vk2 e6e7 cells  (Millipore)


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    Structured Review

    Millipore vk2 e6e7 cells
    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and <t>VK2/E6E7</t> cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Vk2 E6e7 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 4288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    vk2 e6e7 cells - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance"

    Article Title: Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171084

    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Figure Legend Snippet: Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).

    Techniques Used: Expressing, Staining

    Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells. Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).
    Figure Legend Snippet: Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells. Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).

    Techniques Used:

    Immunofluorescence localization of cytokeratin-13. Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).
    Figure Legend Snippet: Immunofluorescence localization of cytokeratin-13. Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).

    Techniques Used: Immunofluorescence, Staining

    TLR4 expression in hPVECs and VK2/E6E7 cells. (A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene Gapdh (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of TLR4 mRNA reported in Fig 10A. The expression of TLR4 mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.
    Figure Legend Snippet: TLR4 expression in hPVECs and VK2/E6E7 cells. (A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene Gapdh (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of TLR4 mRNA reported in Fig 10A. The expression of TLR4 mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.

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

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.

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

    Immunofluorescence localization of vimentin. hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).
    Figure Legend Snippet: Immunofluorescence localization of vimentin. hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).

    Techniques Used: Immunofluorescence, Positive Control, Staining

    Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p
    Figure Legend Snippet: Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .
    Figure Legend Snippet: RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.
    Figure Legend Snippet: Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.

    Techniques Used: Western Blot, Biomarker Assay, Expressing, Positive Control

    RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.
    Figure Legend Snippet: RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.

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

    Cytokoine ELISA of hPVECs and VK2/E6E7 cells. IL-6 (A) and IL-10 (B) in the culture supernatants of un-stimulated and LPS stimulated hPVECs and VK2/E6E7 cells at indicated time points (6, 12, 24 hrs). Cells were seeded at a density of 10 6 /well in 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Levels of IL-6 were up-regulated in LPS-induced cells. Values represent the mean ± SD of three independent experiments performed on different days. Level of significance (*p
    Figure Legend Snippet: Cytokoine ELISA of hPVECs and VK2/E6E7 cells. IL-6 (A) and IL-10 (B) in the culture supernatants of un-stimulated and LPS stimulated hPVECs and VK2/E6E7 cells at indicated time points (6, 12, 24 hrs). Cells were seeded at a density of 10 6 /well in 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Levels of IL-6 were up-regulated in LPS-induced cells. Values represent the mean ± SD of three independent experiments performed on different days. Level of significance (*p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p
    Figure Legend Snippet: qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    2) Product Images from "LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118"

    Article Title: LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118

    Journal: Oncotarget

    doi:

    LDL down-regulates HIF-1α, HIF-2α, and HIF-1β in hCMEC/D3 cells in both hypoxia and normoxia A. hCMEC/D3 cells were exposed to the indicated concentrations of native LDL (25–100 μg/ml) under hypoxic (1% O 2 ) condition for 72 hr, after which Western blot analysis was performed to monitor protein levels of HIF-1α and HIF-2α. B–D. hCMEC/D3 cells were exposed to the indicated concentrations of LDL for 24 (B), 48 (C), and 72 hr (D) under normoxic (21% O 2 ) condition, after which Western blot analysis was performed to monitor protein levels of HIF-1β. E. hCMEC/D3 cells were treated with 1 μM DMOG for 4 hr, followed by 50–100 μg/ml LDL for additional 72 hr, after which expression of HIF-1α, HIF-2α, and HIF-1β in the cytoplasmic and nuclear fractions was assessed by Western blot analysis. Blots re-probed for β-actin and laminin A were used as loading controls for cytoplasmic and nuclear fractions, respectively. All blots were quantified densitometrically using ImageJ software. The relative protein abundance was calculated by comparing to either β-actin or Laminin A and expressed as fold increase over controls (without LDL treatment). Values for controls were arbitrarily set to 1.0. At least three independent experiments ( n ≥ 3) were performed. * p
    Figure Legend Snippet: LDL down-regulates HIF-1α, HIF-2α, and HIF-1β in hCMEC/D3 cells in both hypoxia and normoxia A. hCMEC/D3 cells were exposed to the indicated concentrations of native LDL (25–100 μg/ml) under hypoxic (1% O 2 ) condition for 72 hr, after which Western blot analysis was performed to monitor protein levels of HIF-1α and HIF-2α. B–D. hCMEC/D3 cells were exposed to the indicated concentrations of LDL for 24 (B), 48 (C), and 72 hr (D) under normoxic (21% O 2 ) condition, after which Western blot analysis was performed to monitor protein levels of HIF-1β. E. hCMEC/D3 cells were treated with 1 μM DMOG for 4 hr, followed by 50–100 μg/ml LDL for additional 72 hr, after which expression of HIF-1α, HIF-2α, and HIF-1β in the cytoplasmic and nuclear fractions was assessed by Western blot analysis. Blots re-probed for β-actin and laminin A were used as loading controls for cytoplasmic and nuclear fractions, respectively. All blots were quantified densitometrically using ImageJ software. The relative protein abundance was calculated by comparing to either β-actin or Laminin A and expressed as fold increase over controls (without LDL treatment). Values for controls were arbitrarily set to 1.0. At least three independent experiments ( n ≥ 3) were performed. * p

    Techniques Used: Western Blot, Expressing, Software

    3) Product Images from "Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance"

    Article Title: Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171084

    Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).
    Figure Legend Snippet: Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Figure Legend Snippet: Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).

    Techniques Used: Expressing, Staining

    Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p
    Figure Legend Snippet: Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.
    Figure Legend Snippet: Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.

    Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Binding Assay, Mutagenesis

    4) Product Images from "Anti-ribosomal phosphoprotein autoantibody triggers interleukin-10 overproduction via phosphatidylinositol 3-kinase-dependent signalling pathways in lipopolysaccharide-activated macrophages"

    Article Title: Anti-ribosomal phosphoprotein autoantibody triggers interleukin-10 overproduction via phosphatidylinositol 3-kinase-dependent signalling pathways in lipopolysaccharide-activated macrophages

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02925.x

    The molecular model illustrates the augmentation of interleukin (IL)-10 expression by anti-ribosomal phosphoprotein monoclonal antibody (anti-P mAb). In macrophages, anti-P mAb (9B6) augments lipopolysaccharide (LPS)-induced IL-10 production via binding to the Fc γ receptor (FcγR) and then activates spleen tyrosine kinase (Syk) and phosphatidylinositol 3-kinase (PI3K). Subsequently, anti-P mAb activates Akt (PKB; protein kinase B), extracellular signal regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), while it decreases glycogen synthase kinase 3 (GSK3) and nuclear factor (NF)-κB activities. Increased activity of JNK and ERK, accompanied by decreased GSK3 activity, results in activation of cAMP-enhanced activation protein 1 (AP-1), serum response element (SRE) and cyclic AMP response element (CRE). These signalling effects contribute to the increase in IL-10 production. LBP, LPS-binding protein; ITAM, immunoreceptor tyrosine-based activation motif; PKC, protein kinase B.
    Figure Legend Snippet: The molecular model illustrates the augmentation of interleukin (IL)-10 expression by anti-ribosomal phosphoprotein monoclonal antibody (anti-P mAb). In macrophages, anti-P mAb (9B6) augments lipopolysaccharide (LPS)-induced IL-10 production via binding to the Fc γ receptor (FcγR) and then activates spleen tyrosine kinase (Syk) and phosphatidylinositol 3-kinase (PI3K). Subsequently, anti-P mAb activates Akt (PKB; protein kinase B), extracellular signal regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), while it decreases glycogen synthase kinase 3 (GSK3) and nuclear factor (NF)-κB activities. Increased activity of JNK and ERK, accompanied by decreased GSK3 activity, results in activation of cAMP-enhanced activation protein 1 (AP-1), serum response element (SRE) and cyclic AMP response element (CRE). These signalling effects contribute to the increase in IL-10 production. LBP, LPS-binding protein; ITAM, immunoreceptor tyrosine-based activation motif; PKC, protein kinase B.

    Techniques Used: Expressing, Binding Assay, Activity Assay, Activation Assay

    5) Product Images from "CXCR2-Driven Ovarian Cancer Progression Involves Upregulation of Proinflammatory Chemokines by Potentiating NF-?B Activation via EGFR-Transactivated Akt Signaling"

    Article Title: CXCR2-Driven Ovarian Cancer Progression Involves Upregulation of Proinflammatory Chemokines by Potentiating NF-?B Activation via EGFR-Transactivated Akt Signaling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0083789

    Confirmation of CXCR2-potentiated NF-κB signaling in OVCAR-3 cells. (A) CXCR2 protein expression in OVA versus OVCXCR2 cells. Western blot and immunofluorescent staining were carried out using antibodies specific to CXCR2 and β-actin as a loading control. (B) Comparison of growth rates in OVA and OVCXCR2 cells. Cells were incubated for 0, 24, 48 and 72 h and growth rates normalized to 0 h densities in each cell line. Experiments were performed in triplicate and all data are shown as mean ± S.E. * and ** (p≤0.05) in each group by ANOVA and Tukey’s pairwise comparisons. # (p≤0.05) between OVA and OVCXCR2 cells by Student’s t -test. (C) Effect of TNF (10 ng/ml) effects over time (0-120 min) on NF-κB activation in OVA and OVCXCR2 cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to IκB, IKK and their phosphorylated forms (pIκB and pIKK). β-actin was used as a loading control. (D) Comparison of EGFR activation in OVA and OVCXCR2 cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to EGFR, Akt, Erk and the phosphorylated forms (pEGFR, pAkt and pErk). The non-phosphorylated forms were used as loading controls. (E) Effects of EGFR downstream inhibitors on NF-κB luciferase activity in OVA and OVCXCR2 cells. After transfection with NF-κB luciferase vector overnight, cells were treated with vehicle (C), AG-1478 (AG, 2 µM), LY294002 (LY, 2 µM) or PD98059 (PD, 20 µM) for 4 h. (F) Effect of CXCL1/2/3 pan specific antibody for neutralization on cell proliferation in OVA and OVCXCR2 cells. Cells were incubated with normal IgG (C) and antibody (1∶100 dilution) for 48 h. The cell proliferation assay was performed using MTT and values were normalized to untreated controls. (G) Inhibitory effects of Bay11-7082 (2 µM) on CXCL1-induced cell invasion in OVA and OVCXCR2 cells. All experiments were performed at least in triplicate and data are shown as mean ± S.E. * and # (p≤0.05) as calculated by Student’s t -test.
    Figure Legend Snippet: Confirmation of CXCR2-potentiated NF-κB signaling in OVCAR-3 cells. (A) CXCR2 protein expression in OVA versus OVCXCR2 cells. Western blot and immunofluorescent staining were carried out using antibodies specific to CXCR2 and β-actin as a loading control. (B) Comparison of growth rates in OVA and OVCXCR2 cells. Cells were incubated for 0, 24, 48 and 72 h and growth rates normalized to 0 h densities in each cell line. Experiments were performed in triplicate and all data are shown as mean ± S.E. * and ** (p≤0.05) in each group by ANOVA and Tukey’s pairwise comparisons. # (p≤0.05) between OVA and OVCXCR2 cells by Student’s t -test. (C) Effect of TNF (10 ng/ml) effects over time (0-120 min) on NF-κB activation in OVA and OVCXCR2 cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to IκB, IKK and their phosphorylated forms (pIκB and pIKK). β-actin was used as a loading control. (D) Comparison of EGFR activation in OVA and OVCXCR2 cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to EGFR, Akt, Erk and the phosphorylated forms (pEGFR, pAkt and pErk). The non-phosphorylated forms were used as loading controls. (E) Effects of EGFR downstream inhibitors on NF-κB luciferase activity in OVA and OVCXCR2 cells. After transfection with NF-κB luciferase vector overnight, cells were treated with vehicle (C), AG-1478 (AG, 2 µM), LY294002 (LY, 2 µM) or PD98059 (PD, 20 µM) for 4 h. (F) Effect of CXCL1/2/3 pan specific antibody for neutralization on cell proliferation in OVA and OVCXCR2 cells. Cells were incubated with normal IgG (C) and antibody (1∶100 dilution) for 48 h. The cell proliferation assay was performed using MTT and values were normalized to untreated controls. (G) Inhibitory effects of Bay11-7082 (2 µM) on CXCL1-induced cell invasion in OVA and OVCXCR2 cells. All experiments were performed at least in triplicate and data are shown as mean ± S.E. * and # (p≤0.05) as calculated by Student’s t -test.

    Techniques Used: Expressing, Western Blot, Staining, Incubation, Activation Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Neutralization, Proliferation Assay, MTT Assay

    CXCR2 transactivates EGFR which contributes to NF-κB signaling via Akt activation. (A) Comparison of EGFR activation in SKA and SKCXCR2 cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to EGFR, Akt, Erk and the phosphorylated forms (pEGFR, pAkt and pErk). The non-phosphorylated forms were used as loading controls. (B) Representative immunofluorescent staining patterns indicating Akt activation and CXCR2 protein expression levels in SKA and SKCXCR2 cells. (C) Comparative effects of AG-1478, LY294002 and PD98059 on cell proliferation in SKA and SKCXCR2 cells. Cells were incubated with vehicle (Control), AG-1478 (AG, 2 µM), LY294002 (LY, 2 µM) or PD98059 (PD, 20 µM) for 48 h. The cell proliferation assay was performed using MTT and values were normalized to untreated controls. * and # (p≤0.05) when compared to Controls (C) and SKA cells, respectively, by Student’s t -test. (D) Dose-dependent effects of EGFR downstream inhibitors on NF-κB luciferase activities in SKA and SKCXCR2 cells. After transfection with NF-κB luciferase vector overnight, cells were treated with AG-1478 (EGFR inhibitor, 0, 0.5, 1 and 2 µM), LY294002 (Akt inhibitor, 0, 0.5, 1 and 2 µM) or PD98059 (Erk inhibitor, 0, 5, 10 and 20 µM) for 4 h. * and # (p≤0.05) when compared to Controls (0 h) and SKA cells, respectively, by Student’s t -test. All experiments were performed at least in triplicate and data are shown as mean ± S.E. (E) Confirmation of specific inhibitors on EGFR, Akt and Erk activation in SKA and SKCXCR2 cells. Cells were treated with AG-1478 (2 µM), LY294002 (2 µM) and PD98059 (20 µM) for 4 h. Whole cell lysates were prepared and a western blot was carried out using antibodies specific to EGFR, Akt, Erk and their phosphorylated forms (pEGFR, pAkt and pErk). Non-phosphorylated forms were used as loading controls.
    Figure Legend Snippet: CXCR2 transactivates EGFR which contributes to NF-κB signaling via Akt activation. (A) Comparison of EGFR activation in SKA and SKCXCR2 cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to EGFR, Akt, Erk and the phosphorylated forms (pEGFR, pAkt and pErk). The non-phosphorylated forms were used as loading controls. (B) Representative immunofluorescent staining patterns indicating Akt activation and CXCR2 protein expression levels in SKA and SKCXCR2 cells. (C) Comparative effects of AG-1478, LY294002 and PD98059 on cell proliferation in SKA and SKCXCR2 cells. Cells were incubated with vehicle (Control), AG-1478 (AG, 2 µM), LY294002 (LY, 2 µM) or PD98059 (PD, 20 µM) for 48 h. The cell proliferation assay was performed using MTT and values were normalized to untreated controls. * and # (p≤0.05) when compared to Controls (C) and SKA cells, respectively, by Student’s t -test. (D) Dose-dependent effects of EGFR downstream inhibitors on NF-κB luciferase activities in SKA and SKCXCR2 cells. After transfection with NF-κB luciferase vector overnight, cells were treated with AG-1478 (EGFR inhibitor, 0, 0.5, 1 and 2 µM), LY294002 (Akt inhibitor, 0, 0.5, 1 and 2 µM) or PD98059 (Erk inhibitor, 0, 5, 10 and 20 µM) for 4 h. * and # (p≤0.05) when compared to Controls (0 h) and SKA cells, respectively, by Student’s t -test. All experiments were performed at least in triplicate and data are shown as mean ± S.E. (E) Confirmation of specific inhibitors on EGFR, Akt and Erk activation in SKA and SKCXCR2 cells. Cells were treated with AG-1478 (2 µM), LY294002 (2 µM) and PD98059 (20 µM) for 4 h. Whole cell lysates were prepared and a western blot was carried out using antibodies specific to EGFR, Akt, Erk and their phosphorylated forms (pEGFR, pAkt and pErk). Non-phosphorylated forms were used as loading controls.

    Techniques Used: Activation Assay, Western Blot, Staining, Expressing, Incubation, Proliferation Assay, MTT Assay, Luciferase, Transfection, Plasmid Preparation

    Inhibitory effect of CXCR2 shRNA on CXCR2-deriven cancer progression in SKCXCR2 cells. (A) Knockdown of CXCR2 protein expression and comparison of EGFR activation after transfection of Control and CXCR2 shRNA in SKCXCR2 cells. After transfection with shRNAs for control and CXCR2 for 72 h, a Western blot was carried out using antibodies specific to CXCR2, EGFR, Akt, Erk and the phosphorylated forms (pEGFR, pAkt and pErk). The non-phosphorylated forms and β-actin were used as loading controls. (B) Comparison of growth rates in Control (black bars) and CXCR2 shRNA (gray bars) transfected SKCXCR2 cells. Cells were incubated for 0, 24 and 48 h and growth rates normalized to 0 h densities in each cell line. Experiments were performed in triplicate and all data are shown as mean ± S.E. * and ** (p≤0.05) in each group by ANOVA and Tukey’s pairwise comparisons. # (p≤0.05) between Control and CXCR2 shRNA transfected cells by Student’s t -test. (C) Cellular CXCL1 and CXCL2 concentrations in Control and CXCR2 shRNA transfected cells. Whole cell lysates were prepared, an ELISA carried out using antibodies specific to CXCL1 and CXCL2 and values normalized to total protein. (D) Effect of TNF (10 ng/ml) over time (0–120 min) on NF-κB activation in Control and CXCR2 shRNA transfected cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to IκB, IKK and their phosphorylated forms (pIκB and pIKK). β-actin was used as a loading control. (E) Effect of CXCL1/2/3 antibody on NF-κB luciferase activities in Control and CXCR2 shRNA transfected cells. After transfection of shRNA for 48 h followed by transfection of NF-κB luciferase vector overnight, cells were incubated with normal IgG and the CXCL1/2/3 antibody (Ab, 1∶100 dilution) for 4 h. (F) Effects of EGRF downstream inhibitors on NF-κB luciferase activity in Control and CXCR2 shRNA transfected cells. After transfection of shRNA for 48 h followed by transfection of NF-κB luciferase vector overnight, cells were treated with vehicle (C), AG-1478 (AG, 2 µM), LY294002 (LY, 2 µM) or PD98059 (PD, 20 µM) for 4 h. (G) Effect of CXCL1/2/3 antibody on cell proliferation in Control and CXCR2 shRNA transfected cells. Cells were incubated with normal IgG and CXCL1/2/3 antibody (Ab, 1∶100 dilution) for 48 h. The cell proliferation assay was performed using MTT and values were normalized to untreated controls. (H) Effect of CXCL1 on cell proliferation in Control and CXCR2 shRNA transfected cells for 48 h incubation. (I) Comparison of CXCL1-induced cell migration and invasion in Control and CXCR2 shRNA transfected cells. (J) Effect of TNF on NF-κB and mCXCL1 promoter luciferase activities in Control and CXCR2 shRNA transfected cells. After transfection of shRNA for 48 h followed by transfection of NF-κB or CXCL1 promoter luciferase vector overnight, cells were treated with TNF (10 ng/ml) for 4 h. All experiments were performed at least in triplicate and data are shown as mean ± S.E. * and # (p≤0.05) as calculated by Student’s t -test.
    Figure Legend Snippet: Inhibitory effect of CXCR2 shRNA on CXCR2-deriven cancer progression in SKCXCR2 cells. (A) Knockdown of CXCR2 protein expression and comparison of EGFR activation after transfection of Control and CXCR2 shRNA in SKCXCR2 cells. After transfection with shRNAs for control and CXCR2 for 72 h, a Western blot was carried out using antibodies specific to CXCR2, EGFR, Akt, Erk and the phosphorylated forms (pEGFR, pAkt and pErk). The non-phosphorylated forms and β-actin were used as loading controls. (B) Comparison of growth rates in Control (black bars) and CXCR2 shRNA (gray bars) transfected SKCXCR2 cells. Cells were incubated for 0, 24 and 48 h and growth rates normalized to 0 h densities in each cell line. Experiments were performed in triplicate and all data are shown as mean ± S.E. * and ** (p≤0.05) in each group by ANOVA and Tukey’s pairwise comparisons. # (p≤0.05) between Control and CXCR2 shRNA transfected cells by Student’s t -test. (C) Cellular CXCL1 and CXCL2 concentrations in Control and CXCR2 shRNA transfected cells. Whole cell lysates were prepared, an ELISA carried out using antibodies specific to CXCL1 and CXCL2 and values normalized to total protein. (D) Effect of TNF (10 ng/ml) over time (0–120 min) on NF-κB activation in Control and CXCR2 shRNA transfected cells. Whole cell lysates were prepared and Western blots carried out using antibodies specific to IκB, IKK and their phosphorylated forms (pIκB and pIKK). β-actin was used as a loading control. (E) Effect of CXCL1/2/3 antibody on NF-κB luciferase activities in Control and CXCR2 shRNA transfected cells. After transfection of shRNA for 48 h followed by transfection of NF-κB luciferase vector overnight, cells were incubated with normal IgG and the CXCL1/2/3 antibody (Ab, 1∶100 dilution) for 4 h. (F) Effects of EGRF downstream inhibitors on NF-κB luciferase activity in Control and CXCR2 shRNA transfected cells. After transfection of shRNA for 48 h followed by transfection of NF-κB luciferase vector overnight, cells were treated with vehicle (C), AG-1478 (AG, 2 µM), LY294002 (LY, 2 µM) or PD98059 (PD, 20 µM) for 4 h. (G) Effect of CXCL1/2/3 antibody on cell proliferation in Control and CXCR2 shRNA transfected cells. Cells were incubated with normal IgG and CXCL1/2/3 antibody (Ab, 1∶100 dilution) for 48 h. The cell proliferation assay was performed using MTT and values were normalized to untreated controls. (H) Effect of CXCL1 on cell proliferation in Control and CXCR2 shRNA transfected cells for 48 h incubation. (I) Comparison of CXCL1-induced cell migration and invasion in Control and CXCR2 shRNA transfected cells. (J) Effect of TNF on NF-κB and mCXCL1 promoter luciferase activities in Control and CXCR2 shRNA transfected cells. After transfection of shRNA for 48 h followed by transfection of NF-κB or CXCL1 promoter luciferase vector overnight, cells were treated with TNF (10 ng/ml) for 4 h. All experiments were performed at least in triplicate and data are shown as mean ± S.E. * and # (p≤0.05) as calculated by Student’s t -test.

    Techniques Used: shRNA, Expressing, Activation Assay, Transfection, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Luciferase, Plasmid Preparation, Activity Assay, Proliferation Assay, MTT Assay, Migration

    6) Product Images from "Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences"

    Article Title: Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01978

    Canonical and non-canonical NF-κB signaling in humans. Activation of the canonical NF-κB pathway is triggered by a broad range of proinflammatory cytokines such as TNFα or IL-1, bacterial pattern recognition molecules such as LPS, or antigen stimulation. Non-canonical signaling is triggered by TNF family receptors and their ligands, resulting in activation of NIK kinase activity. Both pathways cumulate in the activation of IKK (IκB-kinases) which phosphorylate inhibitory IκB binding partners for their poly-ubiquitination and proteosomal degradation (canonical axis) or the processing of p100 into its active form (non-canonical axis). Resulting NF-κB dimers translocate to the nucleus. Depending on their assembly into activating hetero- or repressive homo-dimeric conformations, NF-κB signaling regulates the expression of hundreds of target genes. TNF(R), tumor necrosis factor (receptor); IL-1(R), interleukin-1 (receptor); LPS, lipopolysaccharide; BAFF(-R), B-cell activating factor (receptor); LTβ(R), lymphotoxin β (receptor); TLR, toll-like receptor; TCR/BCR, T-cell/B-cell receptor; NIK, NF-κB inducing kinase; NEMO, NF-κB essential modulator; IKK, IκB kinase; IκB, Inhibitor of NF-κB; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.
    Figure Legend Snippet: Canonical and non-canonical NF-κB signaling in humans. Activation of the canonical NF-κB pathway is triggered by a broad range of proinflammatory cytokines such as TNFα or IL-1, bacterial pattern recognition molecules such as LPS, or antigen stimulation. Non-canonical signaling is triggered by TNF family receptors and their ligands, resulting in activation of NIK kinase activity. Both pathways cumulate in the activation of IKK (IκB-kinases) which phosphorylate inhibitory IκB binding partners for their poly-ubiquitination and proteosomal degradation (canonical axis) or the processing of p100 into its active form (non-canonical axis). Resulting NF-κB dimers translocate to the nucleus. Depending on their assembly into activating hetero- or repressive homo-dimeric conformations, NF-κB signaling regulates the expression of hundreds of target genes. TNF(R), tumor necrosis factor (receptor); IL-1(R), interleukin-1 (receptor); LPS, lipopolysaccharide; BAFF(-R), B-cell activating factor (receptor); LTβ(R), lymphotoxin β (receptor); TLR, toll-like receptor; TCR/BCR, T-cell/B-cell receptor; NIK, NF-κB inducing kinase; NEMO, NF-κB essential modulator; IKK, IκB kinase; IκB, Inhibitor of NF-κB; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.

    Techniques Used: Activation Assay, Activity Assay, Binding Assay, Expressing

    7) Product Images from "KIOM-79 Protects AGE-Induced Retinal Pericyte Apoptosis via Inhibition of NF-kappaB Activation In Vitro and In Vivo"

    Article Title: KIOM-79 Protects AGE-Induced Retinal Pericyte Apoptosis via Inhibition of NF-kappaB Activation In Vitro and In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043591

    Effect of KIOM-79 on IκB kinase (IKK) complex activation. KIOM-79, parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhized radix and Euphorbiae radix, and the IKK-2 inhibitor IV supplied by the manufacturer in this kit were tested for their ability to inhibit IKK-b activity using an ELISA-based kinase activity assay. Inhibition by a compound was defined by the 50% inhibition concentration (IC 50 ) of the IKK activity. IC 50 values were calculated from the dose inhibition curve. Values in the graphs represent means ± SE, n = 6.
    Figure Legend Snippet: Effect of KIOM-79 on IκB kinase (IKK) complex activation. KIOM-79, parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhized radix and Euphorbiae radix, and the IKK-2 inhibitor IV supplied by the manufacturer in this kit were tested for their ability to inhibit IKK-b activity using an ELISA-based kinase activity assay. Inhibition by a compound was defined by the 50% inhibition concentration (IC 50 ) of the IKK activity. IC 50 values were calculated from the dose inhibition curve. Values in the graphs represent means ± SE, n = 6.

    Techniques Used: Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Kinase Assay, Inhibition, Concentration Assay

    The effect of KIOM-79 on NF-κB activity in AGE-BSA-treated retinal pericytes. The pericytes were exposed for 6 hours to AGE-BSA (10 to 200 µg/mL) or BSA (A). The pericytes were pretreated with KIOM-79 or PDTC for 1 hour, followed by treatment with 100 µg/mL AGE-BSA for 6 hours (B–D). Apoptotic cells were detected using an FITC-labelled Annexin V protein and flow cytometry (A and B). Electrophoretic mobility shift assay for NF-κB (C). Western blot analysis was used to detect phospho-IκB-α and IκB-α (D). Each bar represents the mean ± SE from four independent experiments (*p
    Figure Legend Snippet: The effect of KIOM-79 on NF-κB activity in AGE-BSA-treated retinal pericytes. The pericytes were exposed for 6 hours to AGE-BSA (10 to 200 µg/mL) or BSA (A). The pericytes were pretreated with KIOM-79 or PDTC for 1 hour, followed by treatment with 100 µg/mL AGE-BSA for 6 hours (B–D). Apoptotic cells were detected using an FITC-labelled Annexin V protein and flow cytometry (A and B). Electrophoretic mobility shift assay for NF-κB (C). Western blot analysis was used to detect phospho-IκB-α and IκB-α (D). Each bar represents the mean ± SE from four independent experiments (*p

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Electrophoretic Mobility Shift Assay, Western Blot

    8) Product Images from "Estrogen receptor expression in chronic hepatitis C and hepatocellular carcinoma pathogenesis"

    Article Title: Estrogen receptor expression in chronic hepatitis C and hepatocellular carcinoma pathogenesis

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v23.i37.6802

    Expression of cyclin D1 in nuclear and cytoplasmic tissue lysates from normal and diseased subjects. A: Lysates prepared from nuclear and cytoplasmic fractions of liver tissues from normal, HCV and HCC subjects were subjected to Western blotting and probed with antibodies against cyclin D1, β-actin, histones (nuclear lysates only) and GAPDH (cytoplasmic lysates only). Representative blots for each group are depicted. B and C: The bands corresponding to cyclin D1 and β-actin were quantified by densitometric analyses using ImageJ. Expression of cyclin D1 was normalized to the expression of β-actin in nuclear (B) and cytoplasmic (C) lysates and plotted. a P
    Figure Legend Snippet: Expression of cyclin D1 in nuclear and cytoplasmic tissue lysates from normal and diseased subjects. A: Lysates prepared from nuclear and cytoplasmic fractions of liver tissues from normal, HCV and HCC subjects were subjected to Western blotting and probed with antibodies against cyclin D1, β-actin, histones (nuclear lysates only) and GAPDH (cytoplasmic lysates only). Representative blots for each group are depicted. B and C: The bands corresponding to cyclin D1 and β-actin were quantified by densitometric analyses using ImageJ. Expression of cyclin D1 was normalized to the expression of β-actin in nuclear (B) and cytoplasmic (C) lysates and plotted. a P

    Techniques Used: Expressing, Western Blot

    Expression of estrogen receptor subtypes in whole tissue lysates from normal male and female subjects. A: Whole tissue lysates from liver tissues of normal donors were subjected to Western blotting and probed with antibodies against ERα, ERβ and β-actin. A representative blot is shown. B-D: The bands corresponding to ERα, ERβ and β-actin were quantified by densitometric analyses using ImageJ. Each symbol represents one individual. Expression of the ER subtypes was normalized to the expression of β-actin and plotted (B). Gender-based ER subtype expression was evaluated by segregating the gender and plotted (C). The ERα:ERβ expression ratio was also plotted for each gender group (D). a P
    Figure Legend Snippet: Expression of estrogen receptor subtypes in whole tissue lysates from normal male and female subjects. A: Whole tissue lysates from liver tissues of normal donors were subjected to Western blotting and probed with antibodies against ERα, ERβ and β-actin. A representative blot is shown. B-D: The bands corresponding to ERα, ERβ and β-actin were quantified by densitometric analyses using ImageJ. Each symbol represents one individual. Expression of the ER subtypes was normalized to the expression of β-actin and plotted (B). Gender-based ER subtype expression was evaluated by segregating the gender and plotted (C). The ERα:ERβ expression ratio was also plotted for each gender group (D). a P

    Techniques Used: Expressing, Western Blot

    Expression of estrogen receptor subtypes in nuclear and cytoplasmic tissue lysates from normal and diseased subjects. A: Lysates prepared from nuclear and cytoplasmic fractions of liver tissues from normal, HCV and HCC subjects were subjected to Western blotting and probed with antibodies against ERα, ERβ, β-actin, histone (nuclear lysates only) and GAPDH (cytoplasmic lysates only). Representative blots for each group are depicted. B-G: The bands corresponding to ERα, ERβ and β-actin were quantified by densitometric analyses using ImageJ. Expression of the ERα and ERβ was normalized to the expression of β-actin and plotted with (B and C). The ERα:ERβ expression ratio was also plotted for normal, HCV and HCC group (D). Expression of ER subtypes and ERα:ERβ expression ratio in the male population of normal, HCV and HCC groups was plotted separately (E-G). a P
    Figure Legend Snippet: Expression of estrogen receptor subtypes in nuclear and cytoplasmic tissue lysates from normal and diseased subjects. A: Lysates prepared from nuclear and cytoplasmic fractions of liver tissues from normal, HCV and HCC subjects were subjected to Western blotting and probed with antibodies against ERα, ERβ, β-actin, histone (nuclear lysates only) and GAPDH (cytoplasmic lysates only). Representative blots for each group are depicted. B-G: The bands corresponding to ERα, ERβ and β-actin were quantified by densitometric analyses using ImageJ. Expression of the ERα and ERβ was normalized to the expression of β-actin and plotted with (B and C). The ERα:ERβ expression ratio was also plotted for normal, HCV and HCC group (D). Expression of ER subtypes and ERα:ERβ expression ratio in the male population of normal, HCV and HCC groups was plotted separately (E-G). a P

    Techniques Used: Expressing, Western Blot

    Expression of pNF-κB and pIKK in nuclear and cytoplasmic tissue lysates from normal and diseased subjects. A: Lysates prepared from nuclear and cytoplasmic fractions of liver tissues from normal, HCV and HCC subjects were subjected to Western blotting. Nuclear tissue lysates were probed with antibodies against pNF-κB, NF-κB, β-actin, and histones while cytoplasmic tissue lysates were probed with antibodies against pIKK, IKK, β-actin and GAPDH. Representative blots for each group are depicted. B and C: The bands corresponding to pNF-κB, NF-κB, pIKK, IKK and β-actin were quantified by densitometric analyses using ImageJ. The ratio of pNF-κB:NF-κB was normalized to the expression of β-actin and plotted (B). The ratio of pIKK:IKK was normalized to the expression of β-actin and plotted with gender pooled (C) subjects. a P
    Figure Legend Snippet: Expression of pNF-κB and pIKK in nuclear and cytoplasmic tissue lysates from normal and diseased subjects. A: Lysates prepared from nuclear and cytoplasmic fractions of liver tissues from normal, HCV and HCC subjects were subjected to Western blotting. Nuclear tissue lysates were probed with antibodies against pNF-κB, NF-κB, β-actin, and histones while cytoplasmic tissue lysates were probed with antibodies against pIKK, IKK, β-actin and GAPDH. Representative blots for each group are depicted. B and C: The bands corresponding to pNF-κB, NF-κB, pIKK, IKK and β-actin were quantified by densitometric analyses using ImageJ. The ratio of pNF-κB:NF-κB was normalized to the expression of β-actin and plotted (B). The ratio of pIKK:IKK was normalized to the expression of β-actin and plotted with gender pooled (C) subjects. a P

    Techniques Used: Expressing, Western Blot

    9) Product Images from "Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase Activation"

    Article Title: Polyenylpyrrole Derivatives Inhibit NLRP3 Inflammasome Activation and Inflammatory Mediator Expression by Reducing Reactive Oxygen Species Production and Mitogen-Activated Protein Kinase Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076754

    Effect of compound 1h on ROS production and MAPK phosphorylation in LPS-activated macrophages. In ( A ), RAW 264.7 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with compound 1h (20 µM), N-acetyl cysteine (NAC; 10 mM), or DMSO (vehicle), then 2’, 7’-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by LPS (1 µg/ml) stimulation for the indicated time, then ROS levels were measured by detection of the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In ( B ), RAW 264.7 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with compound 1h (20 µM) or DMSO, then LPS (1 µg/ml) was added and incubation continued for 0-60 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed by Western blotting and expressed relative to actin expression and as a fold increase compared to the control group at 0 time. In ( C ), J774A.1 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with 10-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 20 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed as in B. In ( A ), the data are expressed as the mean ± SD for three separate experiments, while, in ( B ) and ( C ), the results are representative of those obtained in three different experiments. * indicates a significant difference at the level of p
    Figure Legend Snippet: Effect of compound 1h on ROS production and MAPK phosphorylation in LPS-activated macrophages. In ( A ), RAW 264.7 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with compound 1h (20 µM), N-acetyl cysteine (NAC; 10 mM), or DMSO (vehicle), then 2’, 7’-dichlorofluorescein diacetate (2 µM) was added for 30 min, followed by LPS (1 µg/ml) stimulation for the indicated time, then ROS levels were measured by detection of the mean fluorescence intensity (MFI) of the fluorophore carboxyl-DCF and expressing this value relative to that at time zero. In ( B ), RAW 264.7 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with compound 1h (20 µM) or DMSO, then LPS (1 µg/ml) was added and incubation continued for 0-60 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed by Western blotting and expressed relative to actin expression and as a fold increase compared to the control group at 0 time. In ( C ), J774A.1 macrophages (5 × 10 5 /ml; 1 ml) were incubated for 30 min with 10-40 µM compound 1h or DMSO, then LPS (1 µg/ml) was added and incubation continued for 20 min, then phosphorylation of ERK1/2, JNK1/2, and p38 was analyzed as in B. In ( A ), the data are expressed as the mean ± SD for three separate experiments, while, in ( B ) and ( C ), the results are representative of those obtained in three different experiments. * indicates a significant difference at the level of p

    Techniques Used: Incubation, Fluorescence, Expressing, Western Blot

    10) Product Images from "Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene"

    Article Title: Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2018.12.016

    Effect of inhibition of NF-κB pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) expression in mouse enterocytes. A: NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. B: NF-κB inhibitor Bay-11 also prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or /LPS-treated mice. Densitometry of MLCK protein levels. C: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. n = 3 experiments. ∗∗ P
    Figure Legend Snippet: Effect of inhibition of NF-κB pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) expression in mouse enterocytes. A: NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. B: NF-κB inhibitor Bay-11 also prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or /LPS-treated mice. Densitometry of MLCK protein levels. C: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. n = 3 experiments. ∗∗ P

    Techniques Used: Inhibition, Activation Assay, Expressing, Mouse Assay

    Activation of p65/p50 canonical pathway by lipopolysaccharide (LPS) in mice enterocytes and effect of NF-κB inhibitors on LPS-induced increase in mouse intestinal epithelial tight junction permeability. A: LPS i.p. injections (0.1 mg/kg body weight) in mice caused activation of canonical p65/p50 pathway, as assessed by degradation of inhibitory κ B (IκB)-α protein expression on day 3 in mice enterocytes. Densitometry of IκB-α protein levels. B: The immunoblot analysis from LPS-treated mice enterocytes revealed significant increase in nuclear p65 protein expression on day 3 compared with untreated mice enterocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Lamin B were used as loading controls for cytoplasmic (cyto) and nuclear (nuc) fractions, respectively C: Confocal immunofluorescence of mouse intestines treated with LPS (0.1 mg/kg body weight) on day 3 indicated p65 (red) ( arrowheads ) translocation to the nucleus (blue) compared with control (C) mouse enterocytes. D: NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC; 10 mg/kg body weight), and Bay-11 (5 mg/kg body weight) pretreatment prevented the LPS-induced increase in 10K dextran flux. PDTC and Bay-11 were dissolved in dimethyl sulfoxide and injected 1 hour before LPS treatment. Data are expressed as means ± SEM. n = 4 experiments ( A and C ); n = 3 experiments ( B ). ∗∗ P
    Figure Legend Snippet: Activation of p65/p50 canonical pathway by lipopolysaccharide (LPS) in mice enterocytes and effect of NF-κB inhibitors on LPS-induced increase in mouse intestinal epithelial tight junction permeability. A: LPS i.p. injections (0.1 mg/kg body weight) in mice caused activation of canonical p65/p50 pathway, as assessed by degradation of inhibitory κ B (IκB)-α protein expression on day 3 in mice enterocytes. Densitometry of IκB-α protein levels. B: The immunoblot analysis from LPS-treated mice enterocytes revealed significant increase in nuclear p65 protein expression on day 3 compared with untreated mice enterocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Lamin B were used as loading controls for cytoplasmic (cyto) and nuclear (nuc) fractions, respectively C: Confocal immunofluorescence of mouse intestines treated with LPS (0.1 mg/kg body weight) on day 3 indicated p65 (red) ( arrowheads ) translocation to the nucleus (blue) compared with control (C) mouse enterocytes. D: NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC; 10 mg/kg body weight), and Bay-11 (5 mg/kg body weight) pretreatment prevented the LPS-induced increase in 10K dextran flux. PDTC and Bay-11 were dissolved in dimethyl sulfoxide and injected 1 hour before LPS treatment. Data are expressed as means ± SEM. n = 4 experiments ( A and C ); n = 3 experiments ( B ). ∗∗ P

    Techniques Used: Activation Assay, Mouse Assay, Permeability, Expressing, Immunofluorescence, Translocation Assay, Injection

    11) Product Images from "IKBKE inhibits TSC1 to activate the mTOR/S6K pathway for oncogenic transformation"

    Article Title: IKBKE inhibits TSC1 to activate the mTOR/S6K pathway for oncogenic transformation

    Journal: Turkish Journal of Biology

    doi: 10.3906/biy-1801-57

    S6K inactivation and TSC1 stabilization correlate upon IKBKE silencing. SW480 cells were silenced with IKKε siRNA (please refer to Göktuna et al., 2016 for depletion controls such as IKKε and α-tubulin western blots; data are obtained from a similar experimental setup) for 48 h and then they were treated with 10 ng/mL LPS for 0 to 5 h for the activation of mTOR/S6K and IKKε. According to WB results, only S6K (A) activation, not mTOR (B), was diminished in the absence of IKKε (as quantified by phospho- S6K/S6K densitometric band intensities) and S6K inactivation was inversely correlating with the stabilization of TSC1 (as quantified in (C)).
    Figure Legend Snippet: S6K inactivation and TSC1 stabilization correlate upon IKBKE silencing. SW480 cells were silenced with IKKε siRNA (please refer to Göktuna et al., 2016 for depletion controls such as IKKε and α-tubulin western blots; data are obtained from a similar experimental setup) for 48 h and then they were treated with 10 ng/mL LPS for 0 to 5 h for the activation of mTOR/S6K and IKKε. According to WB results, only S6K (A) activation, not mTOR (B), was diminished in the absence of IKKε (as quantified by phospho- S6K/S6K densitometric band intensities) and S6K inactivation was inversely correlating with the stabilization of TSC1 (as quantified in (C)).

    Techniques Used: Western Blot, Activation Assay

    S6K cannot be activated in the absence of IKKε. SW480 cells were first silenced with IKKε siRNA for 48 h and later treated with either no serum, 10 ng/mL LPS, or 100 ng/mL TNFα for an additional 24 h for the activation of the mTOR/S6K pathway. Upon IKKε silencing, p70-S6K phosphorylations were seriously diminished in all stress conditions relative to the control. A) WB results for IKKε, TBK1, phospho-S6K, and S6K proteins by the use of specific antibodies. B) Quantification of phospho-S6K/S6K band intensities in WB (for p-S6K quantifications only heavy isoform has been used since total antibody only recognizes this isoform).
    Figure Legend Snippet: S6K cannot be activated in the absence of IKKε. SW480 cells were first silenced with IKKε siRNA for 48 h and later treated with either no serum, 10 ng/mL LPS, or 100 ng/mL TNFα for an additional 24 h for the activation of the mTOR/S6K pathway. Upon IKKε silencing, p70-S6K phosphorylations were seriously diminished in all stress conditions relative to the control. A) WB results for IKKε, TBK1, phospho-S6K, and S6K proteins by the use of specific antibodies. B) Quantification of phospho-S6K/S6K band intensities in WB (for p-S6K quantifications only heavy isoform has been used since total antibody only recognizes this isoform).

    Techniques Used: Activation Assay, Western Blot

    12) Product Images from "Troglitazone Inhibits Matrix Metalloproteinase-9 Expression and Invasion of Breast Cancer Cell through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism"

    Article Title: Troglitazone Inhibits Matrix Metalloproteinase-9 Expression and Invasion of Breast Cancer Cell through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism

    Journal: Journal of Breast Cancer

    doi: 10.4048/jbc.2018.21.1.28

    Troglitazone inhibits 12- O -tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-9 (MMP-9) expression in MCF-7 cells. MCF-7 cells were pretreated with troglitazone and then TPA was added for 24 hours. (A) MMP-9 secretion by gelatin zymography (top). MMP-9 protein expression was determined by western blot. β-Actin was the internal control (bottom). (B) MMP-9 mRNA was analyzed by real-time polymerase chain reaction with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. (C) Wild type MMP-9-luc reporters and a Renilla luciferase thymidine kinase reporter vector were co-transfected into MCF-7 cells. Cells were treated with troglitazone and TPA and MMP-9 promoter activity was measured with dual-luciferase reporter assays. (D) Peroxisome proliferator-activated receptor γ antagonist GW9662 was added to cells for 30 minutes before troglitazone treatment. Lysates were analyzed by western blot with anti-MMP-9. β-Actin was the loading control. Values are mean±standard error of the mean of three independent experiments. * p
    Figure Legend Snippet: Troglitazone inhibits 12- O -tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-9 (MMP-9) expression in MCF-7 cells. MCF-7 cells were pretreated with troglitazone and then TPA was added for 24 hours. (A) MMP-9 secretion by gelatin zymography (top). MMP-9 protein expression was determined by western blot. β-Actin was the internal control (bottom). (B) MMP-9 mRNA was analyzed by real-time polymerase chain reaction with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. (C) Wild type MMP-9-luc reporters and a Renilla luciferase thymidine kinase reporter vector were co-transfected into MCF-7 cells. Cells were treated with troglitazone and TPA and MMP-9 promoter activity was measured with dual-luciferase reporter assays. (D) Peroxisome proliferator-activated receptor γ antagonist GW9662 was added to cells for 30 minutes before troglitazone treatment. Lysates were analyzed by western blot with anti-MMP-9. β-Actin was the loading control. Values are mean±standard error of the mean of three independent experiments. * p

    Techniques Used: Expressing, Zymography, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Plasmid Preparation, Transfection, Activity Assay

    13) Product Images from "Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1"

    Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01865

    p38 MAPK signaling pathway is common to IL-17A- and TGF-β-induced IL-6 production. Healthy donors fibroblasts were treated with the indicated concentrations of SB203580 (A) or 20 µM SB203580 (B) for 1 h prior to the addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml) in triplicates. After 48 h, culture SNs were collected and IL-6 levels were assessed by ELISA. (A) Results are shown as fold change to untreated cells, mean + SEM is indicated, N = 3. Please note the log 2 scale. (B) Results are shown as the percentage of IL-6 production induced by IL-17A and/or TGF- β in the absence of inhibitor (levels of IL-6 were: 14.7 ± 7.4 pg/ml for IL-17A, 11.4 ± 6.1 pg/ml for TGF-β, and 48.9 ± 13.7 pg/ml for IL-17A + TGF-β). Bars represent the mean + SEM of three experiments. Significant differences versus control were assessed by paired t -test.
    Figure Legend Snippet: p38 MAPK signaling pathway is common to IL-17A- and TGF-β-induced IL-6 production. Healthy donors fibroblasts were treated with the indicated concentrations of SB203580 (A) or 20 µM SB203580 (B) for 1 h prior to the addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml) in triplicates. After 48 h, culture SNs were collected and IL-6 levels were assessed by ELISA. (A) Results are shown as fold change to untreated cells, mean + SEM is indicated, N = 3. Please note the log 2 scale. (B) Results are shown as the percentage of IL-6 production induced by IL-17A and/or TGF- β in the absence of inhibitor (levels of IL-6 were: 14.7 ± 7.4 pg/ml for IL-17A, 11.4 ± 6.1 pg/ml for TGF-β, and 48.9 ± 13.7 pg/ml for IL-17A + TGF-β). Bars represent the mean + SEM of three experiments. Significant differences versus control were assessed by paired t -test.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Phosphorylation of MAPK p38 is enhanced by the combined action of IL-17A and TGF-β. (A) Western blot (WB) of healthy donors fibroblasts treated with 1 µM TPCA-1 and/or 10 µM Ly294002 for 1 h prior to addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml) and cultured for an additional 10 min. Results are representative of three experiments with inhibitors and two additional experiments with cytokines only. (B) Quantification of Western blot (WB) analysis was performed with ImageJ software ( http://rsbweb.nih.gov/ij ) and values were normalized to β-actin, N = 5. Results are shown as fold change to IL-17A-treated cells (for p-p38, p-IκBα, and p-NF-κB p65) or to untreated cells (for p-Akt), N = 5. Significance assessed by paired t test.
    Figure Legend Snippet: Phosphorylation of MAPK p38 is enhanced by the combined action of IL-17A and TGF-β. (A) Western blot (WB) of healthy donors fibroblasts treated with 1 µM TPCA-1 and/or 10 µM Ly294002 for 1 h prior to addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml) and cultured for an additional 10 min. Results are representative of three experiments with inhibitors and two additional experiments with cytokines only. (B) Quantification of Western blot (WB) analysis was performed with ImageJ software ( http://rsbweb.nih.gov/ij ) and values were normalized to β-actin, N = 5. Results are shown as fold change to IL-17A-treated cells (for p-p38, p-IκBα, and p-NF-κB p65) or to untreated cells (for p-Akt), N = 5. Significance assessed by paired t test.

    Techniques Used: Western Blot, Cell Culture, Software

    14) Product Images from "Schizandrin B inhibits the cis-DDP-induced apoptosis of HK-2 cells by activating ERK/NF-κB signaling to regulate the expression of surviving"

    Article Title: Schizandrin B inhibits the cis-DDP-induced apoptosis of HK-2 cells by activating ERK/NF-κB signaling to regulate the expression of surviving

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3409

    Effects of SchB on the cis-DDP-induced apoptosis of HK-2 cells. (A) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with 10 µ M cis-DDP for 24 h and then stained with DAPI to observe nuclear morphology (magnification, ×400). (B) Following co-staining of the HK-2 cells with Annexin V-FITC and PI, the apoptotic rates of the HK-2 cells were determined by flow cytometry. (C) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with cis-DDP for 24 h. The activation of caspase-3 was then determined by in-cell western blot analysis. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Effects of SchB on the cis-DDP-induced apoptosis of HK-2 cells. (A) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with 10 µ M cis-DDP for 24 h and then stained with DAPI to observe nuclear morphology (magnification, ×400). (B) Following co-staining of the HK-2 cells with Annexin V-FITC and PI, the apoptotic rates of the HK-2 cells were determined by flow cytometry. (C) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with cis-DDP for 24 h. The activation of caspase-3 was then determined by in-cell western blot analysis. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry, Activation Assay, In-Cell ELISA, Standard Deviation

    Effects of SchB and cis-DDP on the apoptosis/survival signaling pathway of HK-2 cells. HK-2 cells were used to establish a SchB (40 µ M) incubation group, a cis-DDP (10 µ M) incubation group and a SchB (40 µ M) + cis-DDP (10 µ M) co-incubation group and the cells were incubated for 1, 2, 4, 8 and 16 h. (A) The activation of associated signal transduction pathways was determined using protein chips. (B) Heat-maps of the protein chip results. (C) The predicted signaling pathways in the cis-DDP-induced apoptosis and SchB-induced survival of HK-2 cells. SchB, schizandrin B; cis-DDP, cis-dichlorodiammine platinum; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κBα; PARP, poly(ADP-ribose) polymerase; p, phosphorylated.
    Figure Legend Snippet: Effects of SchB and cis-DDP on the apoptosis/survival signaling pathway of HK-2 cells. HK-2 cells were used to establish a SchB (40 µ M) incubation group, a cis-DDP (10 µ M) incubation group and a SchB (40 µ M) + cis-DDP (10 µ M) co-incubation group and the cells were incubated for 1, 2, 4, 8 and 16 h. (A) The activation of associated signal transduction pathways was determined using protein chips. (B) Heat-maps of the protein chip results. (C) The predicted signaling pathways in the cis-DDP-induced apoptosis and SchB-induced survival of HK-2 cells. SchB, schizandrin B; cis-DDP, cis-dichlorodiammine platinum; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κBα; PARP, poly(ADP-ribose) polymerase; p, phosphorylated.

    Techniques Used: Incubation, Activation Assay, Transduction, Chromatin Immunoprecipitation

    Signaling via ERK/NF-κB activation in the cis-DDP-induced apoptosis of HK-2 cells. (A) HK-2 cells were incubated with 40 µ M SchB for 0, 0.5, 1, 2, 4 and 8 h, and the cell lysates were collected. Western blotting was performed to determine the phosphorylation of ERK, IKKα/β, IκBα and p65. (B) HK-2 cells were incubated with 10, 20 and 40 µ M SchB for 0.5, 2, 2 and 8 h, and the phosphorylation of ERK, IKKα/β, IκBα and p65 was determined, respectively. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Signaling via ERK/NF-κB activation in the cis-DDP-induced apoptosis of HK-2 cells. (A) HK-2 cells were incubated with 40 µ M SchB for 0, 0.5, 1, 2, 4 and 8 h, and the cell lysates were collected. Western blotting was performed to determine the phosphorylation of ERK, IKKα/β, IκBα and p65. (B) HK-2 cells were incubated with 10, 20 and 40 µ M SchB for 0.5, 2, 2 and 8 h, and the phosphorylation of ERK, IKKα/β, IκBα and p65 was determined, respectively. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Activation Assay, Incubation, Western Blot, Standard Deviation

    Effects of survivin on the cis-DDP-induced apoptosis of HK-2 cells. (A) The hSurvivin RNAi sequence was transfected into HK-2 cells. Transfected and untransfected cells were then incubated with 40 µ M SchB alone or with PDTC or U0126 for 4 h and stimulated with cis-DDP for 24 h. The activation of cleaved-caspase-3 protein was determined. (B) Survivin RNAi sequences were synthesized and transfected into HK-2 cells, which were incubated with 40 µ M SchB for 16 h. The expression of survivin was then determined. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Effects of survivin on the cis-DDP-induced apoptosis of HK-2 cells. (A) The hSurvivin RNAi sequence was transfected into HK-2 cells. Transfected and untransfected cells were then incubated with 40 µ M SchB alone or with PDTC or U0126 for 4 h and stimulated with cis-DDP for 24 h. The activation of cleaved-caspase-3 protein was determined. (B) Survivin RNAi sequences were synthesized and transfected into HK-2 cells, which were incubated with 40 µ M SchB for 16 h. The expression of survivin was then determined. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Sequencing, Transfection, Incubation, Activation Assay, Synthesized, Expressing, Standard Deviation

    Effects of SchB on the cis-DDP-induced loss of viability of HK-2 cells. (A) HK-2 cells were incubated with SchB at different concentrations (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 24 h. Cell viability was then determined using a Cell Counting kit-8 assay. (B) HK-2 cells were incubated with 40 µ M SchB for 0, 6, 12, 18, 24, 30, 36 and 42 h, and the cell viability was then determined. (C) HK-2 cells were incubated with cis-DDP at different concentrations (0, 2.5, 5, 10, 20 and 30 µ M) for 24 h, and cell viability was then determined. (D) Cells were pre-incubated with different concentrations of SchB (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 2 h and then stimulated with 10 µ M cis-DDP for 24 h. The viability of the cells was determined. Data are presented as the mean ± standard deviation (n=3). (A-C) * P
    Figure Legend Snippet: Effects of SchB on the cis-DDP-induced loss of viability of HK-2 cells. (A) HK-2 cells were incubated with SchB at different concentrations (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 24 h. Cell viability was then determined using a Cell Counting kit-8 assay. (B) HK-2 cells were incubated with 40 µ M SchB for 0, 6, 12, 18, 24, 30, 36 and 42 h, and the cell viability was then determined. (C) HK-2 cells were incubated with cis-DDP at different concentrations (0, 2.5, 5, 10, 20 and 30 µ M) for 24 h, and cell viability was then determined. (D) Cells were pre-incubated with different concentrations of SchB (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 2 h and then stimulated with 10 µ M cis-DDP for 24 h. The viability of the cells was determined. Data are presented as the mean ± standard deviation (n=3). (A-C) * P

    Techniques Used: Incubation, Cell Counting, Standard Deviation

    15) Product Images from "KIOM-79 Protects AGE-Induced Retinal Pericyte Apoptosis via Inhibition of NF-kappaB Activation In Vitro and In Vivo"

    Article Title: KIOM-79 Protects AGE-Induced Retinal Pericyte Apoptosis via Inhibition of NF-kappaB Activation In Vitro and In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043591

    Effect of KIOM-79 on IκB kinase (IKK) complex activation. KIOM-79, parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhized radix and Euphorbiae radix, and the IKK-2 inhibitor IV supplied by the manufacturer in this kit were tested for their ability to inhibit IKK-b activity using an ELISA-based kinase activity assay. Inhibition by a compound was defined by the 50% inhibition concentration (IC 50 ) of the IKK activity. IC 50 values were calculated from the dose inhibition curve. Values in the graphs represent means ± SE, n = 6.
    Figure Legend Snippet: Effect of KIOM-79 on IκB kinase (IKK) complex activation. KIOM-79, parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhized radix and Euphorbiae radix, and the IKK-2 inhibitor IV supplied by the manufacturer in this kit were tested for their ability to inhibit IKK-b activity using an ELISA-based kinase activity assay. Inhibition by a compound was defined by the 50% inhibition concentration (IC 50 ) of the IKK activity. IC 50 values were calculated from the dose inhibition curve. Values in the graphs represent means ± SE, n = 6.

    Techniques Used: Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Kinase Assay, Inhibition, Concentration Assay

    16) Product Images from "Schizandrin B inhibits the cis-DDP-induced apoptosis of HK-2 cells by activating ERK/NF-κB signaling to regulate the expression of surviving"

    Article Title: Schizandrin B inhibits the cis-DDP-induced apoptosis of HK-2 cells by activating ERK/NF-κB signaling to regulate the expression of surviving

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3409

    Effects of SchB on the cis-DDP-induced apoptosis of HK-2 cells. (A) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with 10 µ M cis-DDP for 24 h and then stained with DAPI to observe nuclear morphology (magnification, ×400). (B) Following co-staining of the HK-2 cells with Annexin V-FITC and PI, the apoptotic rates of the HK-2 cells were determined by flow cytometry. (C) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with cis-DDP for 24 h. The activation of caspase-3 was then determined by in-cell western blot analysis. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Effects of SchB on the cis-DDP-induced apoptosis of HK-2 cells. (A) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with 10 µ M cis-DDP for 24 h and then stained with DAPI to observe nuclear morphology (magnification, ×400). (B) Following co-staining of the HK-2 cells with Annexin V-FITC and PI, the apoptotic rates of the HK-2 cells were determined by flow cytometry. (C) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with cis-DDP for 24 h. The activation of caspase-3 was then determined by in-cell western blot analysis. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry, Activation Assay, In-Cell ELISA, Standard Deviation

    Effects of SchB and cis-DDP on the apoptosis/survival signaling pathway of HK-2 cells. HK-2 cells were used to establish a SchB (40 µ M) incubation group, a cis-DDP (10 µ M) incubation group and a SchB (40 µ M) + cis-DDP (10 µ M) co-incubation group and the cells were incubated for 1, 2, 4, 8 and 16 h. (A) The activation of associated signal transduction pathways was determined using protein chips. (B) Heat-maps of the protein chip results. (C) The predicted signaling pathways in the cis-DDP-induced apoptosis and SchB-induced survival of HK-2 cells. SchB, schizandrin B; cis-DDP, cis-dichlorodiammine platinum; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κBα; PARP, poly(ADP-ribose) polymerase; p, phosphorylated.
    Figure Legend Snippet: Effects of SchB and cis-DDP on the apoptosis/survival signaling pathway of HK-2 cells. HK-2 cells were used to establish a SchB (40 µ M) incubation group, a cis-DDP (10 µ M) incubation group and a SchB (40 µ M) + cis-DDP (10 µ M) co-incubation group and the cells were incubated for 1, 2, 4, 8 and 16 h. (A) The activation of associated signal transduction pathways was determined using protein chips. (B) Heat-maps of the protein chip results. (C) The predicted signaling pathways in the cis-DDP-induced apoptosis and SchB-induced survival of HK-2 cells. SchB, schizandrin B; cis-DDP, cis-dichlorodiammine platinum; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κBα; PARP, poly(ADP-ribose) polymerase; p, phosphorylated.

    Techniques Used: Incubation, Activation Assay, Transduction, Chromatin Immunoprecipitation

    Signaling via ERK/NF-κB activation in the cis-DDP-induced apoptosis of HK-2 cells. (A) HK-2 cells were incubated with 40 µ M SchB for 0, 0.5, 1, 2, 4 and 8 h, and the cell lysates were collected. Western blotting was performed to determine the phosphorylation of ERK, IKKα/β, IκBα and p65. (B) HK-2 cells were incubated with 10, 20 and 40 µ M SchB for 0.5, 2, 2 and 8 h, and the phosphorylation of ERK, IKKα/β, IκBα and p65 was determined, respectively. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Signaling via ERK/NF-κB activation in the cis-DDP-induced apoptosis of HK-2 cells. (A) HK-2 cells were incubated with 40 µ M SchB for 0, 0.5, 1, 2, 4 and 8 h, and the cell lysates were collected. Western blotting was performed to determine the phosphorylation of ERK, IKKα/β, IκBα and p65. (B) HK-2 cells were incubated with 10, 20 and 40 µ M SchB for 0.5, 2, 2 and 8 h, and the phosphorylation of ERK, IKKα/β, IκBα and p65 was determined, respectively. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Activation Assay, Incubation, Western Blot, Standard Deviation

    Effects of survivin on the cis-DDP-induced apoptosis of HK-2 cells. (A) The hSurvivin RNAi sequence was transfected into HK-2 cells. Transfected and untransfected cells were then incubated with 40 µ M SchB alone or with PDTC or U0126 for 4 h and stimulated with cis-DDP for 24 h. The activation of cleaved-caspase-3 protein was determined. (B) Survivin RNAi sequences were synthesized and transfected into HK-2 cells, which were incubated with 40 µ M SchB for 16 h. The expression of survivin was then determined. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Effects of survivin on the cis-DDP-induced apoptosis of HK-2 cells. (A) The hSurvivin RNAi sequence was transfected into HK-2 cells. Transfected and untransfected cells were then incubated with 40 µ M SchB alone or with PDTC or U0126 for 4 h and stimulated with cis-DDP for 24 h. The activation of cleaved-caspase-3 protein was determined. (B) Survivin RNAi sequences were synthesized and transfected into HK-2 cells, which were incubated with 40 µ M SchB for 16 h. The expression of survivin was then determined. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Sequencing, Transfection, Incubation, Activation Assay, Synthesized, Expressing, Standard Deviation

    Effects of SchB on the cis-DDP-induced loss of viability of HK-2 cells. (A) HK-2 cells were incubated with SchB at different concentrations (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 24 h. Cell viability was then determined using a Cell Counting kit-8 assay. (B) HK-2 cells were incubated with 40 µ M SchB for 0, 6, 12, 18, 24, 30, 36 and 42 h, and the cell viability was then determined. (C) HK-2 cells were incubated with cis-DDP at different concentrations (0, 2.5, 5, 10, 20 and 30 µ M) for 24 h, and cell viability was then determined. (D) Cells were pre-incubated with different concentrations of SchB (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 2 h and then stimulated with 10 µ M cis-DDP for 24 h. The viability of the cells was determined. Data are presented as the mean ± standard deviation (n=3). (A-C) * P
    Figure Legend Snippet: Effects of SchB on the cis-DDP-induced loss of viability of HK-2 cells. (A) HK-2 cells were incubated with SchB at different concentrations (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 24 h. Cell viability was then determined using a Cell Counting kit-8 assay. (B) HK-2 cells were incubated with 40 µ M SchB for 0, 6, 12, 18, 24, 30, 36 and 42 h, and the cell viability was then determined. (C) HK-2 cells were incubated with cis-DDP at different concentrations (0, 2.5, 5, 10, 20 and 30 µ M) for 24 h, and cell viability was then determined. (D) Cells were pre-incubated with different concentrations of SchB (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 2 h and then stimulated with 10 µ M cis-DDP for 24 h. The viability of the cells was determined. Data are presented as the mean ± standard deviation (n=3). (A-C) * P

    Techniques Used: Incubation, Cell Counting, Standard Deviation

    17) Product Images from "Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance"

    Article Title: Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171084

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.

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

    RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .
    Figure Legend Snippet: RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Hypothesized mechanism of Hb-α and Hb-β expression and their role in the function vaginal cells. Stimulation of hPVECs with LPS up-regulates the expression TLR4, NF-κB, Hb-α, Hb-β and pro-inflammatory cytokine (e.g. IL-6). The co-receptor MD2 triggers interactions between the cell surface domain of TLR4 and LPS. Activated TLR4 interacts with cytoplasmic adaptor molecules MyD88 or TRIF which subsequently activates IKK complex. The IKK complex induces phosphorylation of IκB and its subsequent degradation liberates NF-κB and allows it to translocate into the nucleus where it can induce target gene expression including Hb-α, Hb-β and IL-6 etc and may contribute to pathogen clearance by enhancing local host immune responses in hPVECs. This figure represents the best-fit model for these effects.
    Figure Legend Snippet: Hypothesized mechanism of Hb-α and Hb-β expression and their role in the function vaginal cells. Stimulation of hPVECs with LPS up-regulates the expression TLR4, NF-κB, Hb-α, Hb-β and pro-inflammatory cytokine (e.g. IL-6). The co-receptor MD2 triggers interactions between the cell surface domain of TLR4 and LPS. Activated TLR4 interacts with cytoplasmic adaptor molecules MyD88 or TRIF which subsequently activates IKK complex. The IKK complex induces phosphorylation of IκB and its subsequent degradation liberates NF-κB and allows it to translocate into the nucleus where it can induce target gene expression including Hb-α, Hb-β and IL-6 etc and may contribute to pathogen clearance by enhancing local host immune responses in hPVECs. This figure represents the best-fit model for these effects.

    Techniques Used: Expressing

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p
    Figure Legend Snippet: qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    18) Product Images from "Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance"

    Article Title: Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171084

    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Figure Legend Snippet: Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).

    Techniques Used: Expressing, Staining

    Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells. Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).
    Figure Legend Snippet: Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells. Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).

    Techniques Used:

    Immunofluorescence localization of cytokeratin-13. Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).
    Figure Legend Snippet: Immunofluorescence localization of cytokeratin-13. Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).

    Techniques Used: Immunofluorescence, Staining

    TLR4 expression in hPVECs and VK2/E6E7 cells. (A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene Gapdh (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of TLR4 mRNA reported in Fig 10A. The expression of TLR4 mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.
    Figure Legend Snippet: TLR4 expression in hPVECs and VK2/E6E7 cells. (A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene Gapdh (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of TLR4 mRNA reported in Fig 10A. The expression of TLR4 mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.

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

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.

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

    Immunofluorescence localization of vimentin. hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).
    Figure Legend Snippet: Immunofluorescence localization of vimentin. hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).

    Techniques Used: Immunofluorescence, Positive Control, Staining

    Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p
    Figure Legend Snippet: Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .
    Figure Legend Snippet: RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.
    Figure Legend Snippet: Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.

    Techniques Used: Western Blot, Biomarker Assay, Expressing, Positive Control

    RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.
    Figure Legend Snippet: RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.

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

    Cytokoine ELISA of hPVECs and VK2/E6E7 cells. IL-6 (A) and IL-10 (B) in the culture supernatants of un-stimulated and LPS stimulated hPVECs and VK2/E6E7 cells at indicated time points (6, 12, 24 hrs). Cells were seeded at a density of 10 6 /well in 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Levels of IL-6 were up-regulated in LPS-induced cells. Values represent the mean ± SD of three independent experiments performed on different days. Level of significance (*p
    Figure Legend Snippet: Cytokoine ELISA of hPVECs and VK2/E6E7 cells. IL-6 (A) and IL-10 (B) in the culture supernatants of un-stimulated and LPS stimulated hPVECs and VK2/E6E7 cells at indicated time points (6, 12, 24 hrs). Cells were seeded at a density of 10 6 /well in 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Levels of IL-6 were up-regulated in LPS-induced cells. Values represent the mean ± SD of three independent experiments performed on different days. Level of significance (*p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p
    Figure Legend Snippet: qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).
    Figure Legend Snippet: Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Figure Legend Snippet: Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).

    Techniques Used: Expressing, Staining

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.

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

    Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p
    Figure Legend Snippet: Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .
    Figure Legend Snippet: RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.
    Figure Legend Snippet: Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.

    Techniques Used: Western Blot, Biomarker Assay, Expressing, Positive Control

    RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.
    Figure Legend Snippet: RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.

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

    Hypothesized mechanism of Hb-α and Hb-β expression and their role in the function vaginal cells. Stimulation of hPVECs with LPS up-regulates the expression TLR4, NF-κB, Hb-α, Hb-β and pro-inflammatory cytokine (e.g. IL-6). The co-receptor MD2 triggers interactions between the cell surface domain of TLR4 and LPS. Activated TLR4 interacts with cytoplasmic adaptor molecules MyD88 or TRIF which subsequently activates IKK complex. The IKK complex induces phosphorylation of IκB and its subsequent degradation liberates NF-κB and allows it to translocate into the nucleus where it can induce target gene expression including Hb-α, Hb-β and IL-6 etc and may contribute to pathogen clearance by enhancing local host immune responses in hPVECs. This figure represents the best-fit model for these effects.
    Figure Legend Snippet: Hypothesized mechanism of Hb-α and Hb-β expression and their role in the function vaginal cells. Stimulation of hPVECs with LPS up-regulates the expression TLR4, NF-κB, Hb-α, Hb-β and pro-inflammatory cytokine (e.g. IL-6). The co-receptor MD2 triggers interactions between the cell surface domain of TLR4 and LPS. Activated TLR4 interacts with cytoplasmic adaptor molecules MyD88 or TRIF which subsequently activates IKK complex. The IKK complex induces phosphorylation of IκB and its subsequent degradation liberates NF-κB and allows it to translocate into the nucleus where it can induce target gene expression including Hb-α, Hb-β and IL-6 etc and may contribute to pathogen clearance by enhancing local host immune responses in hPVECs. This figure represents the best-fit model for these effects.

    Techniques Used: Expressing

    Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.
    Figure Legend Snippet: Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.

    Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Binding Assay, Mutagenesis

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p
    Figure Legend Snippet: qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    19) Product Images from "Schizandrin B inhibits the cis-DDP-induced apoptosis of HK-2 cells by activating ERK/NF-κB signaling to regulate the expression of surviving"

    Article Title: Schizandrin B inhibits the cis-DDP-induced apoptosis of HK-2 cells by activating ERK/NF-κB signaling to regulate the expression of surviving

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3409

    Effects of SchB on the cis-DDP-induced apoptosis of HK-2 cells. (A) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with 10 µ M cis-DDP for 24 h and then stained with DAPI to observe nuclear morphology (magnification, ×400). (B) Following co-staining of the HK-2 cells with Annexin V-FITC and PI, the apoptotic rates of the HK-2 cells were determined by flow cytometry. (C) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with cis-DDP for 24 h. The activation of caspase-3 was then determined by in-cell western blot analysis. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Effects of SchB on the cis-DDP-induced apoptosis of HK-2 cells. (A) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with 10 µ M cis-DDP for 24 h and then stained with DAPI to observe nuclear morphology (magnification, ×400). (B) Following co-staining of the HK-2 cells with Annexin V-FITC and PI, the apoptotic rates of the HK-2 cells were determined by flow cytometry. (C) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with cis-DDP for 24 h. The activation of caspase-3 was then determined by in-cell western blot analysis. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry, Activation Assay, In-Cell ELISA, Standard Deviation

    Effects of SchB and cis-DDP on the apoptosis/survival signaling pathway of HK-2 cells. HK-2 cells were used to establish a SchB (40 µ M) incubation group, a cis-DDP (10 µ M) incubation group and a SchB (40 µ M) + cis-DDP (10 µ M) co-incubation group and the cells were incubated for 1, 2, 4, 8 and 16 h. (A) The activation of associated signal transduction pathways was determined using protein chips. (B) Heat-maps of the protein chip results. (C) The predicted signaling pathways in the cis-DDP-induced apoptosis and SchB-induced survival of HK-2 cells. SchB, schizandrin B; cis-DDP, cis-dichlorodiammine platinum; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κBα; PARP, poly(ADP-ribose) polymerase; p, phosphorylated.
    Figure Legend Snippet: Effects of SchB and cis-DDP on the apoptosis/survival signaling pathway of HK-2 cells. HK-2 cells were used to establish a SchB (40 µ M) incubation group, a cis-DDP (10 µ M) incubation group and a SchB (40 µ M) + cis-DDP (10 µ M) co-incubation group and the cells were incubated for 1, 2, 4, 8 and 16 h. (A) The activation of associated signal transduction pathways was determined using protein chips. (B) Heat-maps of the protein chip results. (C) The predicted signaling pathways in the cis-DDP-induced apoptosis and SchB-induced survival of HK-2 cells. SchB, schizandrin B; cis-DDP, cis-dichlorodiammine platinum; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κBα; PARP, poly(ADP-ribose) polymerase; p, phosphorylated.

    Techniques Used: Incubation, Activation Assay, Transduction, Chromatin Immunoprecipitation

    Signaling via ERK/NF-κB activation in the cis-DDP-induced apoptosis of HK-2 cells. (A) HK-2 cells were incubated with 40 µ M SchB for 0, 0.5, 1, 2, 4 and 8 h, and the cell lysates were collected. Western blotting was performed to determine the phosphorylation of ERK, IKKα/β, IκBα and p65. (B) HK-2 cells were incubated with 10, 20 and 40 µ M SchB for 0.5, 2, 2 and 8 h, and the phosphorylation of ERK, IKKα/β, IκBα and p65 was determined, respectively. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Signaling via ERK/NF-κB activation in the cis-DDP-induced apoptosis of HK-2 cells. (A) HK-2 cells were incubated with 40 µ M SchB for 0, 0.5, 1, 2, 4 and 8 h, and the cell lysates were collected. Western blotting was performed to determine the phosphorylation of ERK, IKKα/β, IκBα and p65. (B) HK-2 cells were incubated with 10, 20 and 40 µ M SchB for 0.5, 2, 2 and 8 h, and the phosphorylation of ERK, IKKα/β, IκBα and p65 was determined, respectively. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Activation Assay, Incubation, Western Blot, Standard Deviation

    Effects of survivin on the cis-DDP-induced apoptosis of HK-2 cells. (A) The hSurvivin RNAi sequence was transfected into HK-2 cells. Transfected and untransfected cells were then incubated with 40 µ M SchB alone or with PDTC or U0126 for 4 h and stimulated with cis-DDP for 24 h. The activation of cleaved-caspase-3 protein was determined. (B) Survivin RNAi sequences were synthesized and transfected into HK-2 cells, which were incubated with 40 µ M SchB for 16 h. The expression of survivin was then determined. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Effects of survivin on the cis-DDP-induced apoptosis of HK-2 cells. (A) The hSurvivin RNAi sequence was transfected into HK-2 cells. Transfected and untransfected cells were then incubated with 40 µ M SchB alone or with PDTC or U0126 for 4 h and stimulated with cis-DDP for 24 h. The activation of cleaved-caspase-3 protein was determined. (B) Survivin RNAi sequences were synthesized and transfected into HK-2 cells, which were incubated with 40 µ M SchB for 16 h. The expression of survivin was then determined. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Sequencing, Transfection, Incubation, Activation Assay, Synthesized, Expressing, Standard Deviation

    Effects of SchB on the cis-DDP-induced loss of viability of HK-2 cells. (A) HK-2 cells were incubated with SchB at different concentrations (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 24 h. Cell viability was then determined using a Cell Counting kit-8 assay. (B) HK-2 cells were incubated with 40 µ M SchB for 0, 6, 12, 18, 24, 30, 36 and 42 h, and the cell viability was then determined. (C) HK-2 cells were incubated with cis-DDP at different concentrations (0, 2.5, 5, 10, 20 and 30 µ M) for 24 h, and cell viability was then determined. (D) Cells were pre-incubated with different concentrations of SchB (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 2 h and then stimulated with 10 µ M cis-DDP for 24 h. The viability of the cells was determined. Data are presented as the mean ± standard deviation (n=3). (A-C) * P
    Figure Legend Snippet: Effects of SchB on the cis-DDP-induced loss of viability of HK-2 cells. (A) HK-2 cells were incubated with SchB at different concentrations (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 24 h. Cell viability was then determined using a Cell Counting kit-8 assay. (B) HK-2 cells were incubated with 40 µ M SchB for 0, 6, 12, 18, 24, 30, 36 and 42 h, and the cell viability was then determined. (C) HK-2 cells were incubated with cis-DDP at different concentrations (0, 2.5, 5, 10, 20 and 30 µ M) for 24 h, and cell viability was then determined. (D) Cells were pre-incubated with different concentrations of SchB (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 2 h and then stimulated with 10 µ M cis-DDP for 24 h. The viability of the cells was determined. Data are presented as the mean ± standard deviation (n=3). (A-C) * P

    Techniques Used: Incubation, Cell Counting, Standard Deviation

    20) Product Images from "TNF-α Modulation of Intestinal Tight Junction Permeability Is Mediated by NIK/IKK-α Axis Activation of the Canonical NF-κB Pathway"

    Article Title: TNF-α Modulation of Intestinal Tight Junction Permeability Is Mediated by NIK/IKK-α Axis Activation of the Canonical NF-κB Pathway

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2015.12.016

    Effect of siRNA-induced MAP3 kinase knockdown on TNF-α activation of NF-κB p65. A: NIK siRNA transfection prevents the TNF-α–induced degradation of IκB-α as assessed by Western blot analysis. B: NIK silencing inhibits the TNF-α–induced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. C: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced degradation of IκB-α. D: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. Data are expressed as means ± SEM. **** P
    Figure Legend Snippet: Effect of siRNA-induced MAP3 kinase knockdown on TNF-α activation of NF-κB p65. A: NIK siRNA transfection prevents the TNF-α–induced degradation of IκB-α as assessed by Western blot analysis. B: NIK silencing inhibits the TNF-α–induced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. C: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced degradation of IκB-α. D: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. Data are expressed as means ± SEM. **** P

    Techniques Used: Activation Assay, Transfection, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

    Effect of siRNA-induced NIK and MEKK-1 knockdown on TNF-α–induced increase in Caco-2 TJ permeability. A: NIK siRNA transfection results in a near complete depletion in NIK protein expression. B: NIK silencing prevents the TNF-α–induced drop in Caco-2 TER. C: NIK silencing by siRNA transfection prevents the TNF-α–induced increase in inulin flux. D: MEKK-1 siRNA transfection results in a near complete depletion in MEKK-1 protein expression. E: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced drop in Caco-2 TER. F: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced increase in inulin flux. G: NIK inhibitor (4H-isoquinoline-1,3-dione; 50 μmol/L) prevents the TNF-phosphorylation of NIK. H: NIK inhibitor prevents the TNF-α–induced drop in Caco-2 TER. Data are expressed as means ± SEM. n = 4. ** P
    Figure Legend Snippet: Effect of siRNA-induced NIK and MEKK-1 knockdown on TNF-α–induced increase in Caco-2 TJ permeability. A: NIK siRNA transfection results in a near complete depletion in NIK protein expression. B: NIK silencing prevents the TNF-α–induced drop in Caco-2 TER. C: NIK silencing by siRNA transfection prevents the TNF-α–induced increase in inulin flux. D: MEKK-1 siRNA transfection results in a near complete depletion in MEKK-1 protein expression. E: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced drop in Caco-2 TER. F: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced increase in inulin flux. G: NIK inhibitor (4H-isoquinoline-1,3-dione; 50 μmol/L) prevents the TNF-phosphorylation of NIK. H: NIK inhibitor prevents the TNF-α–induced drop in Caco-2 TER. Data are expressed as means ± SEM. n = 4. ** P

    Techniques Used: Permeability, Transfection, Expressing

    Effect of siRNA-induced silencing of NIK and MEKK-1 on NF-κB signaling pathway and mouse intestinal permeability. A: NIK siRNA transfection in vivo prevents the TNF-α–induced degradation of IκB-α in mouse intestinal tissues. B: MEKK-1 siRNA transfection in vivo does not inhibit the TNF-α–induced degradation of IκB-α in mouse intestinal tissues. C: NF-κB p65 siRNA transfection in vivo prevents the TNF–α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. D: NF-κB p65 siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. *** P
    Figure Legend Snippet: Effect of siRNA-induced silencing of NIK and MEKK-1 on NF-κB signaling pathway and mouse intestinal permeability. A: NIK siRNA transfection in vivo prevents the TNF-α–induced degradation of IκB-α in mouse intestinal tissues. B: MEKK-1 siRNA transfection in vivo does not inhibit the TNF-α–induced degradation of IκB-α in mouse intestinal tissues. C: NF-κB p65 siRNA transfection in vivo prevents the TNF–α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. D: NF-κB p65 siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. *** P

    Techniques Used: Permeability, Transfection, In Vivo, Expressing, Western Blot

    Effect of TNF-α on MLCK expression in vivo . A: TNF-α causes an increase in mouse intestinal tissue MLCK mRNA transcript as assessed by real-time PCR; TNF-α 24 hours of treatment. B: TNF-α causes a time-dependent increase in mouse intestinal tissue MLCK protein expression as assessed by Western blot analysis. C: NIK siRNA transfection in vivo results in a near-complete knockdown of NIK expression in mouse intestinal tissue. D: NIK siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. E: NIK siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. F: MEKK-1 siRNA transfection in vivo results in a near-complete knockdown of MEKK-1 expression in mouse intestinal tissue. G: siRNA-induced knockdown of MEKK-1 in vivo does not prevent the TNF-α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. H: MEKK-1 siRNA transfection in vivo does not prevent the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. ** P
    Figure Legend Snippet: Effect of TNF-α on MLCK expression in vivo . A: TNF-α causes an increase in mouse intestinal tissue MLCK mRNA transcript as assessed by real-time PCR; TNF-α 24 hours of treatment. B: TNF-α causes a time-dependent increase in mouse intestinal tissue MLCK protein expression as assessed by Western blot analysis. C: NIK siRNA transfection in vivo results in a near-complete knockdown of NIK expression in mouse intestinal tissue. D: NIK siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. E: NIK siRNA transfection in vivo prevents the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. F: MEKK-1 siRNA transfection in vivo results in a near-complete knockdown of MEKK-1 expression in mouse intestinal tissue. G: siRNA-induced knockdown of MEKK-1 in vivo does not prevent the TNF-α–induced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. H: MEKK-1 siRNA transfection in vivo does not prevent the TNF-α–induced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. ** P

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

    Effect of siRNA-induced MEKK-1 and NIK knockdown on TNF-α activation of IKK-α. A: siRNA-induced knockdown of NIK prevents the TNF-α–induced phosphorylation of IKK-α as assessed by Western blot analysis. B: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced phosphorylation of IKK-α. Effect of siRNA induced knockdown of NIK and MEKK-1 on TNF-α–induced increase in MLCK gene activity and protein expression. C: siRNA-induced knockdown of NIK results in a complete inhibition of TNF-α–induced increase in MLCK promoter activity. D: siRNA-induced knockdown of NIK prevents the TNF-α–induced increase in MLCK mRNA levels. E: NIK silencing by siRNA transfection prevents the TNF-α–induced increase in MLCK protein expression. F: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced increase in MLCK promoter activity. G: Knocking-down MEKK-1 by siRNA does not prevent the TNF-α–induced increase in MLCK mRNA levels. H: Knocking-down MEKK-1 by siRNA does not affect the TNF-α–induced increase in MLCK protein expression. I: Knocking-down NIK by siRNA prevents the TNF-α–induced phosphorylation of p38 kinase (siNIK: siRNA NIK transfection). J: Knocking-down NIK by siRNA does not inhibit the TNF-α–induced phosphorylation of ERK1/2. K: Knocking-down p38 kinase by siRNA does not inhibit the TNF-α–induced degradation of IκB-α. * P
    Figure Legend Snippet: Effect of siRNA-induced MEKK-1 and NIK knockdown on TNF-α activation of IKK-α. A: siRNA-induced knockdown of NIK prevents the TNF-α–induced phosphorylation of IKK-α as assessed by Western blot analysis. B: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced phosphorylation of IKK-α. Effect of siRNA induced knockdown of NIK and MEKK-1 on TNF-α–induced increase in MLCK gene activity and protein expression. C: siRNA-induced knockdown of NIK results in a complete inhibition of TNF-α–induced increase in MLCK promoter activity. D: siRNA-induced knockdown of NIK prevents the TNF-α–induced increase in MLCK mRNA levels. E: NIK silencing by siRNA transfection prevents the TNF-α–induced increase in MLCK protein expression. F: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-α–induced increase in MLCK promoter activity. G: Knocking-down MEKK-1 by siRNA does not prevent the TNF-α–induced increase in MLCK mRNA levels. H: Knocking-down MEKK-1 by siRNA does not affect the TNF-α–induced increase in MLCK protein expression. I: Knocking-down NIK by siRNA prevents the TNF-α–induced phosphorylation of p38 kinase (siNIK: siRNA NIK transfection). J: Knocking-down NIK by siRNA does not inhibit the TNF-α–induced phosphorylation of ERK1/2. K: Knocking-down p38 kinase by siRNA does not inhibit the TNF-α–induced degradation of IκB-α. * P

    Techniques Used: Activation Assay, Western Blot, Activity Assay, Expressing, Inhibition, Transfection

    Effect of TNF-α activation of NF-κB pathway in mouse intestinal permeability. A: TNF-α (5 μg) causes an increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. B: TNF-α causes a time-dependent increase in IκB-α degradation in mouse intestinal tissue, starting at 2 hours and continuing up to 24 hours as assessed by Western blot analysis. C: TNF-α caused a time-dependent increase in phosphorylation of NIK and MEKK-1 in mouse intestinal tissue as assessed by Western blot analysis. ** P
    Figure Legend Snippet: Effect of TNF-α activation of NF-κB pathway in mouse intestinal permeability. A: TNF-α (5 μg) causes an increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. B: TNF-α causes a time-dependent increase in IκB-α degradation in mouse intestinal tissue, starting at 2 hours and continuing up to 24 hours as assessed by Western blot analysis. C: TNF-α caused a time-dependent increase in phosphorylation of NIK and MEKK-1 in mouse intestinal tissue as assessed by Western blot analysis. ** P

    Techniques Used: Activation Assay, Permeability, Western Blot

    Time course effect of TNF-α on Caco-2 NIK and MEKK-1 activation. A: TNF-α (10 ng/mL) causes a time-dependent increase in Caco-2 NIK phosphorylation (total NIK was used for equal protein loading). B: TNF-α causes a time-dependent increase in MEKK-1 phosphorylation (total MEKK-1 was used for equal protein loading). MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-κB-inducing kinase; TNF, tumor necrosis factor.
    Figure Legend Snippet: Time course effect of TNF-α on Caco-2 NIK and MEKK-1 activation. A: TNF-α (10 ng/mL) causes a time-dependent increase in Caco-2 NIK phosphorylation (total NIK was used for equal protein loading). B: TNF-α causes a time-dependent increase in MEKK-1 phosphorylation (total MEKK-1 was used for equal protein loading). MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-κB-inducing kinase; TNF, tumor necrosis factor.

    Techniques Used: Activation Assay

    21) Product Images from "Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1"

    Article Title: Interleukin-6 and Type-I Collagen Production by Systemic Sclerosis Fibroblasts Are Differentially Regulated by Interleukin-17A in the Presence of Transforming Growth Factor-Beta 1

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01865

    p38 MAPK signaling pathway is common to IL-17A- and TGF-β-induced IL-6 production. Healthy donors fibroblasts were treated with the indicated concentrations of SB203580 (A) or 20 µM SB203580 (B) for 1 h prior to the addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml) in triplicates. After 48 h, culture SNs were collected and IL-6 levels were assessed by ELISA. (A) Results are shown as fold change to untreated cells, mean + SEM is indicated, N = 3. Please note the log 2 scale. (B) Results are shown as the percentage of IL-6 production induced by IL-17A and/or TGF- β in the absence of inhibitor (levels of IL-6 were: 14.7 ± 7.4 pg/ml for IL-17A, 11.4 ± 6.1 pg/ml for TGF-β, and 48.9 ± 13.7 pg/ml for IL-17A + TGF-β). Bars represent the mean + SEM of three experiments. Significant differences versus control were assessed by paired t -test.
    Figure Legend Snippet: p38 MAPK signaling pathway is common to IL-17A- and TGF-β-induced IL-6 production. Healthy donors fibroblasts were treated with the indicated concentrations of SB203580 (A) or 20 µM SB203580 (B) for 1 h prior to the addition of IL-17A (25 ng/ml) and/or TGF-β (2.5 ng/ml) in triplicates. After 48 h, culture SNs were collected and IL-6 levels were assessed by ELISA. (A) Results are shown as fold change to untreated cells, mean + SEM is indicated, N = 3. Please note the log 2 scale. (B) Results are shown as the percentage of IL-6 production induced by IL-17A and/or TGF- β in the absence of inhibitor (levels of IL-6 were: 14.7 ± 7.4 pg/ml for IL-17A, 11.4 ± 6.1 pg/ml for TGF-β, and 48.9 ± 13.7 pg/ml for IL-17A + TGF-β). Bars represent the mean + SEM of three experiments. Significant differences versus control were assessed by paired t -test.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    22) Product Images from "Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene"

    Article Title: Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2018.12.016

    Effect of inhibition of NF-κB pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) expression in mouse enterocytes. A: NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. B: NF-κB inhibitor Bay-11 also prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or /LPS-treated mice. Densitometry of MLCK protein levels. C: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. n = 3 experiments. ∗∗ P
    Figure Legend Snippet: Effect of inhibition of NF-κB pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) expression in mouse enterocytes. A: NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. B: NF-κB inhibitor Bay-11 also prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or /LPS-treated mice. Densitometry of MLCK protein levels. C: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. n = 3 experiments. ∗∗ P

    Techniques Used: Inhibition, Activation Assay, Expressing, Mouse Assay

    Activation of p65/p50 canonical pathway by lipopolysaccharide (LPS) in mice enterocytes and effect of NF-κB inhibitors on LPS-induced increase in mouse intestinal epithelial tight junction permeability. A: LPS i.p. injections (0.1 mg/kg body weight) in mice caused activation of canonical p65/p50 pathway, as assessed by degradation of inhibitory κ B (IκB)-α protein expression on day 3 in mice enterocytes. Densitometry of IκB-α protein levels. B: The immunoblot analysis from LPS-treated mice enterocytes revealed significant increase in nuclear p65 protein expression on day 3 compared with untreated mice enterocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Lamin B were used as loading controls for cytoplasmic (cyto) and nuclear (nuc) fractions, respectively C: Confocal immunofluorescence of mouse intestines treated with LPS (0.1 mg/kg body weight) on day 3 indicated p65 (red) ( arrowheads ) translocation to the nucleus (blue) compared with control (C) mouse enterocytes. D: NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC; 10 mg/kg body weight), and Bay-11 (5 mg/kg body weight) pretreatment prevented the LPS-induced increase in 10K dextran flux. PDTC and Bay-11 were dissolved in dimethyl sulfoxide and injected 1 hour before LPS treatment. Data are expressed as means ± SEM. n = 4 experiments ( A and C ); n = 3 experiments ( B ). ∗∗ P
    Figure Legend Snippet: Activation of p65/p50 canonical pathway by lipopolysaccharide (LPS) in mice enterocytes and effect of NF-κB inhibitors on LPS-induced increase in mouse intestinal epithelial tight junction permeability. A: LPS i.p. injections (0.1 mg/kg body weight) in mice caused activation of canonical p65/p50 pathway, as assessed by degradation of inhibitory κ B (IκB)-α protein expression on day 3 in mice enterocytes. Densitometry of IκB-α protein levels. B: The immunoblot analysis from LPS-treated mice enterocytes revealed significant increase in nuclear p65 protein expression on day 3 compared with untreated mice enterocytes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Lamin B were used as loading controls for cytoplasmic (cyto) and nuclear (nuc) fractions, respectively C: Confocal immunofluorescence of mouse intestines treated with LPS (0.1 mg/kg body weight) on day 3 indicated p65 (red) ( arrowheads ) translocation to the nucleus (blue) compared with control (C) mouse enterocytes. D: NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC; 10 mg/kg body weight), and Bay-11 (5 mg/kg body weight) pretreatment prevented the LPS-induced increase in 10K dextran flux. PDTC and Bay-11 were dissolved in dimethyl sulfoxide and injected 1 hour before LPS treatment. Data are expressed as means ± SEM. n = 4 experiments ( A and C ); n = 3 experiments ( B ). ∗∗ P

    Techniques Used: Activation Assay, Mouse Assay, Permeability, Expressing, Immunofluorescence, Translocation Assay, Injection

    Effect of pharmacologic inhibition of NF-κB on lipopolysaccharide (LPS)-induced increase in intestinal epithelial tight junction permeability. A: Filter-grown Caco-2 monolayers were treated with 300 pg/mL LPS for a 5-day experimental period. Pharmacologic inhibition of NF-κB by 10 μm ammonium pyrrolidinedithiocarbamate (PDTC) inhibited the LPS (physiological dose of 300 pg/mL)-induced drop in Caco-2 transepithelial electrical resistance. B: Pharmacologic inhibition of NF-κB by PDTC prevented the LPS-induced increase in inulin flux. NF-κB inhibitor PDTC was added 1 hour before LPS treatment. All experimental treatments were renewed every 24 hours for the 5-day experimental period. Data are expressed as means ± SEM. n = 4 independent experiments. ∗∗∗ P
    Figure Legend Snippet: Effect of pharmacologic inhibition of NF-κB on lipopolysaccharide (LPS)-induced increase in intestinal epithelial tight junction permeability. A: Filter-grown Caco-2 monolayers were treated with 300 pg/mL LPS for a 5-day experimental period. Pharmacologic inhibition of NF-κB by 10 μm ammonium pyrrolidinedithiocarbamate (PDTC) inhibited the LPS (physiological dose of 300 pg/mL)-induced drop in Caco-2 transepithelial electrical resistance. B: Pharmacologic inhibition of NF-κB by PDTC prevented the LPS-induced increase in inulin flux. NF-κB inhibitor PDTC was added 1 hour before LPS treatment. All experimental treatments were renewed every 24 hours for the 5-day experimental period. Data are expressed as means ± SEM. n = 4 independent experiments. ∗∗∗ P

    Techniques Used: Inhibition, Permeability

    23) Product Images from "LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118"

    Article Title: LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118

    Journal: Oncotarget

    doi:

    LDL induces HIF-1α hydroxylation at Pro402 and Pro564 sties, while increases 20S proteasome activity in hCMEC/D3 cells A–B. hCMEC/D3 cells were exposed to LDL (100 μg/ml) in hypoxia, after which Western blot analysis was performed to monitor hydroxylation of HIF-1α using antibodies specifically recognizing hydroxylated HIF-1α at Pro402 (A) and Pro564 (B) respectively. C–D. CT-L activity of 20S proteasome was analysed in hCMEC/D3 after exposed (24 hr) to the indicated concentrations of LDL in either hypoxia (C) or normoxia (D). Cells were treated with MG132 as control. E–F. hCMEC/D3 cells were exposed to 100 μg/ml LDL for 48 hr in the absence or presence of pre-treatment with 100 nM BSc2118 (4 hr prior to LDL) in normoxia (E) or hypoxia (F), after which Western blot analysis was performed to assess expression of NF-κB p65 and HIF-1β. At least three independent experiments ( n ≥ 3) were performed. * p
    Figure Legend Snippet: LDL induces HIF-1α hydroxylation at Pro402 and Pro564 sties, while increases 20S proteasome activity in hCMEC/D3 cells A–B. hCMEC/D3 cells were exposed to LDL (100 μg/ml) in hypoxia, after which Western blot analysis was performed to monitor hydroxylation of HIF-1α using antibodies specifically recognizing hydroxylated HIF-1α at Pro402 (A) and Pro564 (B) respectively. C–D. CT-L activity of 20S proteasome was analysed in hCMEC/D3 after exposed (24 hr) to the indicated concentrations of LDL in either hypoxia (C) or normoxia (D). Cells were treated with MG132 as control. E–F. hCMEC/D3 cells were exposed to 100 μg/ml LDL for 48 hr in the absence or presence of pre-treatment with 100 nM BSc2118 (4 hr prior to LDL) in normoxia (E) or hypoxia (F), after which Western blot analysis was performed to assess expression of NF-κB p65 and HIF-1β. At least three independent experiments ( n ≥ 3) were performed. * p

    Techniques Used: Activity Assay, Western Blot, Expressing

    24) Product Images from "Troglitazone Inhibits Matrix Metalloproteinase-9 Expression and Invasion of Breast Cancer Cell through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism"

    Article Title: Troglitazone Inhibits Matrix Metalloproteinase-9 Expression and Invasion of Breast Cancer Cell through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism

    Journal: Journal of Breast Cancer

    doi: 10.4048/jbc.2018.21.1.28

    Troglitazone inhibits 12- O -tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-9 (MMP-9) expression in MCF-7 cells. MCF-7 cells were pretreated with troglitazone and then TPA was added for 24 hours. (A) MMP-9 secretion by gelatin zymography (top). MMP-9 protein expression was determined by western blot. β-Actin was the internal control (bottom). (B) MMP-9 mRNA was analyzed by real-time polymerase chain reaction with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. (C) Wild type MMP-9-luc reporters and a Renilla luciferase thymidine kinase reporter vector were co-transfected into MCF-7 cells. Cells were treated with troglitazone and TPA and MMP-9 promoter activity was measured with dual-luciferase reporter assays. (D) Peroxisome proliferator-activated receptor γ antagonist GW9662 was added to cells for 30 minutes before troglitazone treatment. Lysates were analyzed by western blot with anti-MMP-9. β-Actin was the loading control. Values are mean±standard error of the mean of three independent experiments. * p
    Figure Legend Snippet: Troglitazone inhibits 12- O -tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-9 (MMP-9) expression in MCF-7 cells. MCF-7 cells were pretreated with troglitazone and then TPA was added for 24 hours. (A) MMP-9 secretion by gelatin zymography (top). MMP-9 protein expression was determined by western blot. β-Actin was the internal control (bottom). (B) MMP-9 mRNA was analyzed by real-time polymerase chain reaction with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. (C) Wild type MMP-9-luc reporters and a Renilla luciferase thymidine kinase reporter vector were co-transfected into MCF-7 cells. Cells were treated with troglitazone and TPA and MMP-9 promoter activity was measured with dual-luciferase reporter assays. (D) Peroxisome proliferator-activated receptor γ antagonist GW9662 was added to cells for 30 minutes before troglitazone treatment. Lysates were analyzed by western blot with anti-MMP-9. β-Actin was the loading control. Values are mean±standard error of the mean of three independent experiments. * p

    Techniques Used: Expressing, Zymography, Western Blot, Real-time Polymerase Chain Reaction, Luciferase, Plasmid Preparation, Transfection, Activity Assay

    25) Product Images from "Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by Inhibition of NF-?B and Src Protein Kinase Signaling Pathways"

    Article Title: Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by Inhibition of NF-?B and Src Protein Kinase Signaling Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0057218

    Effect of 5-FU and/or curcumin or PI-3K inhibitor wortmannin on activation of IκBα kinase (IKK) in HCT116 and HCT116+ch3 colon cancer cells. A: HCT116 cells were treated with 5-FU (5 µM) for 0, 5, 10, 20, 40, or 60 minutes or were pretreated with curcumin (5 µM) or wortmannin (10 nM) for 1 h and then co-treated with 1 µM 5-FU for 0, 5, 10, 20, 40, or 60 minutes. B: HCT116+ch3 cells were treated with 5-FU (1 µM) for 0, 5, 10, 20, 40, or 60 minutes or were pretreated with curcumin (5 µM) or wortmannin (10 nM) for 1 h and then co-treated with 0.1 µM 5-FU for 0, 5, 10, 20, 40, or 60 minutes. Cells were lysed and immune complex kinase assays were performed as described in Materials and Methods. Equal amounts of total protein (500 ng protein per lane) were separated by SDS-PAGE under reducing conditions and then analyzed by immunoblotting using antibodies against phosphospecific IκBα (lane I), IKK-α (lane II), and IKK-β (lane III).
    Figure Legend Snippet: Effect of 5-FU and/or curcumin or PI-3K inhibitor wortmannin on activation of IκBα kinase (IKK) in HCT116 and HCT116+ch3 colon cancer cells. A: HCT116 cells were treated with 5-FU (5 µM) for 0, 5, 10, 20, 40, or 60 minutes or were pretreated with curcumin (5 µM) or wortmannin (10 nM) for 1 h and then co-treated with 1 µM 5-FU for 0, 5, 10, 20, 40, or 60 minutes. B: HCT116+ch3 cells were treated with 5-FU (1 µM) for 0, 5, 10, 20, 40, or 60 minutes or were pretreated with curcumin (5 µM) or wortmannin (10 nM) for 1 h and then co-treated with 0.1 µM 5-FU for 0, 5, 10, 20, 40, or 60 minutes. Cells were lysed and immune complex kinase assays were performed as described in Materials and Methods. Equal amounts of total protein (500 ng protein per lane) were separated by SDS-PAGE under reducing conditions and then analyzed by immunoblotting using antibodies against phosphospecific IκBα (lane I), IKK-α (lane II), and IKK-β (lane III).

    Techniques Used: Activation Assay, Immune Complex Kinase Assay, SDS Page

    26) Product Images from "Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance"

    Article Title: Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171084

    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Figure Legend Snippet: Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).

    Techniques Used: Expressing, Staining

    Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells. Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).
    Figure Legend Snippet: Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells. Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).

    Techniques Used:

    Immunofluorescence localization of cytokeratin-13. Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).
    Figure Legend Snippet: Immunofluorescence localization of cytokeratin-13. Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).

    Techniques Used: Immunofluorescence, Staining

    TLR4 expression in hPVECs and VK2/E6E7 cells. (A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene Gapdh (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of TLR4 mRNA reported in Fig 10A. The expression of TLR4 mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.
    Figure Legend Snippet: TLR4 expression in hPVECs and VK2/E6E7 cells. (A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene Gapdh (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of TLR4 mRNA reported in Fig 10A. The expression of TLR4 mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.

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

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.

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

    Immunofluorescence localization of vimentin. hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).
    Figure Legend Snippet: Immunofluorescence localization of vimentin. hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).

    Techniques Used: Immunofluorescence, Positive Control, Staining

    Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p
    Figure Legend Snippet: Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .
    Figure Legend Snippet: RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.
    Figure Legend Snippet: Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.

    Techniques Used: Western Blot, Biomarker Assay, Expressing, Positive Control

    RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.
    Figure Legend Snippet: RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.

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

    Cytokoine ELISA of hPVECs and VK2/E6E7 cells. IL-6 (A) and IL-10 (B) in the culture supernatants of un-stimulated and LPS stimulated hPVECs and VK2/E6E7 cells at indicated time points (6, 12, 24 hrs). Cells were seeded at a density of 10 6 /well in 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Levels of IL-6 were up-regulated in LPS-induced cells. Values represent the mean ± SD of three independent experiments performed on different days. Level of significance (*p
    Figure Legend Snippet: Cytokoine ELISA of hPVECs and VK2/E6E7 cells. IL-6 (A) and IL-10 (B) in the culture supernatants of un-stimulated and LPS stimulated hPVECs and VK2/E6E7 cells at indicated time points (6, 12, 24 hrs). Cells were seeded at a density of 10 6 /well in 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Levels of IL-6 were up-regulated in LPS-induced cells. Values represent the mean ± SD of three independent experiments performed on different days. Level of significance (*p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p
    Figure Legend Snippet: qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).
    Figure Legend Snippet: Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Figure Legend Snippet: Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).

    Techniques Used: Expressing, Staining

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.

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

    Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p
    Figure Legend Snippet: Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .
    Figure Legend Snippet: RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.
    Figure Legend Snippet: Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.

    Techniques Used: Western Blot, Biomarker Assay, Expressing, Positive Control

    RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.
    Figure Legend Snippet: RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.

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

    Hypothesized mechanism of Hb-α and Hb-β expression and their role in the function vaginal cells. Stimulation of hPVECs with LPS up-regulates the expression TLR4, NF-κB, Hb-α, Hb-β and pro-inflammatory cytokine (e.g. IL-6). The co-receptor MD2 triggers interactions between the cell surface domain of TLR4 and LPS. Activated TLR4 interacts with cytoplasmic adaptor molecules MyD88 or TRIF which subsequently activates IKK complex. The IKK complex induces phosphorylation of IκB and its subsequent degradation liberates NF-κB and allows it to translocate into the nucleus where it can induce target gene expression including Hb-α, Hb-β and IL-6 etc and may contribute to pathogen clearance by enhancing local host immune responses in hPVECs. This figure represents the best-fit model for these effects.
    Figure Legend Snippet: Hypothesized mechanism of Hb-α and Hb-β expression and their role in the function vaginal cells. Stimulation of hPVECs with LPS up-regulates the expression TLR4, NF-κB, Hb-α, Hb-β and pro-inflammatory cytokine (e.g. IL-6). The co-receptor MD2 triggers interactions between the cell surface domain of TLR4 and LPS. Activated TLR4 interacts with cytoplasmic adaptor molecules MyD88 or TRIF which subsequently activates IKK complex. The IKK complex induces phosphorylation of IκB and its subsequent degradation liberates NF-κB and allows it to translocate into the nucleus where it can induce target gene expression including Hb-α, Hb-β and IL-6 etc and may contribute to pathogen clearance by enhancing local host immune responses in hPVECs. This figure represents the best-fit model for these effects.

    Techniques Used: Expressing

    Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.
    Figure Legend Snippet: Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.

    Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Binding Assay, Mutagenesis

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p
    Figure Legend Snippet: qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    27) Product Images from "Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences"

    Article Title: Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01978

    Canonical and non-canonical NF-κB signaling in humans. Activation of the canonical NF-κB pathway is triggered by a broad range of proinflammatory cytokines such as TNFα or IL-1, bacterial pattern recognition molecules such as LPS, or antigen stimulation. Non-canonical signaling is triggered by TNF family receptors and their ligands, resulting in activation of NIK kinase activity. Both pathways cumulate in the activation of IKK (IκB-kinases) which phosphorylate inhibitory IκB binding partners for their poly-ubiquitination and proteosomal degradation (canonical axis) or the processing of p100 into its active form (non-canonical axis). Resulting NF-κB dimers translocate to the nucleus. Depending on their assembly into activating hetero- or repressive homo-dimeric conformations, NF-κB signaling regulates the expression of hundreds of target genes. TNF(R), tumor necrosis factor (receptor); IL-1(R), interleukin-1 (receptor); LPS, lipopolysaccharide; BAFF(-R), B-cell activating factor (receptor); LTβ(R), lymphotoxin β (receptor); TLR, toll-like receptor; TCR/BCR, T-cell/B-cell receptor; NIK, NF-κB inducing kinase; NEMO, NF-κB essential modulator; IKK, IκB kinase; IκB, Inhibitor of NF-κB; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.
    Figure Legend Snippet: Canonical and non-canonical NF-κB signaling in humans. Activation of the canonical NF-κB pathway is triggered by a broad range of proinflammatory cytokines such as TNFα or IL-1, bacterial pattern recognition molecules such as LPS, or antigen stimulation. Non-canonical signaling is triggered by TNF family receptors and their ligands, resulting in activation of NIK kinase activity. Both pathways cumulate in the activation of IKK (IκB-kinases) which phosphorylate inhibitory IκB binding partners for their poly-ubiquitination and proteosomal degradation (canonical axis) or the processing of p100 into its active form (non-canonical axis). Resulting NF-κB dimers translocate to the nucleus. Depending on their assembly into activating hetero- or repressive homo-dimeric conformations, NF-κB signaling regulates the expression of hundreds of target genes. TNF(R), tumor necrosis factor (receptor); IL-1(R), interleukin-1 (receptor); LPS, lipopolysaccharide; BAFF(-R), B-cell activating factor (receptor); LTβ(R), lymphotoxin β (receptor); TLR, toll-like receptor; TCR/BCR, T-cell/B-cell receptor; NIK, NF-κB inducing kinase; NEMO, NF-κB essential modulator; IKK, IκB kinase; IκB, Inhibitor of NF-κB; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.

    Techniques Used: Activation Assay, Activity Assay, Binding Assay, Expressing

    28) Product Images from "KIOM-79 Protects AGE-Induced Retinal Pericyte Apoptosis via Inhibition of NF-kappaB Activation In Vitro and In Vivo"

    Article Title: KIOM-79 Protects AGE-Induced Retinal Pericyte Apoptosis via Inhibition of NF-kappaB Activation In Vitro and In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043591

    Effect of KIOM-79 on the formation of advanced glycation end products (AGEs). AGEs levels in (A) blood and (B) vitreous were obtained using ELISA-based assays. The values in the graph represent means ± SE, n = 8. *p
    Figure Legend Snippet: Effect of KIOM-79 on the formation of advanced glycation end products (AGEs). AGEs levels in (A) blood and (B) vitreous were obtained using ELISA-based assays. The values in the graph represent means ± SE, n = 8. *p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Protein expression of Bax (A), TNF-α (B) and cIAP-2 (C). Western blot analysis in retina tissue from a normal Zucker lean rat (ZL), vehicle-treated ZDF rat (ZDF) and ZDF rat treated with KIOM-79 (KIOM-79). Values in the bar graphs represent means ± SE, n = 8. *p
    Figure Legend Snippet: Protein expression of Bax (A), TNF-α (B) and cIAP-2 (C). Western blot analysis in retina tissue from a normal Zucker lean rat (ZL), vehicle-treated ZDF rat (ZDF) and ZDF rat treated with KIOM-79 (KIOM-79). Values in the bar graphs represent means ± SE, n = 8. *p

    Techniques Used: Expressing, Western Blot

    Effect of KIOM-79 on histopathological changes. (A) Trypsin-digested retinal vessel pattern analysis. Representative whole mount of retinal vessel from a normal Zucker lean rat (ZL), vehicle-treated ZDF rat (ZDF) and ZDF rat treated with KIOM-79 (KIOM-79) was stained with periodic acid-Schiff. Acellular capillary (black arrow) were observed in vehicle-treated ZDF rats. X200 magnification. (B) Immunofluorescence staining for NG2 (green) in trypsin-digested retinal vessels. (C) In fluorescein-dextran microscopy, the white arrowhead and arrow indicate the fluorescein leakage areas and vessel narrowing (magnified inset), respectively. For quantitative analysis, (D) acellular formation of retinal vessel were counted, and (E) the number of pericytes was determined by counting the number of NG2 positive cells per mm 2 of capillary area. (F) The changes in retinal angiography lesion scores obtained using the retinal scoring methods described in Materials and Methods . Values in the bar graphs represent means ± SE, n = 8. *p
    Figure Legend Snippet: Effect of KIOM-79 on histopathological changes. (A) Trypsin-digested retinal vessel pattern analysis. Representative whole mount of retinal vessel from a normal Zucker lean rat (ZL), vehicle-treated ZDF rat (ZDF) and ZDF rat treated with KIOM-79 (KIOM-79) was stained with periodic acid-Schiff. Acellular capillary (black arrow) were observed in vehicle-treated ZDF rats. X200 magnification. (B) Immunofluorescence staining for NG2 (green) in trypsin-digested retinal vessels. (C) In fluorescein-dextran microscopy, the white arrowhead and arrow indicate the fluorescein leakage areas and vessel narrowing (magnified inset), respectively. For quantitative analysis, (D) acellular formation of retinal vessel were counted, and (E) the number of pericytes was determined by counting the number of NG2 positive cells per mm 2 of capillary area. (F) The changes in retinal angiography lesion scores obtained using the retinal scoring methods described in Materials and Methods . Values in the bar graphs represent means ± SE, n = 8. *p

    Techniques Used: Staining, Immunofluorescence, Microscopy

    Analysis of KIOM-79 by HPLC. HPLC chromatogram of KIOM-79 by Agilent 1200 HPLC system with MWD detection at 254 nm (A) and 3D-HPLC chromatogram of KIOM-79 by Shimadzu HPLC system with DAD detection at 200–400 nm (B).
    Figure Legend Snippet: Analysis of KIOM-79 by HPLC. HPLC chromatogram of KIOM-79 by Agilent 1200 HPLC system with MWD detection at 254 nm (A) and 3D-HPLC chromatogram of KIOM-79 by Shimadzu HPLC system with DAD detection at 200–400 nm (B).

    Techniques Used: High Performance Liquid Chromatography

    Apoptosis of retinal pericytes. (A) Representative trypsin-digested retinal vessels from a normal Zucker lean rat (ZL), vehicle-treated ZDF rat (ZDF) and ZDF rat treated with KIOM-79 (KIOM-79) were stained with TUNEL (brown). Apoptotic pericytes (arrow) were observed in vehicle-treated ZDF rats. X200 magnification. (B) Quantitative analysis of TUNEL-positive cells in trypsin-digested retinal vessel. All data are expressed as mean ± SE, n = 8. *p
    Figure Legend Snippet: Apoptosis of retinal pericytes. (A) Representative trypsin-digested retinal vessels from a normal Zucker lean rat (ZL), vehicle-treated ZDF rat (ZDF) and ZDF rat treated with KIOM-79 (KIOM-79) were stained with TUNEL (brown). Apoptotic pericytes (arrow) were observed in vehicle-treated ZDF rats. X200 magnification. (B) Quantitative analysis of TUNEL-positive cells in trypsin-digested retinal vessel. All data are expressed as mean ± SE, n = 8. *p

    Techniques Used: Staining, TUNEL Assay

    Effect of KIOM-79 on NF-κB activation. (A) Representative photomicrographs of retinal vessels from a normal Zucker lean rat (ZL), vehicle-treated ZDF rat (ZDF) and ZDF rat treated with KIOM-79 (KIOM-79). NF-κB activation (arrow) was determined by southwestern histochemistry. The sections were visualized by nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate staining and counterstained with methyl green X200 magnification. (B) Analysis of NF-κB DNA binding activity by ELISA-based assay. KIOM-79 significantly inhibited nuclear NF-κB DNA binding activity (p
    Figure Legend Snippet: Effect of KIOM-79 on NF-κB activation. (A) Representative photomicrographs of retinal vessels from a normal Zucker lean rat (ZL), vehicle-treated ZDF rat (ZDF) and ZDF rat treated with KIOM-79 (KIOM-79). NF-κB activation (arrow) was determined by southwestern histochemistry. The sections were visualized by nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) substrate staining and counterstained with methyl green X200 magnification. (B) Analysis of NF-κB DNA binding activity by ELISA-based assay. KIOM-79 significantly inhibited nuclear NF-κB DNA binding activity (p

    Techniques Used: Activation Assay, Staining, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    Effect of KIOM-79 on IκB kinase (IKK) complex activation. KIOM-79, parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhized radix and Euphorbiae radix, and the IKK-2 inhibitor IV supplied by the manufacturer in this kit were tested for their ability to inhibit IKK-b activity using an ELISA-based kinase activity assay. Inhibition by a compound was defined by the 50% inhibition concentration (IC 50 ) of the IKK activity. IC 50 values were calculated from the dose inhibition curve. Values in the graphs represent means ± SE, n = 6.
    Figure Legend Snippet: Effect of KIOM-79 on IκB kinase (IKK) complex activation. KIOM-79, parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhized radix and Euphorbiae radix, and the IKK-2 inhibitor IV supplied by the manufacturer in this kit were tested for their ability to inhibit IKK-b activity using an ELISA-based kinase activity assay. Inhibition by a compound was defined by the 50% inhibition concentration (IC 50 ) of the IKK activity. IC 50 values were calculated from the dose inhibition curve. Values in the graphs represent means ± SE, n = 6.

    Techniques Used: Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Kinase Assay, Inhibition, Concentration Assay

    The effect of KIOM-79 on NF-κB activity in AGE-BSA-treated retinal pericytes. The pericytes were exposed for 6 hours to AGE-BSA (10 to 200 µg/mL) or BSA (A). The pericytes were pretreated with KIOM-79 or PDTC for 1 hour, followed by treatment with 100 µg/mL AGE-BSA for 6 hours (B–D). Apoptotic cells were detected using an FITC-labelled Annexin V protein and flow cytometry (A and B). Electrophoretic mobility shift assay for NF-κB (C). Western blot analysis was used to detect phospho-IκB-α and IκB-α (D). Each bar represents the mean ± SE from four independent experiments (*p
    Figure Legend Snippet: The effect of KIOM-79 on NF-κB activity in AGE-BSA-treated retinal pericytes. The pericytes were exposed for 6 hours to AGE-BSA (10 to 200 µg/mL) or BSA (A). The pericytes were pretreated with KIOM-79 or PDTC for 1 hour, followed by treatment with 100 µg/mL AGE-BSA for 6 hours (B–D). Apoptotic cells were detected using an FITC-labelled Annexin V protein and flow cytometry (A and B). Electrophoretic mobility shift assay for NF-κB (C). Western blot analysis was used to detect phospho-IκB-α and IκB-α (D). Each bar represents the mean ± SE from four independent experiments (*p

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Electrophoretic Mobility Shift Assay, Western Blot

    29) Product Images from "Auranofin displays anticancer activity against ovarian cancer cells through FOXO3 activation independent of p53"

    Article Title: Auranofin displays anticancer activity against ovarian cancer cells through FOXO3 activation independent of p53

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2014.2579

    Chemical structure of auranofin.
    Figure Legend Snippet: Chemical structure of auranofin.

    Techniques Used:

    A diagram represents the model for the FOXO3-dependent anticancer function of auranofin. A schematic shows that auranofin inhibits IKK-β and leads to FOXO3 translocation from the cytoplasm into the nucleus to upregulate the expression of Bax and Bim pro-apoptotic proteins, as well as to downregulate of the expression of Bcl-2 anti-apoptotic protein. In addition, auranofin induces the activation of caspase-3 protein in a FOXO3-dependent manner. As a result of this FOXO3-mediated apoptotic pathway, auranofin promotes cancer cell apoptosis.
    Figure Legend Snippet: A diagram represents the model for the FOXO3-dependent anticancer function of auranofin. A schematic shows that auranofin inhibits IKK-β and leads to FOXO3 translocation from the cytoplasm into the nucleus to upregulate the expression of Bax and Bim pro-apoptotic proteins, as well as to downregulate of the expression of Bcl-2 anti-apoptotic protein. In addition, auranofin induces the activation of caspase-3 protein in a FOXO3-dependent manner. As a result of this FOXO3-mediated apoptotic pathway, auranofin promotes cancer cell apoptosis.

    Techniques Used: Translocation Assay, Expressing, Activation Assay

    Auranofin downregulates IKK-β expression and promotes the nuclear translocation of FOXO3 protein in SKOV3 cells. SKOV3 cells were treated with auranofin (25, 50 and 100 nM) or control (0 nM) for 48 h and cytoplasmic and nuclear extracts that had been fractionated from cells were analyzed by western blot analysis with specific antibodies as indicated. β-actin and PARP1 represent the loading controls of cytoplasmic and nuclear extracts, respectively.
    Figure Legend Snippet: Auranofin downregulates IKK-β expression and promotes the nuclear translocation of FOXO3 protein in SKOV3 cells. SKOV3 cells were treated with auranofin (25, 50 and 100 nM) or control (0 nM) for 48 h and cytoplasmic and nuclear extracts that had been fractionated from cells were analyzed by western blot analysis with specific antibodies as indicated. β-actin and PARP1 represent the loading controls of cytoplasmic and nuclear extracts, respectively.

    Techniques Used: Expressing, Translocation Assay, Western Blot

    Auranofin inhibits cell survival or growth of SKOV3 cells. The cell numbers were determined by the cell counting assay after SKOV3 cells (1×10 4 cells/plate) were treated with auranofin (100 nM) or the control (DMSO) for 0, 24, 72 and 120 h. The significant P-values between the auranofin group versus the control group are indicated ( * P
    Figure Legend Snippet: Auranofin inhibits cell survival or growth of SKOV3 cells. The cell numbers were determined by the cell counting assay after SKOV3 cells (1×10 4 cells/plate) were treated with auranofin (100 nM) or the control (DMSO) for 0, 24, 72 and 120 h. The significant P-values between the auranofin group versus the control group are indicated ( * P

    Techniques Used: Cell Counting

    The cytotoxic effect of auranofin on SKOV3 cells. (A) The dose-dependent effect of auranofin (0, 50, 100, 200 and 400 nM) on SKOV3 cells after 72-h incubation. (B) The time-dependent effect of auranofin (100 nM) on SKOV3 cells after 0, 24, 72 and 120 h. The cell viability was determined by the MTT assay and the relative cell survival rate percentage was calculated by dividing the optical density of each auranofin treatment by the optical density of the control (DMSO) treatment. The significant P-values between the auranofin group versus the control group are indicated ( * P
    Figure Legend Snippet: The cytotoxic effect of auranofin on SKOV3 cells. (A) The dose-dependent effect of auranofin (0, 50, 100, 200 and 400 nM) on SKOV3 cells after 72-h incubation. (B) The time-dependent effect of auranofin (100 nM) on SKOV3 cells after 0, 24, 72 and 120 h. The cell viability was determined by the MTT assay and the relative cell survival rate percentage was calculated by dividing the optical density of each auranofin treatment by the optical density of the control (DMSO) treatment. The significant P-values between the auranofin group versus the control group are indicated ( * P

    Techniques Used: Incubation, MTT Assay

    Auranofin suppresses the colony-forming ability of SKOV3 cells. The colony numbers were determined by the colony formation assay. (A) SKOV3 cells (500 cells/plate) were treated with auranofin (100 nM) or the control (DMSO) for 7 days and stained with crystal violet solution. The representative images of the assays are shown. (B) The numbers of colonies in the auranofin-treated plates were compared with those of the controltreated plates. The results are the mean ± SEM numbers of cell colonies of three replicates. * P
    Figure Legend Snippet: Auranofin suppresses the colony-forming ability of SKOV3 cells. The colony numbers were determined by the colony formation assay. (A) SKOV3 cells (500 cells/plate) were treated with auranofin (100 nM) or the control (DMSO) for 7 days and stained with crystal violet solution. The representative images of the assays are shown. (B) The numbers of colonies in the auranofin-treated plates were compared with those of the controltreated plates. The results are the mean ± SEM numbers of cell colonies of three replicates. * P

    Techniques Used: Colony Assay, Staining

    Auranofin induces cellular apoptosis in SKOV3 cells. (A) SKOV3 cells were treated with auranofin (100 nM) or control (DMSO) for 48 h. Total lysates of cells were analyzed by western blot analysis with specific antibodies against apoptosis-related proteins as indicated. β-actin represents the loading controls. (B) DNA samples extracted from SKOV3 cells, which were treated with auranofin or control as described above, and subjected to DNA fragmentation assay. Equal amounts of the extracted DNA (2 μg/lane) and size markers (1-kb ladder) were subjected to electrophoresis on 2% agarose gels, which were stained with ethidium bromide and photographed.
    Figure Legend Snippet: Auranofin induces cellular apoptosis in SKOV3 cells. (A) SKOV3 cells were treated with auranofin (100 nM) or control (DMSO) for 48 h. Total lysates of cells were analyzed by western blot analysis with specific antibodies against apoptosis-related proteins as indicated. β-actin represents the loading controls. (B) DNA samples extracted from SKOV3 cells, which were treated with auranofin or control as described above, and subjected to DNA fragmentation assay. Equal amounts of the extracted DNA (2 μg/lane) and size markers (1-kb ladder) were subjected to electrophoresis on 2% agarose gels, which were stained with ethidium bromide and photographed.

    Techniques Used: Western Blot, DNA Fragmentation Assay, Electrophoresis, Staining

    FOXO3 is essential for auranofin-mediated apoptosis in SKOV3 cells. SKOV3 cells were transfected with control-siRNA or FOXO3-siRNA for 24 h as described in Materials and methods. Subsequently, the transfected cells were treated with auranofin (100 nM) or control (DMSO) for 48 h and total lysates of cells were analyzed by western blot analysis with specific antibodies against apoptosis-related proteins as indicated. β-actin represents the loading controls.
    Figure Legend Snippet: FOXO3 is essential for auranofin-mediated apoptosis in SKOV3 cells. SKOV3 cells were transfected with control-siRNA or FOXO3-siRNA for 24 h as described in Materials and methods. Subsequently, the transfected cells were treated with auranofin (100 nM) or control (DMSO) for 48 h and total lysates of cells were analyzed by western blot analysis with specific antibodies against apoptosis-related proteins as indicated. β-actin represents the loading controls.

    Techniques Used: Transfection, Western Blot

    30) Product Images from "Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats"

    Article Title: Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats

    Journal: Critical care medicine

    doi: 10.1097/CCM.0b013e3181c027ae

    Effects of recombinant osteopontin (OPN) treatment on matrix metalloproteinase (MMP)-9 induction and tissue inhibitor of MMP (TIMP)-1 reduction in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). Western blots for MMP-9 (
    Figure Legend Snippet: Effects of recombinant osteopontin (OPN) treatment on matrix metalloproteinase (MMP)-9 induction and tissue inhibitor of MMP (TIMP)-1 reduction in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). Western blots for MMP-9 (

    Techniques Used: Recombinant, Western Blot

    31) Product Images from "Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance"

    Article Title: Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171084

    Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).
    Figure Legend Snippet: Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Figure Legend Snippet: Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).

    Techniques Used: Expressing, Staining

    Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p
    Figure Legend Snippet: Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.
    Figure Legend Snippet: Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.

    Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Binding Assay, Mutagenesis

    32) Product Images from "IL-1?-Induced Increase in Intestinal Epithelial Tight Junction Permeability Is Mediated by MEKK-1 Activation of Canonical NF-?B Pathway"

    Article Title: IL-1?-Induced Increase in Intestinal Epithelial Tight Junction Permeability Is Mediated by MEKK-1 Activation of Canonical NF-?B Pathway

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2010.100371

    Effect of siRNA-induced MEKK-1 and NIK knockdown of IL-1β–induced increase in Caco-2 TJ permeability. A: MEKK-1 siRNA transfection resulted in a near complete depletion in MEKK-1 protein expression as determined by Western blot analysis. B: MEKK-1 siRNA transfection prevented the IL-1β–induced drop in Caco-2 TER (means ± SE, n = 4). * P
    Figure Legend Snippet: Effect of siRNA-induced MEKK-1 and NIK knockdown of IL-1β–induced increase in Caco-2 TJ permeability. A: MEKK-1 siRNA transfection resulted in a near complete depletion in MEKK-1 protein expression as determined by Western blot analysis. B: MEKK-1 siRNA transfection prevented the IL-1β–induced drop in Caco-2 TER (means ± SE, n = 4). * P

    Techniques Used: Permeability, Transfection, Expressing, Western Blot

    Effect of siRNA-induced MEKK-1 knockdown of IL-1β-activation of NF-κB p65 and p52. A: MEKK-1 siRNA transfection prevented the IL-1β–induced degradation of IκB-α as assessed by Western blot analysis. B: MEKK-1 silencing inhibited the IL-1β–induced binding of p65 to its binding site on the DNA probe as measured by DNA ELISA-binding assay. * P
    Figure Legend Snippet: Effect of siRNA-induced MEKK-1 knockdown of IL-1β-activation of NF-κB p65 and p52. A: MEKK-1 siRNA transfection prevented the IL-1β–induced degradation of IκB-α as assessed by Western blot analysis. B: MEKK-1 silencing inhibited the IL-1β–induced binding of p65 to its binding site on the DNA probe as measured by DNA ELISA-binding assay. * P

    Techniques Used: Activation Assay, Transfection, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

    Effect of siRNA induced knockdown of MEKK-1 on IL-1β–induced increase in MLCK gene activity and protein expression. A: MEKK-1 siRNA transfection resulted in a complete inhibition of IL-1β–induced increase in MLCK promoter activity. * P
    Figure Legend Snippet: Effect of siRNA induced knockdown of MEKK-1 on IL-1β–induced increase in MLCK gene activity and protein expression. A: MEKK-1 siRNA transfection resulted in a complete inhibition of IL-1β–induced increase in MLCK promoter activity. * P

    Techniques Used: Activity Assay, Expressing, Transfection, Inhibition

    Proposed scheme of the intracellular pathways involved in IL-1β–induced increase in intestinal epithelial tight junction (TJ) permeability. IL-1β treatment resulted in activation of the MEKK-1 and NIK signaling cascades. MEKK-1 activation resulted in a step-wise activation of IKK and the canonical NF-κB pathway and activation of the MLCK gene, culminating in the opening of the TJ barrier.
    Figure Legend Snippet: Proposed scheme of the intracellular pathways involved in IL-1β–induced increase in intestinal epithelial tight junction (TJ) permeability. IL-1β treatment resulted in activation of the MEKK-1 and NIK signaling cascades. MEKK-1 activation resulted in a step-wise activation of IKK and the canonical NF-κB pathway and activation of the MLCK gene, culminating in the opening of the TJ barrier.

    Techniques Used: Permeability, Activation Assay

    Time-course effect of IL-1β on Caco-2 IKK catalytic subunit activation. A: Time-course effect of IL-1β (10 ng/ml) on Caco-2 IKK-α and IKK-β phosphorylation. IL-1β caused a time-dependent increase in Caco-2 IKK-α and IKK-β activation. B: Time-course effect of IL-1β on IκB-α degradation (β-actin was used for equal protein loading). C: Graph of IKK-α/IKK-β activation versus IκB-α degradation ( r = 0.905). D: MEKK-1 siRNA transfection prevented the IL-1β–induced phosphorylation of IKK-α and IKK-β as assessed by Western blot analysis. E: NIK siRNA transfection did not prevent the IL-1β–induced phosphorylation of IKK-α and IKK-β.
    Figure Legend Snippet: Time-course effect of IL-1β on Caco-2 IKK catalytic subunit activation. A: Time-course effect of IL-1β (10 ng/ml) on Caco-2 IKK-α and IKK-β phosphorylation. IL-1β caused a time-dependent increase in Caco-2 IKK-α and IKK-β activation. B: Time-course effect of IL-1β on IκB-α degradation (β-actin was used for equal protein loading). C: Graph of IKK-α/IKK-β activation versus IκB-α degradation ( r = 0.905). D: MEKK-1 siRNA transfection prevented the IL-1β–induced phosphorylation of IKK-α and IKK-β as assessed by Western blot analysis. E: NIK siRNA transfection did not prevent the IL-1β–induced phosphorylation of IKK-α and IKK-β.

    Techniques Used: Activation Assay, Transfection, Western Blot

    Time course effect of IL-1β on Caco-2 MEKK-1 and NIK activation. A: Time course effect of IL-1β (10 ng/ml) on Caco-2 MEKK-1 phosphorylation (total MEKK-1 was used for equal protein loading). B: Time course effect of IL-1β on NIK phosphorylation (total NIK was used for equal protein loading). IL-1β caused a time-dependent increase in Caco-2 MEKK-1 and NIK activation.
    Figure Legend Snippet: Time course effect of IL-1β on Caco-2 MEKK-1 and NIK activation. A: Time course effect of IL-1β (10 ng/ml) on Caco-2 MEKK-1 phosphorylation (total MEKK-1 was used for equal protein loading). B: Time course effect of IL-1β on NIK phosphorylation (total NIK was used for equal protein loading). IL-1β caused a time-dependent increase in Caco-2 MEKK-1 and NIK activation.

    Techniques Used: Activation Assay

    33) Product Images from "Schizandrin B inhibits the cis-DDP-induced apoptosis of HK-2 cells by activating ERK/NF-κB signaling to regulate the expression of surviving"

    Article Title: Schizandrin B inhibits the cis-DDP-induced apoptosis of HK-2 cells by activating ERK/NF-κB signaling to regulate the expression of surviving

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2018.3409

    Effects of SchB on the cis-DDP-induced apoptosis of HK-2 cells. (A) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with 10 µ M cis-DDP for 24 h and then stained with DAPI to observe nuclear morphology (magnification, ×400). (B) Following co-staining of the HK-2 cells with Annexin V-FITC and PI, the apoptotic rates of the HK-2 cells were determined by flow cytometry. (C) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with cis-DDP for 24 h. The activation of caspase-3 was then determined by in-cell western blot analysis. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Effects of SchB on the cis-DDP-induced apoptosis of HK-2 cells. (A) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with 10 µ M cis-DDP for 24 h and then stained with DAPI to observe nuclear morphology (magnification, ×400). (B) Following co-staining of the HK-2 cells with Annexin V-FITC and PI, the apoptotic rates of the HK-2 cells were determined by flow cytometry. (C) Following the pre-incubation of HK-2 cells with 10, 20 and 40 µ M SchB for 2 h, the cells were stimulated with cis-DDP for 24 h. The activation of caspase-3 was then determined by in-cell western blot analysis. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry, Activation Assay, In-Cell ELISA, Standard Deviation

    Effects of SchB and cis-DDP on the apoptosis/survival signaling pathway of HK-2 cells. HK-2 cells were used to establish a SchB (40 µ M) incubation group, a cis-DDP (10 µ M) incubation group and a SchB (40 µ M) + cis-DDP (10 µ M) co-incubation group and the cells were incubated for 1, 2, 4, 8 and 16 h. (A) The activation of associated signal transduction pathways was determined using protein chips. (B) Heat-maps of the protein chip results. (C) The predicted signaling pathways in the cis-DDP-induced apoptosis and SchB-induced survival of HK-2 cells. SchB, schizandrin B; cis-DDP, cis-dichlorodiammine platinum; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κBα; PARP, poly(ADP-ribose) polymerase; p, phosphorylated.
    Figure Legend Snippet: Effects of SchB and cis-DDP on the apoptosis/survival signaling pathway of HK-2 cells. HK-2 cells were used to establish a SchB (40 µ M) incubation group, a cis-DDP (10 µ M) incubation group and a SchB (40 µ M) + cis-DDP (10 µ M) co-incubation group and the cells were incubated for 1, 2, 4, 8 and 16 h. (A) The activation of associated signal transduction pathways was determined using protein chips. (B) Heat-maps of the protein chip results. (C) The predicted signaling pathways in the cis-DDP-induced apoptosis and SchB-induced survival of HK-2 cells. SchB, schizandrin B; cis-DDP, cis-dichlorodiammine platinum; ERK, extracellular signal-regulated kinase; JNK, c-Jun-N-terminal kinase; NF-κB, nuclear factor κB; IκBα, inhibitor of NF-κBα; PARP, poly(ADP-ribose) polymerase; p, phosphorylated.

    Techniques Used: Incubation, Activation Assay, Transduction, Chromatin Immunoprecipitation

    Signaling via ERK/NF-κB activation in the cis-DDP-induced apoptosis of HK-2 cells. (A) HK-2 cells were incubated with 40 µ M SchB for 0, 0.5, 1, 2, 4 and 8 h, and the cell lysates were collected. Western blotting was performed to determine the phosphorylation of ERK, IKKα/β, IκBα and p65. (B) HK-2 cells were incubated with 10, 20 and 40 µ M SchB for 0.5, 2, 2 and 8 h, and the phosphorylation of ERK, IKKα/β, IκBα and p65 was determined, respectively. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Signaling via ERK/NF-κB activation in the cis-DDP-induced apoptosis of HK-2 cells. (A) HK-2 cells were incubated with 40 µ M SchB for 0, 0.5, 1, 2, 4 and 8 h, and the cell lysates were collected. Western blotting was performed to determine the phosphorylation of ERK, IKKα/β, IκBα and p65. (B) HK-2 cells were incubated with 10, 20 and 40 µ M SchB for 0.5, 2, 2 and 8 h, and the phosphorylation of ERK, IKKα/β, IκBα and p65 was determined, respectively. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Activation Assay, Incubation, Western Blot, Standard Deviation

    Effects of survivin on the cis-DDP-induced apoptosis of HK-2 cells. (A) The hSurvivin RNAi sequence was transfected into HK-2 cells. Transfected and untransfected cells were then incubated with 40 µ M SchB alone or with PDTC or U0126 for 4 h and stimulated with cis-DDP for 24 h. The activation of cleaved-caspase-3 protein was determined. (B) Survivin RNAi sequences were synthesized and transfected into HK-2 cells, which were incubated with 40 µ M SchB for 16 h. The expression of survivin was then determined. Data are presented as the mean ± standard deviation (n=3). * P
    Figure Legend Snippet: Effects of survivin on the cis-DDP-induced apoptosis of HK-2 cells. (A) The hSurvivin RNAi sequence was transfected into HK-2 cells. Transfected and untransfected cells were then incubated with 40 µ M SchB alone or with PDTC or U0126 for 4 h and stimulated with cis-DDP for 24 h. The activation of cleaved-caspase-3 protein was determined. (B) Survivin RNAi sequences were synthesized and transfected into HK-2 cells, which were incubated with 40 µ M SchB for 16 h. The expression of survivin was then determined. Data are presented as the mean ± standard deviation (n=3). * P

    Techniques Used: Sequencing, Transfection, Incubation, Activation Assay, Synthesized, Expressing, Standard Deviation

    Effects of SchB on the cis-DDP-induced loss of viability of HK-2 cells. (A) HK-2 cells were incubated with SchB at different concentrations (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 24 h. Cell viability was then determined using a Cell Counting kit-8 assay. (B) HK-2 cells were incubated with 40 µ M SchB for 0, 6, 12, 18, 24, 30, 36 and 42 h, and the cell viability was then determined. (C) HK-2 cells were incubated with cis-DDP at different concentrations (0, 2.5, 5, 10, 20 and 30 µ M) for 24 h, and cell viability was then determined. (D) Cells were pre-incubated with different concentrations of SchB (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 2 h and then stimulated with 10 µ M cis-DDP for 24 h. The viability of the cells was determined. Data are presented as the mean ± standard deviation (n=3). (A-C) * P
    Figure Legend Snippet: Effects of SchB on the cis-DDP-induced loss of viability of HK-2 cells. (A) HK-2 cells were incubated with SchB at different concentrations (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 24 h. Cell viability was then determined using a Cell Counting kit-8 assay. (B) HK-2 cells were incubated with 40 µ M SchB for 0, 6, 12, 18, 24, 30, 36 and 42 h, and the cell viability was then determined. (C) HK-2 cells were incubated with cis-DDP at different concentrations (0, 2.5, 5, 10, 20 and 30 µ M) for 24 h, and cell viability was then determined. (D) Cells were pre-incubated with different concentrations of SchB (0, 2.5, 5, 10, 20, 40 and 80 µ M) for 2 h and then stimulated with 10 µ M cis-DDP for 24 h. The viability of the cells was determined. Data are presented as the mean ± standard deviation (n=3). (A-C) * P

    Techniques Used: Incubation, Cell Counting, Standard Deviation

    34) Product Images from "Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance"

    Article Title: Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171084

    Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).
    Figure Legend Snippet: Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).

    Techniques Used: Expressing, Staining

    Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells. Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).
    Figure Legend Snippet: Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells. Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).

    Techniques Used:

    Immunofluorescence localization of cytokeratin-13. Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).
    Figure Legend Snippet: Immunofluorescence localization of cytokeratin-13. Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).

    Techniques Used: Immunofluorescence, Staining

    TLR4 expression in hPVECs and VK2/E6E7 cells. (A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene Gapdh (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of TLR4 mRNA reported in Fig 10A. The expression of TLR4 mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.
    Figure Legend Snippet: TLR4 expression in hPVECs and VK2/E6E7 cells. (A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene Gapdh (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of TLR4 mRNA reported in Fig 10A. The expression of TLR4 mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.

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

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.

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

    Immunofluorescence localization of vimentin. hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).
    Figure Legend Snippet: Immunofluorescence localization of vimentin. hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).

    Techniques Used: Immunofluorescence, Positive Control, Staining

    Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p
    Figure Legend Snippet: Expression of NF- κ B in hPVECs and VK2/E6E7 cells. (A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10 6 /well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay

    RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .
    Figure Legend Snippet: RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.
    Figure Legend Snippet: Western blot analysis of biomarker expression in hPVECs, VK2/E6E7 cells and RBCs. (a) Cytokeratin-13 (47kDa) expression: Lane-1: RBCs, lane-2: hPVECs and lane-3: VK2/E6E7 cells. (b) Solute carrier family 4 member-1 (SLC4A1) (100 kDa) expression: Lane-1: RBCs (positive control), lane-2: hPVECs, lane-3: VK2/E6E7 cells. (c)Vimentin (54 kDa) expression: Lane-1: vimentin expression in HeLa cells (positive control), Lane-2: hPVECs, Lane-3: VK2/E6E7 cells. (d) β-actin (42 kDa) expression in hPVECs (loading control). The blots shown are one of the representative pictures from three independent experiments performed on three different days.

    Techniques Used: Western Blot, Biomarker Assay, Expressing, Positive Control

    RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.
    Figure Legend Snippet: RT-PCR analysis of human-β defensin-1 ( hBD-1 ). (A) mRNA expression of hBD1 (139bp) was observed in un-stimulated (a1, a3), LPS stimulated (a2, a4) in hPVECs (a1, a2) and VK2/E6E7 cells (a3, a4). Cells were seeded at a density of 10 6 /well in a 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Expression of hBD1 mRNA was up-regulated in LPS-induced cells. Representative image of RT-PCR analysis of hBD-1 mRNAs expression is shown. Gapdh confirmed roughly equivalent loading of RNA samples. 1: hPVECs (Unstimulated); 2:hPVECs induced with LPS; 3:VK2/E6E7 cells (Unstimulated); 4:VK2/E6E7 cells stimulated with LPS (10μg/ml). (B). Densitometric analysis of bands from RT-PCR amplification products of hBD1 mRNA reported in Figure 13A. Expression of hBD-1 was increased in LPS-stimulated hPVECS and VK2/E6E7 cells.

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

    Cytokoine ELISA of hPVECs and VK2/E6E7 cells. IL-6 (A) and IL-10 (B) in the culture supernatants of un-stimulated and LPS stimulated hPVECs and VK2/E6E7 cells at indicated time points (6, 12, 24 hrs). Cells were seeded at a density of 10 6 /well in 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Levels of IL-6 were up-regulated in LPS-induced cells. Values represent the mean ± SD of three independent experiments performed on different days. Level of significance (*p
    Figure Legend Snippet: Cytokoine ELISA of hPVECs and VK2/E6E7 cells. IL-6 (A) and IL-10 (B) in the culture supernatants of un-stimulated and LPS stimulated hPVECs and VK2/E6E7 cells at indicated time points (6, 12, 24 hrs). Cells were seeded at a density of 10 6 /well in 24-well plates and treated with LPS (10 μg/ml for 6 hrs). Levels of IL-6 were up-regulated in LPS-induced cells. Values represent the mean ± SD of three independent experiments performed on different days. Level of significance (*p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p
    Figure Legend Snippet: qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    35) Product Images from "Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance"

    Article Title: Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171084

    Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).
    Figure Legend Snippet: Verification of NF-κB interactions with the binding sites in the Hb-α promoter by Chromatin immune precipitation assay (ChIP). Cross-linked and sheared chromatin from hPVECs was immune-precipitated with p65- NF-κB antibody and analysed by RT-PCR. The results revealed that NF-κB efficiently interacted with the binding sites present in the Hb-α promoter (a: untreated; b: LPS treated (10 μg/ml for 2 hrs); c: LPS treated for 24 hrs; 1: Input sample from control; 2: p65- NF-κB antibody pull down from control and LPS treated (b,c); 3: IgG antibody pull down from control (a) and LPS treated (b, c).

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. (A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, Gapdh (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.

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

    RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .
    Figure Legend Snippet: RT-PCR analysis of Hb-α and Hb-β in presence of LPS and Bay 11–7082. (A) Hb-α (429 bp) (a) and Hb-β (444 bp) (b) expression was observed in hPVECs (1,2) and VK2/E6E7 cells (3,4) before and after the treatment with LPS (10 μg/ml for 6 hrs) and Bay 11–7082 (5μM for 24 hrs). LPS-induced (a1, a3, b1, b3), Bay 11–7082 treated (a2, a4, b2, b4) are shown. Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells, whereas Bay 11–7082 attenuated this expression (c) Gapdh (238 bp) was used as loading control. The gels shown are the representative pictures from three independent experiments. (B). Densitometric analysis of RT-PCR bands shown in Fig 12A. Expression of both Hb-α and Hb-β was elevated in LPS-stimulated cells. In contrast, Bay 11–7082 attenuated the expression of Hb-α and Hb-β .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Hypothesized mechanism of Hb-α and Hb-β expression and their role in the function vaginal cells. Stimulation of hPVECs with LPS up-regulates the expression TLR4, NF-κB, Hb-α, Hb-β and pro-inflammatory cytokine (e.g. IL-6). The co-receptor MD2 triggers interactions between the cell surface domain of TLR4 and LPS. Activated TLR4 interacts with cytoplasmic adaptor molecules MyD88 or TRIF which subsequently activates IKK complex. The IKK complex induces phosphorylation of IκB and its subsequent degradation liberates NF-κB and allows it to translocate into the nucleus where it can induce target gene expression including Hb-α, Hb-β and IL-6 etc and may contribute to pathogen clearance by enhancing local host immune responses in hPVECs. This figure represents the best-fit model for these effects.
    Figure Legend Snippet: Hypothesized mechanism of Hb-α and Hb-β expression and their role in the function vaginal cells. Stimulation of hPVECs with LPS up-regulates the expression TLR4, NF-κB, Hb-α, Hb-β and pro-inflammatory cytokine (e.g. IL-6). The co-receptor MD2 triggers interactions between the cell surface domain of TLR4 and LPS. Activated TLR4 interacts with cytoplasmic adaptor molecules MyD88 or TRIF which subsequently activates IKK complex. The IKK complex induces phosphorylation of IκB and its subsequent degradation liberates NF-κB and allows it to translocate into the nucleus where it can induce target gene expression including Hb-α, Hb-β and IL-6 etc and may contribute to pathogen clearance by enhancing local host immune responses in hPVECs. This figure represents the best-fit model for these effects.

    Techniques Used: Expressing

    Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.
    Figure Legend Snippet: Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS. EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.

    Techniques Used: Electrophoretic Mobility Shift Assay, Incubation, Binding Assay, Mutagenesis

    Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.
    Figure Legend Snippet: Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells at protein level. (A, B). Immunofluorescence localization of Hb-α and Hb-β. Cytoplasmic localization (green) of Hb-α (A) and Hb-β (B) was observed in hPVECs (a,b,c) and VK2/E6E7 cells (d,e,f). DAPI stained nucleus (blue). a, d: Untreated cells; b,e: LPS treated cells; c,f: Primary antibody controls did not show Hb-α and Hb-β localization. Expression was significantly up-regulated when hPVECs and VK2/E6E7 cells stimulated with LPS (10 μg/ml for 6 hrs) (b, e). The figure shown is one of the representative pictures from three independent experiments performed on three different days (Mag. 63X). (C). Western blot analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells. β-actin (42 kDa) (loading control) expression in hPVECs (1: Untreated hPVECs, 2: hPVECs induced with LPS, 3: Untreated VK2/E6E7 cells and 4: VK2/E6E7 cells induced with LPS). (D). Densitometric analysis of bands from western blot of Hb-α and Hb-β shown in Fig 7C.

    Techniques Used: Expressing, Immunofluorescence, Staining, Western Blot

    qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p
    Figure Legend Snippet: qPCR analysis of Hb-α and Hb-β expression in hPVECs and VK2/E6E7 cells before and after induction with LPS. Cells seeded at a density of 10 6 /well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of Hb-α and Hb-β was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    36) Product Images from "Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene"

    Article Title: Lipopolysaccharide-Induced Increase in Intestinal Permeability Is Mediated by TAK-1 Activation of IKK and MLCK/MYLK Gene

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2018.12.016

    Effect of inhibition of NF-κB pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) expression in mouse enterocytes. A: NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. B: NF-κB inhibitor Bay-11 also prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or /LPS-treated mice. Densitometry of MLCK protein levels. C: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. n = 3 experiments. ∗∗ P
    Figure Legend Snippet: Effect of inhibition of NF-κB pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) expression in mouse enterocytes. A: NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC) prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. B: NF-κB inhibitor Bay-11 also prevented LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or /LPS-treated mice. Densitometry of MLCK protein levels. C: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced increase in MLCK protein expression (5 day) compared with vehicle- or LPS-treated mice. Densitometry of MLCK protein levels. n = 3 experiments. ∗∗ P

    Techniques Used: Inhibition, Activation Assay, Expressing, Mouse Assay

    Lipopolysaccharide (LPS)-induced activation of NF-κB canonical pathway was inhibited in toll-like receptor (TLR)-4 −/− and myeloid differentiation primary response (MyD)88 −/− mice and with transforming growth factor-β–activating kinase (TAK)-1 inhibition. A: LPS i.p. injections (0.1 mg/kg body weight) in TLR-4 −/− and MyD88 −/− mice did not induce inhibitory κ B (IκB)-α degradation (day 3) compared with TLR4 +/+ and MyD88 +/+ control (C) mice, respectively. Densitometry of IκB-α protein. B: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced degradation of IκB-α expression (day 3) compared with vehicle- or LPS-treated mice. Densitometry of IκB-α protein levels. n = 3 experiments. ∗∗ P
    Figure Legend Snippet: Lipopolysaccharide (LPS)-induced activation of NF-κB canonical pathway was inhibited in toll-like receptor (TLR)-4 −/− and myeloid differentiation primary response (MyD)88 −/− mice and with transforming growth factor-β–activating kinase (TAK)-1 inhibition. A: LPS i.p. injections (0.1 mg/kg body weight) in TLR-4 −/− and MyD88 −/− mice did not induce inhibitory κ B (IκB)-α degradation (day 3) compared with TLR4 +/+ and MyD88 +/+ control (C) mice, respectively. Densitometry of IκB-α protein. B: Pretreatment with TAK-1 inhibitor (in; inh), oxyzeanol (Oxz; 5 mg/kg body weight), prevented the LPS-induced degradation of IκB-α expression (day 3) compared with vehicle- or LPS-treated mice. Densitometry of IκB-α protein levels. n = 3 experiments. ∗∗ P

    Techniques Used: Activation Assay, Mouse Assay, Inhibition, Expressing

    Proposed scheme of the intracellular pathways involved in lipopolysaccharide (LPS)-induced activation of canonical NF-κB pathway. LPS treatment results in activation of the toll-like receptor (TLR)-4 signal transduction and myeloid differentiation primary response (MyD)88-dependent signaling cascade. Activation of TLR-4/MyD88 signal transduction pathway leads to the activation of IL-1 receptor-associated kinase (IRAK)-4 and phosphorylation of transforming growth factor-β–activating kinase (TAK)-1, leading to the activation of canonical NF-κB pathway of both inhibitory κ B (IκB) kinases (IKKs), IKK-α and IKK-β, which further leads to degradation of IκB-α and nuclear translocation of NF-κB p65/p50. The activation of TAK-1 and canonical NF-κB p65/p50 causes up-regulation of myosin light chain kinase (MLCK) gene and protein expression, ultimately increasing tight junction (TJ) permeability in vitro and in vivo .
    Figure Legend Snippet: Proposed scheme of the intracellular pathways involved in lipopolysaccharide (LPS)-induced activation of canonical NF-κB pathway. LPS treatment results in activation of the toll-like receptor (TLR)-4 signal transduction and myeloid differentiation primary response (MyD)88-dependent signaling cascade. Activation of TLR-4/MyD88 signal transduction pathway leads to the activation of IL-1 receptor-associated kinase (IRAK)-4 and phosphorylation of transforming growth factor-β–activating kinase (TAK)-1, leading to the activation of canonical NF-κB pathway of both inhibitory κ B (IκB) kinases (IKKs), IKK-α and IKK-β, which further leads to degradation of IκB-α and nuclear translocation of NF-κB p65/p50. The activation of TAK-1 and canonical NF-κB p65/p50 causes up-regulation of myosin light chain kinase (MLCK) gene and protein expression, ultimately increasing tight junction (TJ) permeability in vitro and in vivo .

    Techniques Used: Activation Assay, Transduction, Translocation Assay, Expressing, Permeability, In Vitro, In Vivo

    Lipopolysaccharide (LPS) activated transforming growth factor-β–activating kinase (TAK)-1 in vivo , and TAK-1 inhibition prevented the LPS-induced increase in mouse intestinal epithelial tight junction permeability. A: LPS i.p. injections (0.1 mg/kg body weight) in mice caused activation of TAK-1 (phopshoTAK-1) expression by day 3. Densitometry of phosphoTAK-1 protein levels. B: Confocal immunofluorescence showed increase in phopshoTAK-1 expression (green) ( arrowheads ) (nucleus, blue) in the intestines (enterocytes) of mice treated with LPS (0.1 mg/kg body weight). C: Pretreatment with TAK-1 inhibitor (Inh), oxozeaenol (5 mg/kg body weight), prevented the LPS-induced increase in 10K dextran flux. Oxozeaenol was dissolved in dimethyl sulfoxide and injected intraperitoneally, 1 hour before LPS injection. n = 3 experiments ( A and B ). ∗∗ P
    Figure Legend Snippet: Lipopolysaccharide (LPS) activated transforming growth factor-β–activating kinase (TAK)-1 in vivo , and TAK-1 inhibition prevented the LPS-induced increase in mouse intestinal epithelial tight junction permeability. A: LPS i.p. injections (0.1 mg/kg body weight) in mice caused activation of TAK-1 (phopshoTAK-1) expression by day 3. Densitometry of phosphoTAK-1 protein levels. B: Confocal immunofluorescence showed increase in phopshoTAK-1 expression (green) ( arrowheads ) (nucleus, blue) in the intestines (enterocytes) of mice treated with LPS (0.1 mg/kg body weight). C: Pretreatment with TAK-1 inhibitor (Inh), oxozeaenol (5 mg/kg body weight), prevented the LPS-induced increase in 10K dextran flux. Oxozeaenol was dissolved in dimethyl sulfoxide and injected intraperitoneally, 1 hour before LPS injection. n = 3 experiments ( A and B ). ∗∗ P

    Techniques Used: In Vivo, Inhibition, Permeability, Mouse Assay, Activation Assay, Expressing, Immunofluorescence, Injection

    Effect of genetic knockdown of (p65/p50) canonical pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) protein expression. A: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced increase in MLCK mRNA. B: Similarly, TAK-1 siRNA transfection in Caco-2 cells also inhibited LPS-induced increase in MLCK and phosphoMLC protein expression by Western blot analysis and was shown by relative densitometry of MLCK protein. C: p65 siRNA transfection in Caco-2 cells prevented LPS-induced increase in MLCK mRNA. D: p50 and p65 siRNA transfection in Caco-2 cells caused marked inhibition of LPS-induced increase in MLCK protein expression, as analyzed by Western blot analysis and relative analysis of MLCK protein levels by densitometry. n = 3 experiments ( A ); n = 4 experiments ( B and D ). ∗∗ P
    Figure Legend Snippet: Effect of genetic knockdown of (p65/p50) canonical pathway and transforming growth factor-β–activating kinase (TAK)-1 on lipopolysaccharide (LPS)-induced activation of myosin light chain kinase (MLCK) protein expression. A: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced increase in MLCK mRNA. B: Similarly, TAK-1 siRNA transfection in Caco-2 cells also inhibited LPS-induced increase in MLCK and phosphoMLC protein expression by Western blot analysis and was shown by relative densitometry of MLCK protein. C: p65 siRNA transfection in Caco-2 cells prevented LPS-induced increase in MLCK mRNA. D: p50 and p65 siRNA transfection in Caco-2 cells caused marked inhibition of LPS-induced increase in MLCK protein expression, as analyzed by Western blot analysis and relative analysis of MLCK protein levels by densitometry. n = 3 experiments ( A ); n = 4 experiments ( B and D ). ∗∗ P

    Techniques Used: Activation Assay, Expressing, Transfection, Western Blot, Inhibition

    Effect of toll-like receptor (TLR)-4, myeloid differentiation primary response (MyD)88, and transforming growth factor-β–activating kinase (TAK)-1 siRNA on lipopolysaccharide (LPS)-induced activation of NF-κB canonical pathway in Caco-2 cells. A: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced degradation of inhibitory κ B (IκB)-α protein expression compared with nontarget (NT) siRNA-transfected LPS-treated cells. Relative densitometry analysis of IκB-α protein levels. B: TLR-4 siRNA and MyD88 siRNA transfection in Caco-2 cells prevented LPS-induced increase in TAK-1 phosphorylation. Relative densitometry analysis of pTAK-1 protein levels. C: Confocal immunofluorescence showed that TLR-4, MyD88, and TAK-1 siRNA transfection of Caco-2 monolayers prevented the LPS-induced p65 (red) translocation to the nucleus (blue) ( arrowhead ) at 3 day after LPS exposure D: TLR-4 siRNA and MyD88 siRNA transfection in Caco-2 cells prevented LPS-induced degradation of IκB-α protein expression. Densitometry of IκB-α protein levels. E: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced activation of IκB kinase (IKK)-α and IKK-β compared with nontarget (NT) siRNA-transfected LPS-treated cells. Relative densitometry analysis of IKK-α and IKK-β is also shown. n = 4 experiments. ∗∗ P
    Figure Legend Snippet: Effect of toll-like receptor (TLR)-4, myeloid differentiation primary response (MyD)88, and transforming growth factor-β–activating kinase (TAK)-1 siRNA on lipopolysaccharide (LPS)-induced activation of NF-κB canonical pathway in Caco-2 cells. A: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced degradation of inhibitory κ B (IκB)-α protein expression compared with nontarget (NT) siRNA-transfected LPS-treated cells. Relative densitometry analysis of IκB-α protein levels. B: TLR-4 siRNA and MyD88 siRNA transfection in Caco-2 cells prevented LPS-induced increase in TAK-1 phosphorylation. Relative densitometry analysis of pTAK-1 protein levels. C: Confocal immunofluorescence showed that TLR-4, MyD88, and TAK-1 siRNA transfection of Caco-2 monolayers prevented the LPS-induced p65 (red) translocation to the nucleus (blue) ( arrowhead ) at 3 day after LPS exposure D: TLR-4 siRNA and MyD88 siRNA transfection in Caco-2 cells prevented LPS-induced degradation of IκB-α protein expression. Densitometry of IκB-α protein levels. E: TAK-1 siRNA transfection in Caco-2 cells prevented LPS-induced activation of IκB kinase (IKK)-α and IKK-β compared with nontarget (NT) siRNA-transfected LPS-treated cells. Relative densitometry analysis of IKK-α and IKK-β is also shown. n = 4 experiments. ∗∗ P

    Techniques Used: Activation Assay, Transfection, Expressing, Immunofluorescence, Translocation Assay

    Role of transforming growth factor-β–activating kinase (TAK)-1 in the lipopolysaccharide (LPS)-induced activation of canonical NF-κB pathway and Caco-2 tight junction permeability. A: LPS treatment at the concentration of 300 pg/mL caused activation of phospho-TAK-1. B and C: LPS treatment did not induce phosphorylation of NF-κB–inducing kinase (NIK) or mitogen-activated kinase kinase (MEKK)-1 at day 3 after LPS exposure compared with untreated Caco-2 cells. D: Confocal immunofluorescence of Caco-2 cells treated with 300 pg/mL LPS for 5 days indicated increase in pTAK-1 (green) on day 3 and 3.5 after LPS treatment. Nucleus, blue. E: TAK-1, NIK, and MEKK-1 siRNA transfections in Caco-2 cells significantly reduced TAK-1, NIK, and MEKK-1 protein expression, as analyzed by Western blot analysis and relative densitometry, respectively. F: TAK-1 siRNA transfection prevented the LPS-induced drop in Caco-2 cell transepithelial electrical resistance (TER). G: TAK-1 siRNA transfection inhibited the LPS-induced increase in Caco-2 inulin flux. H: NIK or MEKK-1 siRNA in transfection in Caco-2 cells did not prevent the LPS-induced drop in Caco-2 TER. Data are expressed as means ± SEM. n = 4 experiments ( A ). ∗∗∗ P
    Figure Legend Snippet: Role of transforming growth factor-β–activating kinase (TAK)-1 in the lipopolysaccharide (LPS)-induced activation of canonical NF-κB pathway and Caco-2 tight junction permeability. A: LPS treatment at the concentration of 300 pg/mL caused activation of phospho-TAK-1. B and C: LPS treatment did not induce phosphorylation of NF-κB–inducing kinase (NIK) or mitogen-activated kinase kinase (MEKK)-1 at day 3 after LPS exposure compared with untreated Caco-2 cells. D: Confocal immunofluorescence of Caco-2 cells treated with 300 pg/mL LPS for 5 days indicated increase in pTAK-1 (green) on day 3 and 3.5 after LPS treatment. Nucleus, blue. E: TAK-1, NIK, and MEKK-1 siRNA transfections in Caco-2 cells significantly reduced TAK-1, NIK, and MEKK-1 protein expression, as analyzed by Western blot analysis and relative densitometry, respectively. F: TAK-1 siRNA transfection prevented the LPS-induced drop in Caco-2 cell transepithelial electrical resistance (TER). G: TAK-1 siRNA transfection inhibited the LPS-induced increase in Caco-2 inulin flux. H: NIK or MEKK-1 siRNA in transfection in Caco-2 cells did not prevent the LPS-induced drop in Caco-2 TER. Data are expressed as means ± SEM. n = 4 experiments ( A ). ∗∗∗ P

    Techniques Used: Activation Assay, Permeability, Concentration Assay, Immunofluorescence, Transfection, Expressing, Western Blot

    37) Product Images from "Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats"

    Article Title: Protective effects of recombinant osteopontin on early brain injury after subarachnoid hemorrhage in rats

    Journal: Critical care medicine

    doi: 10.1097/CCM.0b013e3181c027ae

    Effects of recombinant osteopontin (OPN) treatment on matrix metalloproteinase (MMP)-9 induction and tissue inhibitor of MMP (TIMP)-1 reduction in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). Western blots for MMP-9 (
    Figure Legend Snippet: Effects of recombinant osteopontin (OPN) treatment on matrix metalloproteinase (MMP)-9 induction and tissue inhibitor of MMP (TIMP)-1 reduction in the left cerebral hemisphere at 24 hours after subarachnoid hemorrhage (SAH). Western blots for MMP-9 (

    Techniques Used: Recombinant, Western Blot

    38) Product Images from "The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration"

    Article Title: The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-014-0046-x

    NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P
    Figure Legend Snippet: NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P

    Techniques Used: Expressing, Migration, Transformation Assay, Flow Cytometry, Cytometry

    Expression of Fascin in B-cell lymphomas. (A) Quantitative PCR (qPCR) of Fascin transcripts in transformed B-cells derived from EBV-transformed lymphoblastoid cell lines (LCL), from Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and from primary effusion lymphoma (PEL) in comparison to Jurkat cells and Fascin-positive, HTLV-1-transformed MT-2 cells. Copy numbers were normalized to those of ß-Actin ( ACTB ) and thereafter normalized to relative Fascin expression in Jurkat cells. The means of two independent experiments are shown. ctrl. indicates control. (B) Detection of Fascin and LMP1 by immunoblot. In addition to the B-cell lines shown in (A) , peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Detection of ACTB served as loading control. Slower migrating bands upon detection of LMP1 reflect HA-LMP1. (C) Immunofluorescence of EBV-transformed LCL-B cells spotted on fibronectin-coated coverslips using phalloidinX-TexasRed for detection of actin and anti-Fascin and secondary anti-mouse Alexa Fluor® 488 antibodies. Nuclei were stained with DAPI. Images were acquired using a LAS AF DMI 6000 fluorescence microscope equipped with a 63 × 1.4 HCX PL APO oil immersion objective lens. Jurkat cells (mock) as shown in Figure 2 B served as negative control.
    Figure Legend Snippet: Expression of Fascin in B-cell lymphomas. (A) Quantitative PCR (qPCR) of Fascin transcripts in transformed B-cells derived from EBV-transformed lymphoblastoid cell lines (LCL), from Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and from primary effusion lymphoma (PEL) in comparison to Jurkat cells and Fascin-positive, HTLV-1-transformed MT-2 cells. Copy numbers were normalized to those of ß-Actin ( ACTB ) and thereafter normalized to relative Fascin expression in Jurkat cells. The means of two independent experiments are shown. ctrl. indicates control. (B) Detection of Fascin and LMP1 by immunoblot. In addition to the B-cell lines shown in (A) , peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Detection of ACTB served as loading control. Slower migrating bands upon detection of LMP1 reflect HA-LMP1. (C) Immunofluorescence of EBV-transformed LCL-B cells spotted on fibronectin-coated coverslips using phalloidinX-TexasRed for detection of actin and anti-Fascin and secondary anti-mouse Alexa Fluor® 488 antibodies. Nuclei were stained with DAPI. Images were acquired using a LAS AF DMI 6000 fluorescence microscope equipped with a 63 × 1.4 HCX PL APO oil immersion objective lens. Jurkat cells (mock) as shown in Figure 2 B served as negative control.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Microscopy, Negative Control

    39) Product Images from "The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration"

    Article Title: The tumor marker Fascin is induced by the Epstein-Barr virus-encoded oncoprotein LMP1 via NF-κB in lymphocytes and contributes to their invasive migration

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/s12964-014-0046-x

    NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P
    Figure Legend Snippet: NF-κB signals are required for maintaining Fascin expression and invasive migration of EBV-transformed LMP1-expressing lymphoblastoid cells (LCL). (A) Viability of LCL-B cells upon treatment with increasing amounts of the IKKβ inhibitor ACHP (1, 2.5, 5, 10, 25 μM) and the JNK-inhibitor SP600125 (10 μM) for 48 h determined by forward-side scatter (FSC/SSC) analysis in flow cytometry. DMSO-treated cells were set as 100%. The means of three independent experiments +/− SE were compared using a paired t-test. *indicates P

    Techniques Used: Expressing, Migration, Transformation Assay, Flow Cytometry, Cytometry

    Expression of Fascin in B-cell lymphomas. (A) Quantitative PCR (qPCR) of Fascin transcripts in transformed B-cells derived from EBV-transformed lymphoblastoid cell lines (LCL), from Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and from primary effusion lymphoma (PEL) in comparison to Jurkat cells and Fascin-positive, HTLV-1-transformed MT-2 cells. Copy numbers were normalized to those of ß-Actin ( ACTB ) and thereafter normalized to relative Fascin expression in Jurkat cells. The means of two independent experiments are shown. ctrl. indicates control. (B) Detection of Fascin and LMP1 by immunoblot. In addition to the B-cell lines shown in (A) , peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Detection of ACTB served as loading control. Slower migrating bands upon detection of LMP1 reflect HA-LMP1. (C) Immunofluorescence of EBV-transformed LCL-B cells spotted on fibronectin-coated coverslips using phalloidinX-TexasRed for detection of actin and anti-Fascin and secondary anti-mouse Alexa Fluor® 488 antibodies. Nuclei were stained with DAPI. Images were acquired using a LAS AF DMI 6000 fluorescence microscope equipped with a 63 × 1.4 HCX PL APO oil immersion objective lens. Jurkat cells (mock) as shown in Figure 2 B served as negative control.
    Figure Legend Snippet: Expression of Fascin in B-cell lymphomas. (A) Quantitative PCR (qPCR) of Fascin transcripts in transformed B-cells derived from EBV-transformed lymphoblastoid cell lines (LCL), from Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and from primary effusion lymphoma (PEL) in comparison to Jurkat cells and Fascin-positive, HTLV-1-transformed MT-2 cells. Copy numbers were normalized to those of ß-Actin ( ACTB ) and thereafter normalized to relative Fascin expression in Jurkat cells. The means of two independent experiments are shown. ctrl. indicates control. (B) Detection of Fascin and LMP1 by immunoblot. In addition to the B-cell lines shown in (A) , peripheral blood mononuclear cells (PBMC) from a healthy donor were analyzed. Detection of ACTB served as loading control. Slower migrating bands upon detection of LMP1 reflect HA-LMP1. (C) Immunofluorescence of EBV-transformed LCL-B cells spotted on fibronectin-coated coverslips using phalloidinX-TexasRed for detection of actin and anti-Fascin and secondary anti-mouse Alexa Fluor® 488 antibodies. Nuclei were stained with DAPI. Images were acquired using a LAS AF DMI 6000 fluorescence microscope equipped with a 63 × 1.4 HCX PL APO oil immersion objective lens. Jurkat cells (mock) as shown in Figure 2 B served as negative control.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Derivative Assay, Immunofluorescence, Staining, Fluorescence, Microscopy, Negative Control

    40) Product Images from "Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences"

    Article Title: Human NF-κB1 Haploinsufficiency and Epstein–Barr Virus-Induced Disease—Molecular Mechanisms and Consequences

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01978

    Canonical and non-canonical NF-κB signaling in humans. Activation of the canonical NF-κB pathway is triggered by a broad range of proinflammatory cytokines such as TNFα or IL-1, bacterial pattern recognition molecules such as LPS, or antigen stimulation. Non-canonical signaling is triggered by TNF family receptors and their ligands, resulting in activation of NIK kinase activity. Both pathways cumulate in the activation of IKK (IκB-kinases) which phosphorylate inhibitory IκB binding partners for their poly-ubiquitination and proteosomal degradation (canonical axis) or the processing of p100 into its active form (non-canonical axis). Resulting NF-κB dimers translocate to the nucleus. Depending on their assembly into activating hetero- or repressive homo-dimeric conformations, NF-κB signaling regulates the expression of hundreds of target genes. TNF(R), tumor necrosis factor (receptor); IL-1(R), interleukin-1 (receptor); LPS, lipopolysaccharide; BAFF(-R), B-cell activating factor (receptor); LTβ(R), lymphotoxin β (receptor); TLR, toll-like receptor; TCR/BCR, T-cell/B-cell receptor; NIK, NF-κB inducing kinase; NEMO, NF-κB essential modulator; IKK, IκB kinase; IκB, Inhibitor of NF-κB; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.
    Figure Legend Snippet: Canonical and non-canonical NF-κB signaling in humans. Activation of the canonical NF-κB pathway is triggered by a broad range of proinflammatory cytokines such as TNFα or IL-1, bacterial pattern recognition molecules such as LPS, or antigen stimulation. Non-canonical signaling is triggered by TNF family receptors and their ligands, resulting in activation of NIK kinase activity. Both pathways cumulate in the activation of IKK (IκB-kinases) which phosphorylate inhibitory IκB binding partners for their poly-ubiquitination and proteosomal degradation (canonical axis) or the processing of p100 into its active form (non-canonical axis). Resulting NF-κB dimers translocate to the nucleus. Depending on their assembly into activating hetero- or repressive homo-dimeric conformations, NF-κB signaling regulates the expression of hundreds of target genes. TNF(R), tumor necrosis factor (receptor); IL-1(R), interleukin-1 (receptor); LPS, lipopolysaccharide; BAFF(-R), B-cell activating factor (receptor); LTβ(R), lymphotoxin β (receptor); TLR, toll-like receptor; TCR/BCR, T-cell/B-cell receptor; NIK, NF-κB inducing kinase; NEMO, NF-κB essential modulator; IKK, IκB kinase; IκB, Inhibitor of NF-κB; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells.

    Techniques Used: Activation Assay, Activity Assay, Binding Assay, Expressing

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    Article Title: Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen- glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss
    Article Snippet: .. For inhibition of MAPK activation, 10 μ mol/L of the MEK1 inhibitor U0126 (BIOMOL) or 20 μ mol/L of the JNK inhibitor SP600125 (Calbiochem, San Diego, CA, USA) was added to the slices during the 1 h slice recovery period after slice preparation and throughout OGD or normoxia. .. Slices were then incubated for 1 h at 37°C in aCSF constantly bubbled with 95% O2 /5% CO2 ( p O2 was at least 760mmHg) to mimic reperfusion for cell death analysis or as indicated.

    Activation Assay:

    Article Title: Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen- glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss
    Article Snippet: .. For inhibition of MAPK activation, 10 μ mol/L of the MEK1 inhibitor U0126 (BIOMOL) or 20 μ mol/L of the JNK inhibitor SP600125 (Calbiochem, San Diego, CA, USA) was added to the slices during the 1 h slice recovery period after slice preparation and throughout OGD or normoxia. .. Slices were then incubated for 1 h at 37°C in aCSF constantly bubbled with 95% O2 /5% CO2 ( p O2 was at least 760mmHg) to mimic reperfusion for cell death analysis or as indicated.

    Incubation:

    Article Title: Glycogen Synthase Kinase-3 (GSK3) Inhibition Induces Prosurvival Autophagic Signals in Human Pancreatic Cancer Cells *
    Article Snippet: .. When indicated, cells were incubated with the JNK inhibitor SP600125 (EMD Millipore, 420128), the mTOR inhibitor Torin1 (Selleckchem, S2827), or the S6K1 inhibitor PF-4708671 (Selleckchem, S2163). .. For evaluation of autophagic flux, cells were incubated with chloroquine (Sigma) or Bafilomycin A1 (LC Laboratories, B-1080).

    Inhibition:

    Article Title: Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen- glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss
    Article Snippet: .. For inhibition of MAPK activation, 10 μ mol/L of the MEK1 inhibitor U0126 (BIOMOL) or 20 μ mol/L of the JNK inhibitor SP600125 (Calbiochem, San Diego, CA, USA) was added to the slices during the 1 h slice recovery period after slice preparation and throughout OGD or normoxia. .. Slices were then incubated for 1 h at 37°C in aCSF constantly bubbled with 95% O2 /5% CO2 ( p O2 was at least 760mmHg) to mimic reperfusion for cell death analysis or as indicated.

    other:

    Article Title: Contrasting Roles of Mitogen-Activated Protein Kinases in Cellular Entry and Replication of Hepatitis C Virus: MKNK1 Facilitates Cell Entry
    Article Snippet: JNK inhibitors SP600125 and AS601245 were purchased from Calbiochem.

    Article Title: Particulate matter Air Pollution induces hypermethylation of the p16 promoter Via a mitochondrial ROS-JNK-DNMT1 pathway
    Article Snippet: SP600125 was purchased from Calbiochem (Darmstadt, Germany).

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