IκB/IKK

(A) Washed human platelets were untreated or pretreated with the PDK1 inhibitor BX-795 (1 μM) for 5 min, and this was followed by stimulation with 2-methylthio-ADP (2MeSADP) (50 nM) under stirring for 5 min. Western blots were then probed for phosphorylated Akt (Thr308), Akt (Ser473), glycogen synthase kinase 3b (GSK3b) (Ser9), mitogen-activated protein kinase kinase 1/2 (MEK1/2) (Ser217/221), extracellular signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204), and cytosolic phospholipase A2 (cPLA2) (Ser505). The western blots shown are representative of three independent experiments.

Immunofluorescence images of Ishikawa cells showing the increase in the nuclear colocalization of ERa (red) and p65 (green) after E2 incubation. Nuclear colocalization is yellow. Treatment with ICI 182,780, PDTC or Bay inhibited the nuclear localization of both ERa and p65. Blue, DAPI-stained nuclei. All pictures were obtained the same day using the same microscope settings. Original magnification 3200, bar =50 um. Arrows, cytoplasmic ERa and p65 staining. ICI, ICI 182,780; Bay, Bay 11-7082.

Reduction in IFN-g production in TcREG by IKK-16 coincides with decrease in T-bet and Eomes expression levels. Flow cytometry plots showing cellular analysis for T-bet and Eomes in CD45+CD3+CD8+ population.

GAB2-induced chemokine expression is dependent on IKKβ-NF-κB activity. (a) Effect of small-molecule inhibitors against PI3K (GDC-0941), PI3K/mTOR (BEZ235), MEK (AZD6288) or IKKβ (TPCA-1, IKK16 and Bay 65–1942) on CXCL1, CXCL2 and CXCL8 mRNA levels in GAB2-overexpressing FTSECs. Cells were treated with 2 μm of each inhibitor or DMSO control for 6 h before collected for quantitative PCR analyses. Data are averages±s.e.m. of four independent experiments. Comparison between DMSO- or inhibitor-treated GAB2-overexpressing FTSECs were used for statistical analyses. n.s., not significant; *P<0.05; **P<0.01. (b) Effect of suppressing IKKβ (encoded by IKBKB) or NF-κB p65 (encoded by RELA) on CXCL1, CXCL2 and CXCL8 mRNA levels in GAB2-overexpressing FTSECs. Cells were infected with a control shRNA targeting LacZ or shRNAs targeting IKBKB or RELA and cultured for 48 h before collected for quantitative PCR analyses. Data are averages±s.e.m. of three independent experiments. *P<0.05; **P<0.01.

(B) Histological examinations of xenograft lesions produced by A549 cells. We stained the lesions with H&E, TUNEL and staining for Ki-67. (C, D) The histogram shows percentage of A549 cells that were positive for proliferation marker Ki-67 and apoptosis marker TUNEL in 3 representative fields. Results are expressed as the mean ± SD. ns. P > 0.05, **P < 0.01 and ***P < 0.001 for the indicated comparisons by 1-way A OVA or Student’s t test.

Renal Nrf2 activity was shown in RTA402-treated acFSGS (RTA402 + acFSGS) mice as early as day 7 (RTA402 + acFSGS) and persisted to day 28, compared to vehicle + acFSGS mice. Kidney in situ ROS production demonstrated by DHE detection.

(E) After transfection with control or catalase siRNAs for 24 h, A375 and G361 cells were treated with vehicle or 2.5 µM BMS-345541, 20 µM PS-1145 and/or 50 µM of fotemustine for an additional 48 h. Cell viability was then assessed. *, p<0.05. (F) A375 and G361 cells were treated with vehicle or 2.5 µM BMS-345541, 20 µM PS-1145 and/or 50 µM of fotemustine, in the presence or absence of catalase inhibitor 3-Amino-1,2,4-triazole (ATZ, 50 µM) for 48 h. Cell viability was then assessed. *, p<0.05.

VSMCs were pretreated with BAY 11-7085 (BAY, 10μmol/L) for 1h and further treated with or without 10^-7mol/L salusin-β for 30min. A, Cytoplasmic extracts (Cy) were obtained and the phosphorylated IκBα, IκBα and HDCA3 protein levels were measured by western blot analysis. B, nuclear extracts (Nu) were obtained and the nuclear p50, p65 and HDAC3 protein levels were measured by western blot analysis. Data are expressed as means ± SD (n=3). *P< 0.05 versus the control with no treatment; #P< 0.05 versus 10^-7mol/L salusin-β treatment alone.

(C) G361 cells were loaded with DCF (ROS probe) and further stimulated with vehicle or indicated IKKβ inhibitors for 3 h. Fluorescence was measured using a fluorescence microplate reader. *, p<0.05 versus control.

A549 cells were treated with NF-kB inhibitor LY2409881 (30 nM) for 2 h, followed by western blot analysis of p-NF-kB.

(E) After transfection with control or catalase siRNAs for 24 h, A375 and G361 cells were treated with vehicle or 2.5 µM BMS-345541, 20 µM PS-1145 and/or 50 µM of fotemustine for an additional 48 h. Cell viability was then assessed. *, p<0.05. (F) A375 and G361 cells were treated with vehicle or 2.5 µM BMS-345541, 20 µM PS-1145 and/or 50 µM of fotemustine, in the presence or absence of catalase inhibitor 3-Amino-1,2,4-triazole (ATZ, 50 µM) for 48 h. Cell viability was then assessed. *, p<0.05.

(A) Washed human platelets were untreated or pretreated with the PDK1 inhibitor BX-795 (1 μM) for 5 min, and this was followed by stimulation with 2-methylthio-ADP (2MeSADP) (50 nM) under stirring for 5 min. Western blots were then probed for phosphorylated Akt (Thr308), Akt (Ser473), glycogen synthase kinase 3b (GSK3b) (Ser9), mitogen-activated protein kinase kinase 1/2 (MEK1/2) (Ser217/221), extracellular signal-regulated kinase 1/2 (ERK1/2) (Thr202/Tyr204), and cytosolic phospholipase A2 (cPLA2) (Ser505). The western blots shown are representative of three independent experiments.

Immunofluorescence images of Ishikawa cells showing the increase in the nuclear colocalization of ERa (red) and p65 (green) after E2 incubation. Nuclear colocalization is yellow. Treatment with ICI 182,780, PDTC or Bay inhibited the nuclear localization of both ERa and p65. Blue, DAPI-stained nuclei. All pictures were obtained the same day using the same microscope settings. Original magnification 3200, bar =50 um. Arrows, cytoplasmic ERa and p65 staining. ICI, ICI 182,780; Bay, Bay 11-7082.

Reduction in IFN-g production in TcREG by IKK-16 coincides with decrease in T-bet and Eomes expression levels. Flow cytometry plots showing cellular analysis for T-bet and Eomes in CD45+CD3+CD8+ population.

GAB2-induced chemokine expression is dependent on IKKβ-NF-κB activity. (a) Effect of small-molecule inhibitors against PI3K (GDC-0941), PI3K/mTOR (BEZ235), MEK (AZD6288) or IKKβ (TPCA-1, IKK16 and Bay 65–1942) on CXCL1, CXCL2 and CXCL8 mRNA levels in GAB2-overexpressing FTSECs. Cells were treated with 2 μm of each inhibitor or DMSO control for 6 h before collected for quantitative PCR analyses. Data are averages±s.e.m. of four independent experiments. Comparison between DMSO- or inhibitor-treated GAB2-overexpressing FTSECs were used for statistical analyses. n.s., not significant; *P<0.05; **P<0.01. (b) Effect of suppressing IKKβ (encoded by IKBKB) or NF-κB p65 (encoded by RELA) on CXCL1, CXCL2 and CXCL8 mRNA levels in GAB2-overexpressing FTSECs. Cells were infected with a control shRNA targeting LacZ or shRNAs targeting IKBKB or RELA and cultured for 48 h before collected for quantitative PCR analyses. Data are averages±s.e.m. of three independent experiments. *P<0.05; **P<0.01.

(B) Histological examinations of xenograft lesions produced by A549 cells. We stained the lesions with H&E, TUNEL and staining for Ki-67. (C, D) The histogram shows percentage of A549 cells that were positive for proliferation marker Ki-67 and apoptosis marker TUNEL in 3 representative fields. Results are expressed as the mean ± SD. ns. P > 0.05, **P < 0.01 and ***P < 0.001 for the indicated comparisons by 1-way A OVA or Student’s t test.

Renal Nrf2 activity was shown in RTA402-treated acFSGS (RTA402 + acFSGS) mice as early as day 7 (RTA402 + acFSGS) and persisted to day 28, compared to vehicle + acFSGS mice. Kidney in situ ROS production demonstrated by DHE detection.

(E) After transfection with control or catalase siRNAs for 24 h, A375 and G361 cells were treated with vehicle or 2.5 µM BMS-345541, 20 µM PS-1145 and/or 50 µM of fotemustine for an additional 48 h. Cell viability was then assessed. *, p<0.05. (F) A375 and G361 cells were treated with vehicle or 2.5 µM BMS-345541, 20 µM PS-1145 and/or 50 µM of fotemustine, in the presence or absence of catalase inhibitor 3-Amino-1,2,4-triazole (ATZ, 50 µM) for 48 h. Cell viability was then assessed. *, p<0.05.

VSMCs were pretreated with BAY 11-7085 (BAY, 10μmol/L) for 1h and further treated with or without 10^-7mol/L salusin-β for 30min. A, Cytoplasmic extracts (Cy) were obtained and the phosphorylated IκBα, IκBα and HDCA3 protein levels were measured by western blot analysis. B, nuclear extracts (Nu) were obtained and the nuclear p50, p65 and HDAC3 protein levels were measured by western blot analysis. Data are expressed as means ± SD (n=3). *P< 0.05 versus the control with no treatment; #P< 0.05 versus 10^-7mol/L salusin-β treatment alone.

(C) G361 cells were loaded with DCF (ROS probe) and further stimulated with vehicle or indicated IKKβ inhibitors for 3 h. Fluorescence was measured using a fluorescence microplate reader. *, p<0.05 versus control.

A549 cells were treated with NF-kB inhibitor LY2409881 (30 nM) for 2 h, followed by western blot analysis of p-NF-kB.

(E) After transfection with control or catalase siRNAs for 24 h, A375 and G361 cells were treated with vehicle or 2.5 µM BMS-345541, 20 µM PS-1145 and/or 50 µM of fotemustine for an additional 48 h. Cell viability was then assessed. *, p<0.05. (F) A375 and G361 cells were treated with vehicle or 2.5 µM BMS-345541, 20 µM PS-1145 and/or 50 µM of fotemustine, in the presence or absence of catalase inhibitor 3-Amino-1,2,4-triazole (ATZ, 50 µM) for 48 h. Cell viability was then assessed. *, p<0.05.