of Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Is Important for Its Interaction with SHP1, a Cytoplasmic Tyrosine Phosphatase with Tumor Suppressor Functions *"

Article Title: The Tyrosine 343 Residue of Nucleophosmin (NPM)-Anaplastic Lymphoma Kinase (ALK) Is Important for Its Interaction with SHP1, a Cytoplasmic Tyrosine Phosphatase with Tumor Suppressor Functions *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.121988

The activation status of NPM-ALK is important for its binding to SHP1. Co-immunoprecipitation ( IP ) experiments using GP293 cells co-transfected with NPM-ALK (or its mutants) and SHP1 revealed binding of SHP1 to NPM-ALK ( lane 2 ), but not the enzymatically inactive NPM-ALK K210R mutant ( lane 3 ) or the NPM-ALK FFF mutant ( lane 4 ). Immunoblotting ( IB ) with anti-SHP1 revealed a relatively equal amount of immunoprecipitated SHP1 proteins. Negative control reactions (−) were performed by omitting the use of anti-SHP1 antibody. Cells co-transfected with SHP1 and an empty vector ( i.e. pcDNA3 ) were used as a negative control. Results shown are representative of three independent experiments.
Figure Legend Snippet: The activation status of NPM-ALK is important for its binding to SHP1. Co-immunoprecipitation ( IP ) experiments using GP293 cells co-transfected with NPM-ALK (or its mutants) and SHP1 revealed binding of SHP1 to NPM-ALK ( lane 2 ), but not the enzymatically inactive NPM-ALK K210R mutant ( lane 3 ) or the NPM-ALK FFF mutant ( lane 4 ). Immunoblotting ( IB ) with anti-SHP1 revealed a relatively equal amount of immunoprecipitated SHP1 proteins. Negative control reactions (−) were performed by omitting the use of anti-SHP1 antibody. Cells co-transfected with SHP1 and an empty vector ( i.e. pcDNA3 ) were used as a negative control. Results shown are representative of three independent experiments.

Techniques Used: Activation Assay, Binding Assay, Immunoprecipitation, Transfection, Mutagenesis, Field Flow Fractionation, Negative Control, Plasmid Preparation

Related Articles

Incubation:

Article Title: An allelic variant in the intergenic region between ERAP1 and ERAP2 correlates with an inverse expression of the two genes
Article Snippet: .. The membranes were incubated ON with mouse anti-ERAP1 mAb antibody (clone B-10, sc-271823 SantaCruz), mouse anti-ERAP2 mAb (clone 3F5, MAB 3830 R & D Systems) and mouse anti-β-Actin mAb (clone C4, sc-477778 SantaCruz). .. The membranes were washed twice in TBST, incubated with horseradish peroxidase–conjugated secondary Ab (Jackson Immunoresearch Laboratories, Inc. West Grove, PA) and revealed by ECL Western blotting detection system (Amersham).

other:

Article Title: The pro-metastasis effect of circANKS1B in breast cancer
Article Snippet: Antibodies and reagents The antibodies we used are as follows: anti-E-cadherin (Abcam # ab40772), anti-Vimentin (Abcam # ab92547), anti-Fibronectin (Proteintech # 15613–1-AP), anti-AGO2 (Abcam # ab57113), anti-USF1 (Santa Cruz # sc-390,027), anti-RNA polymerase II (Santa Cruz # sc-47,701), anti-TGF-β1 (Abcam # ab92486), anti-ESRP1 (Abcam # ab107278), anti-p-Smad2 (Cell Signaling Technology # 8828), anti-p-Smad3 (Cell Signaling Technology # 9520), anti-Smad2/3 (Cell Signaling Technology # 8685), anti-GAPDH (Proteintech # 10494–1-AP) and anti-β-actin (Cell Signaling Technology # 4970).

Article Title: An Aqueous Extract of Herbal Medicine ALWPs Enhances Cognitive Performance and Inhibits LPS-Induced Neuroinflammation via FAK/NF-κB Signaling Pathways
Article Snippet: Antibodies and Chemicals We used the following primary antibodies: mouse anti-β-actin (Cat No: sc-47778, Santa Cruz Biotechnology, Dallas, TX, United States), mouse anti-p-IκBα (B-9, Cat No: sc-8404, Santa Cruz Biotechnology), mouse anti-IκBα (Cat No: sc-1643, Santa Cruz Biotechnology), rabbit anti-NF-κB (P65, Cat No: sc-8008, Santa Cruz Biotechnology), mouse anti-PCNA (Cat No: sc-56, Santa Cruz Biotechnology), rat anti-mouse CD11b (M1/70, Cat No: ab8878, Abcam, Cambridge, MA, United States), rabbit anti-FAK (Cat No: 13009S, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-p-FAK (Tyr397, Cat No: 8556S, Cell Signaling Technology), rabbit anti-p-NF-κB (Ser536, Cat No: 3033L, Cell Signaling Technology), rabbit anti-ERK (Cat No: 9102S, Cell Signaling Technology), rabbit anti-p-ERK (Thr202/Tyr204, Cat No:9101S, Cell Signaling Technology), rabbit anti-JNK (Cat No: MBS8509129, MyBioSource, San Diego, CA, United States), rabbit anti-p-JNK (Thr183/Tyr185, Cat No: MBS8508944, MyBioSource), rabbit anti-P38 (Cat No: 9212S, Cell Signaling Technology), rabbit anti-p-P38 (Cat No: 9211S, Cell Signaling Technology), rabbit anti-TLR4 (Cat No: PA5-11597, Thermo Scientific, Waltham, MA, United States), and rabbit anti-TLR4 (Cat No: NB100-56566, Novus Biologicals, Littleton, CO, United States).

Article Title: ATR/Chk1 signaling induces autophagy through sumoylated RhoB-mediated lysosomal translocation of TSC2 after DNA damage
Article Snippet: Antibodies and chemical reagents Mouse anti-actin (1:2000, sc-47778), anti-Myc (1:2000, sc-40), anti-RhoB (1:1000, sc-8048), anti-GST (1:2000, sc-138), anti-UBC9 (1:2000, sc-271057), anti-LAMP1 (1:200, sc-20011), anti-tuberin (1:1000, sc-271314), rabbit anti-Chk1 (1:1000, sc-7898), and rabbit anti-RhoB (1:1000, sc-180) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse anti-HSP60 (1:200, H3524) and anti-FLAG (M2) (1:2000, F1804) antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA); rabbit anti-TSC2 (1:1000, #4308), anti-ULK1 (1:1000, #8054), anti-AMPKα (1:1000, #2532), anti-PIAS1 (1:1000, #3550), anti-p70S6 kinase (1:1000, #2708), anti-phospho-p70S6 kinase (1:1000, #9205), anti-phospho-ULK1 (1:1000, Ser757#14202), anti-phospho-AMPKα (1:1000, Thr172#2531), anti-HSP60 (1:1000, #4870), anti-phospho-Threonine (1:1000, #9381), anti-Chk2 (1:1000, #2662), anti-Phospho-Chk1 (1:1000, ser345, #2348), and anti-Phospho-Chk2 (1:1000, Thr68, #2661) antibodies were purchased from Cell Signaling Technology; mouse anti-SQSTM1/P62 (1:1000, ab56416) antibody was purchased from Abcam; rat anti-HA (1:2000, #11867431001) monoclonal antibody was purchased from Roche (Mannheim, Germany); rabbit anti-LC3B/MAP1LC3B (1:1000, NB100-2220) was purchased from Novus; mouse anti-phosphoserine (1:1000, #05-1000), mouse anti-phospho-Histone H2A.X (Ser139)(1:200, #05-636) were purchased from Millipore; rabbit anti-RhoB (1:1000, 14326-1-AP) and rabbit anti-ATR (1:1000, 19787-1-AP) were purchased from Proteintech; inhibitors for Chk1 (#681637) and Chk2 (#220485) were purchased from Calbiochem; inhibitors for ATM CP-466722(#11002) and ATR VE-821(#14731) were purchased from MedChemExpress; chloroquine, methyl methanesulphonate, guanidine hydrochloride, and urea were purchased from Sigma-Aldrich (St Louis, MO, USA); Doxorubicin (HY-15142) and Campathecin (HY-16560) were purchased from MedChemExpress.

Article Title: p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity
Article Snippet: Antibodies The following antibodies were used in the present study: anti-δ-catenin (#611537, BD Bioscience, San Jose, CA, USA), anti-GFP (#G1544, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-tubulin (#T9026, Sigma-Aldrich, St Louis, MO, USA), anti-lamin B (SC-6216, Santa Cruz Biotechnology), anti-ß-actin (Santa Cruz Biotechnology), anti-flag (Sigma-Aldrich), anti-PCAF (SC-13124, Santa Cruz Biotechnology), anti-E-cadherin (BD Bioscience), anti-acetylated-Lys (Cell Signaling, Beverly, MA, USA), anti-p120-δ-catenin (BD Bioscience), anti-autophagy-related protein 5 (Atg5) (Cell Signaling), anti-myc (Santa Cruz Biotechnology), and anti-cyclin-D1 (Calbiochem, San Diego, CA, USA).

Article Title: Supervillin promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma in hypoxia via activation of the RhoA/ROCK-ERK/p38 pathway
Article Snippet: Antibodies The primary antibodies described in this article include anti-supervillin (H340 [ ]), anti-ERK1/2 (#4695; Cell Signaling Technology; MA, USA), anti-p-ERK1/2 (#4370; Cell Signaling Technology), anti-p38 (#8690; Cell Signaling Technology), anti-p-p38 (#4511; Cell Signaling Technology), anti-c-Jun N-terminal kinase (JNK)1/2 (#9252; Cell Signaling Technology), anti-p-JNK1/2 (#4668; Cell Signaling Technology), anti-E-cadherin (#sc7870; Santa Cruz Biotechnology, Inc.; CA, USA), anti-Vimentin (#sc73258; Santa Cruz Biotechnology, Inc.; CA, USA), anti-Snail1 (#sc393172; Santa Cruz Biotechnology, Inc), anti-β-actin (#3700; Cell Signaling Technology), and anti-β-tubulin (#TA506805; Origene; China).

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    Santa Cruz Biotechnology mouse anti β actin mab
    ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 <t>β−Actin</t> copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value
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    PCAF-mediated δ-catenin acetylation promotes autophagic degradation of δ-catenin. ( A , B ) The acetyltransferase activity of PCAF is required for the downregulation of δ-catenin. However, proteasome inhibition does not suppress the effect of PCAF on downregulating δ -catenin. HEK293Tcells transfected with GFP-δ-catenin and Flag-PCAF ( A ) and Rv/δ cells transfected with Flag-PCAF ( B ) were treated with the proteasome inhibitor MG132 (10 μM) or the histone acetyltransferase inhibitor Garcinol (5 µM) or Baf A1 (100 nM) or transfected with shPCAF and incubated for 12 h, and then cell lysates were subjected to immunoblotting. ( C ) Autophagy inhibitors attenuate PCAF-mediated δ-catenin degradation. HEK293T cells were transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with the autophagy inhibitors chloroquine (CQ, 100 μM), bafilomycin A1 (BafA1, 100 nM), or 3-methyladenine (3-MA, 5 mM), and cell lysates were subjected to immunoblotting. ( D ) Autophagy inhibitors increase the stability of δ-catenin. HEK293T cells transfected with δ-catenin and treated with 0.2 µM TSA were treated with MG132 (10 μM) or Baf A1 (100 nM) or 3-MA (5 mM) and incubated for 12 h, and then treated with cycloheximide (CHX, 20 ng/ml) for indicated time (h), and then cell lysates were subjected to immunoblotting. α-Tubulin or <t>ß-actin</t> was used as a loading control. Relative δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. **p
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    Santa Cruz Biotechnology anti β actin antibody
    The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to <t>β-actin</t> in 0% FBS group was set to 1. *** p
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    mRNA and protein expression levels of CLDN1 and CLDN4 in immortalized keratinocyte HaCaT cells treated with ATRA. HaCaT cells were incubated with or without 1 µ M ATRA for 36 h. (A) In the cells, CLDN-4, TJP3 and JGB4 were upregulated, while CLDN1 was downregulated. (B) In mice, CLDN1 and FLG were downregulated, while CLDN4 and CLDN2 were upregulated. (C) CLDN1 and CLDN4 protein expression levels were determined by western blotting. <t>β-actin</t> was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments performed in triplicate (n=3). * P
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    ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 β−Actin copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value

    Journal: Scientific Reports

    Article Title: An allelic variant in the intergenic region between ERAP1 and ERAP2 correlates with an inverse expression of the two genes

    doi: 10.1038/s41598-018-28799-8

    Figure Lengend Snippet: ERAP2 but not ERAP1 mRNA is enhanced by NMD inhibition independently from the rs2248374 genotype. ( a ) ERAP1 and ERAP2 mRNA copy numbers normalized to 100000 β−Actin copies before and after emetine treatment for 7 h in samples stratified according to rs2248374 genotype. ( b ) ERAPs mRNA fold change (emetine/untreated ratio) of the respective mRNAs. The arrows indicate the presence of G at rs75862629. p value

    Article Snippet: The membranes were incubated ON with mouse anti-ERAP1 mAb antibody (clone B-10, sc-271823 SantaCruz), mouse anti-ERAP2 mAb (clone 3F5, MAB 3830 R & D Systems) and mouse anti-β-Actin mAb (clone C4, sc-477778 SantaCruz).

    Techniques: Inhibition

    PCAF-mediated δ-catenin acetylation promotes autophagic degradation of δ-catenin. ( A , B ) The acetyltransferase activity of PCAF is required for the downregulation of δ-catenin. However, proteasome inhibition does not suppress the effect of PCAF on downregulating δ -catenin. HEK293Tcells transfected with GFP-δ-catenin and Flag-PCAF ( A ) and Rv/δ cells transfected with Flag-PCAF ( B ) were treated with the proteasome inhibitor MG132 (10 μM) or the histone acetyltransferase inhibitor Garcinol (5 µM) or Baf A1 (100 nM) or transfected with shPCAF and incubated for 12 h, and then cell lysates were subjected to immunoblotting. ( C ) Autophagy inhibitors attenuate PCAF-mediated δ-catenin degradation. HEK293T cells were transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with the autophagy inhibitors chloroquine (CQ, 100 μM), bafilomycin A1 (BafA1, 100 nM), or 3-methyladenine (3-MA, 5 mM), and cell lysates were subjected to immunoblotting. ( D ) Autophagy inhibitors increase the stability of δ-catenin. HEK293T cells transfected with δ-catenin and treated with 0.2 µM TSA were treated with MG132 (10 μM) or Baf A1 (100 nM) or 3-MA (5 mM) and incubated for 12 h, and then treated with cycloheximide (CHX, 20 ng/ml) for indicated time (h), and then cell lysates were subjected to immunoblotting. α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. **p

    Journal: Scientific Reports

    Article Title: p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity

    doi: 10.1038/s41598-019-40238-w

    Figure Lengend Snippet: PCAF-mediated δ-catenin acetylation promotes autophagic degradation of δ-catenin. ( A , B ) The acetyltransferase activity of PCAF is required for the downregulation of δ-catenin. However, proteasome inhibition does not suppress the effect of PCAF on downregulating δ -catenin. HEK293Tcells transfected with GFP-δ-catenin and Flag-PCAF ( A ) and Rv/δ cells transfected with Flag-PCAF ( B ) were treated with the proteasome inhibitor MG132 (10 μM) or the histone acetyltransferase inhibitor Garcinol (5 µM) or Baf A1 (100 nM) or transfected with shPCAF and incubated for 12 h, and then cell lysates were subjected to immunoblotting. ( C ) Autophagy inhibitors attenuate PCAF-mediated δ-catenin degradation. HEK293T cells were transfected with the indicated plasmids. At 12 h post-transfection, cells were treated with the autophagy inhibitors chloroquine (CQ, 100 μM), bafilomycin A1 (BafA1, 100 nM), or 3-methyladenine (3-MA, 5 mM), and cell lysates were subjected to immunoblotting. ( D ) Autophagy inhibitors increase the stability of δ-catenin. HEK293T cells transfected with δ-catenin and treated with 0.2 µM TSA were treated with MG132 (10 μM) or Baf A1 (100 nM) or 3-MA (5 mM) and incubated for 12 h, and then treated with cycloheximide (CHX, 20 ng/ml) for indicated time (h), and then cell lysates were subjected to immunoblotting. α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. **p

    Article Snippet: Antibodies The following antibodies were used in the present study: anti-δ-catenin (#611537, BD Bioscience, San Jose, CA, USA), anti-GFP (#G1544, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-tubulin (#T9026, Sigma-Aldrich, St Louis, MO, USA), anti-lamin B (SC-6216, Santa Cruz Biotechnology), anti-ß-actin (Santa Cruz Biotechnology), anti-flag (Sigma-Aldrich), anti-PCAF (SC-13124, Santa Cruz Biotechnology), anti-E-cadherin (BD Bioscience), anti-acetylated-Lys (Cell Signaling, Beverly, MA, USA), anti-p120-δ-catenin (BD Bioscience), anti-autophagy-related protein 5 (Atg5) (Cell Signaling), anti-myc (Santa Cruz Biotechnology), and anti-cyclin-D1 (Calbiochem, San Diego, CA, USA).

    Techniques: Activity Assay, Inhibition, Transfection, Incubation

    Multiple lysine residues in the N-terminus are responsible for PCAF-mediated δ-catenin downregulation. ( A ) Schematic representation of the triple arginine mutation at Lys360, Lys371, and Lys428 (FL KR), and the deletion/arginine mutation constructs of δ-catenin 1–499, 1–499 KR, 85–499, 85–499 KR, 1–499∆N KR, and 325–499 KR. ( B ) Deletion mutants 85–499 KR and 325–499 KR of δ-catenin were not affected by PCAF. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibody. ( C ) 3-MA restored the downregulated FL KR, 85–499, and 1–499∆N KR mutants of δ-catenin except the 85–499 KR mutation. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs and incubated with 3-MA (1 mM) for 24 h, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibodies. ( D ) PCAF did not acetylate δ-catenin 85–499 KR mutation. HEK293T cells were transfected with full length GFP-δ-catenin or 85–499 KR mutant together with or without Flag-PCAF, and each cell lysates were subjected to immunoprecipitation with anti-acetylated-lysine, followed by immunoblotting of precipitated proteins. α-Tubulin or ß-actin was used as a loading control. Relative values of δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. *p

    Journal: Scientific Reports

    Article Title: p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity

    doi: 10.1038/s41598-019-40238-w

    Figure Lengend Snippet: Multiple lysine residues in the N-terminus are responsible for PCAF-mediated δ-catenin downregulation. ( A ) Schematic representation of the triple arginine mutation at Lys360, Lys371, and Lys428 (FL KR), and the deletion/arginine mutation constructs of δ-catenin 1–499, 1–499 KR, 85–499, 85–499 KR, 1–499∆N KR, and 325–499 KR. ( B ) Deletion mutants 85–499 KR and 325–499 KR of δ-catenin were not affected by PCAF. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibody. ( C ) 3-MA restored the downregulated FL KR, 85–499, and 1–499∆N KR mutants of δ-catenin except the 85–499 KR mutation. HEK293T cells were transfected with the indicated plasmids expressing δ-catenin constructs and incubated with 3-MA (1 mM) for 24 h, and cell lysates were subjected to immunoblotting with anti-GFP and anti-Flag antibodies. ( D ) PCAF did not acetylate δ-catenin 85–499 KR mutation. HEK293T cells were transfected with full length GFP-δ-catenin or 85–499 KR mutant together with or without Flag-PCAF, and each cell lysates were subjected to immunoprecipitation with anti-acetylated-lysine, followed by immunoblotting of precipitated proteins. α-Tubulin or ß-actin was used as a loading control. Relative values of δ-catenin/actin ratios from at least three independent experiments are shown as a bar graph in each panel (ii). Values are presented as the mean ± SEM. *p

    Article Snippet: Antibodies The following antibodies were used in the present study: anti-δ-catenin (#611537, BD Bioscience, San Jose, CA, USA), anti-GFP (#G1544, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-tubulin (#T9026, Sigma-Aldrich, St Louis, MO, USA), anti-lamin B (SC-6216, Santa Cruz Biotechnology), anti-ß-actin (Santa Cruz Biotechnology), anti-flag (Sigma-Aldrich), anti-PCAF (SC-13124, Santa Cruz Biotechnology), anti-E-cadherin (BD Bioscience), anti-acetylated-Lys (Cell Signaling, Beverly, MA, USA), anti-p120-δ-catenin (BD Bioscience), anti-autophagy-related protein 5 (Atg5) (Cell Signaling), anti-myc (Santa Cruz Biotechnology), and anti-cyclin-D1 (Calbiochem, San Diego, CA, USA).

    Techniques: Mutagenesis, Construct, Transfection, Expressing, Incubation, Immunoprecipitation

    PCAF and HDACs regulate the acetylation status and levels of δ-catenin. ( A , B ) PCAF decreases δ-catenin levels. HEK293T ( A ) and δ-catenin-overexpressing CWR22Rv-1 (Rv/δ) ( B ) cells were transfected as indicated, and cell lysates were subjected to immunoblotting. ( C – F ) PCAF interacts with and acetylates δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoprecipitation with anti-δ-catenin ( C ), anti-Flag ( D ), or anti-acetylated-lysine ( E , F ), followed by immunoblotting of precipitated proteins. ( G – I ) HDACs deacetylate and upregulate δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoblotting ( G ) or immunoprecipitation with anti-acetylated-lysine ( H ). At 12 h post-transfection of HEK293T cells with GFP-δ-catenin and HDAC1, cells were treated with 0.2 µM trichostatin A (TSA) or transfected with Flag-PCAF, and incubated for 24 h, followed by immunoblotting with anti-δ-catenin and anti-Flag antibodies ( I ). α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin/HDACs ratios from three different experimental results are shown as a bar graph ( G ). Values are presented as the mean ± SEM. “−”, Mock transfection or vehicle treatment.

    Journal: Scientific Reports

    Article Title: p300/CBP-associated factor promotes autophagic degradation of δ-catenin through acetylation and decreases prostate cancer tumorigenicity

    doi: 10.1038/s41598-019-40238-w

    Figure Lengend Snippet: PCAF and HDACs regulate the acetylation status and levels of δ-catenin. ( A , B ) PCAF decreases δ-catenin levels. HEK293T ( A ) and δ-catenin-overexpressing CWR22Rv-1 (Rv/δ) ( B ) cells were transfected as indicated, and cell lysates were subjected to immunoblotting. ( C – F ) PCAF interacts with and acetylates δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoprecipitation with anti-δ-catenin ( C ), anti-Flag ( D ), or anti-acetylated-lysine ( E , F ), followed by immunoblotting of precipitated proteins. ( G – I ) HDACs deacetylate and upregulate δ-catenin. HEK293T cells were transfected as indicated, and cell lysates were subjected to immunoblotting ( G ) or immunoprecipitation with anti-acetylated-lysine ( H ). At 12 h post-transfection of HEK293T cells with GFP-δ-catenin and HDAC1, cells were treated with 0.2 µM trichostatin A (TSA) or transfected with Flag-PCAF, and incubated for 24 h, followed by immunoblotting with anti-δ-catenin and anti-Flag antibodies ( I ). α-Tubulin or ß-actin was used as a loading control. Relative δ-catenin/actin/HDACs ratios from three different experimental results are shown as a bar graph ( G ). Values are presented as the mean ± SEM. “−”, Mock transfection or vehicle treatment.

    Article Snippet: Antibodies The following antibodies were used in the present study: anti-δ-catenin (#611537, BD Bioscience, San Jose, CA, USA), anti-GFP (#G1544, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-tubulin (#T9026, Sigma-Aldrich, St Louis, MO, USA), anti-lamin B (SC-6216, Santa Cruz Biotechnology), anti-ß-actin (Santa Cruz Biotechnology), anti-flag (Sigma-Aldrich), anti-PCAF (SC-13124, Santa Cruz Biotechnology), anti-E-cadherin (BD Bioscience), anti-acetylated-Lys (Cell Signaling, Beverly, MA, USA), anti-p120-δ-catenin (BD Bioscience), anti-autophagy-related protein 5 (Atg5) (Cell Signaling), anti-myc (Santa Cruz Biotechnology), and anti-cyclin-D1 (Calbiochem, San Diego, CA, USA).

    Techniques: Transfection, Immunoprecipitation, Incubation

    The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Combination of Berberine with Resveratrol Improves the Lipid-Lowering Efficacy

    doi: 10.3390/ijms19123903

    Figure Lengend Snippet: The combination of berberine with resveratrol increased LDLR expression in HepG2 cells. Cells were cultured in 6-well plate for 24 h with a 3 × 10 5 cell density, and then culture medium were replaced by fresh medium containing different concentration of FBS indicated in A, or 1% FBS and drugs indicated in B for another 24 h followed by extraction of the total proteins from the cells. A : the effect of different concentration FBS on LDLR expression were analyzed by western blot assay. Then the band intensity was quantified by grey scanning analysis, and the intensity ratio of LDLR to β-actin in 0% FBS group was set to 1. *** p

    Article Snippet: Goat antibodies directed against LDLR (C-7, sc-18823), HRP-labeled anti-goat IgG (sc-2354) and anti-β-actin antibody (sc-8432) were from Santa Cruz Biotechnology (Heidelberg, German).

    Techniques: Expressing, Cell Culture, Concentration Assay, Western Blot

    mRNA and protein expression levels of CLDN1 and CLDN4 in immortalized keratinocyte HaCaT cells treated with ATRA. HaCaT cells were incubated with or without 1 µ M ATRA for 36 h. (A) In the cells, CLDN-4, TJP3 and JGB4 were upregulated, while CLDN1 was downregulated. (B) In mice, CLDN1 and FLG were downregulated, while CLDN4 and CLDN2 were upregulated. (C) CLDN1 and CLDN4 protein expression levels were determined by western blotting. β-actin was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments performed in triplicate (n=3). * P

    Journal: International Journal of Molecular Medicine

    Article Title: All-trans retinoic acid alters the expression of the tight junction proteins Claudin-1 and -4 and epidermal barrier function-associated genes in the epidermis

    doi: 10.3892/ijmm.2019.4098

    Figure Lengend Snippet: mRNA and protein expression levels of CLDN1 and CLDN4 in immortalized keratinocyte HaCaT cells treated with ATRA. HaCaT cells were incubated with or without 1 µ M ATRA for 36 h. (A) In the cells, CLDN-4, TJP3 and JGB4 were upregulated, while CLDN1 was downregulated. (B) In mice, CLDN1 and FLG were downregulated, while CLDN4 and CLDN2 were upregulated. (C) CLDN1 and CLDN4 protein expression levels were determined by western blotting. β-actin was used as a loading control. Data are presented as the mean ± standard deviation from three independent experiments performed in triplicate (n=3). * P

    Article Snippet: TBS buffer containing Tween-20 (TBST) and 5% non-fat milk was used to block non-specific binding at room temperature for 2 h. Following blocking, primary antibodies were incubated overnight at 4°C: Rabbit anti-human Claudin-1 (cat. no. 13050-1-AP; ProteinTech Group, Inc., Chicago, IL, USA; 1:200), goat anti-human Claudin-4 (cat. no. 17664; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; 1:200) and mouse anti-human β-actin (cat. no. 47778; Santa Cruz Biotechnology, Inc.; 1:1,000).

    Techniques: Expressing, Incubation, Mouse Assay, Western Blot, Standard Deviation