Journal: Journal of cell science

Article Title: The laminin-binding integrins regulate nuclear factor κB-dependent epithelial cell polarity and inflammation.

doi: 10.1242/jcs.259161

Figure Lengend Snippet: Fig. 7. α3−/−α6−/−collecting duct cells exhibit increased NF-κB activation. (A) α3f/fα6f/f and α3−/−α6−/−collecting duct cells were grown to 80% confluence. Then, 24 h later, conditioned medium from these cells was collected and subjected to cytokine multiplex analysis. A subset of cytokines with significant differences between α3−/−α6−/−and α3f/fα6f/f collecting duct cells (P<0.01) are shown. (B-D) α3f/fα6f/f and α3−/−α6−/−collecting duct cells grown to 80% confluence were lysed and subjected to separation of nuclear and cytosolic fractions. Western blot analysis of the fractions (20 µg total protein/lane) showed increased expression of nuclear p65 and cytosolic phospho-p65S536 (p-p65S536) in α3−/−α6−/−compared to α3f/fα6f/f collecting duct cells. Lamin A/C was used as a nuclear marker; β-actin was used as a loading control (B). The expression of p65 (C) or p-p65S536 (D) was normalized to β-actin and quantified as a ratio of α3f/fα6f/f cells. (E) α3f/fα6f/f and α3−/−α6−/−collecting duct cells were grown to 80% confluence. Then, 24 h later, NF-kB activity in these cells was detected using a Nano-Glo Luciferase Assay and quantified as a ratio of α3f/fα6f/f collecting duct cells. (F) α3f/fα6f/f and α3−/−α6−/−collecting duct cells grown to 80% confluence were treated with solvent (3% Tween 80 in regular growth medium) or 10 µM BMS-345541 in solvent for 1 h. NF-kB activity was detected using a Nano-Glo Luciferase Assay and quantified as a ratio of α3f/fα6f/f collecting cells. (G,H) Immunostaining for the collecting duct marker DBA (green) and phospho-p65S276

Article Snippet: In some experiments, the collecting duct cells were treated with IκB kinase inhibitor BMS-34554 (100 μM in water/3% Tween 80) or the vehicle alone for 1 h. To silence NFκB p65, collecting duct cells were transfected with non-silencing siRNA (20 nM, transfection control) or p65-silencing siRNA [NFκB p65 siRNA (m), 29411, Santa Cruz Biotechnology] using Lipofectamine RNAiMAX according to manufacturer’s instructions.

Techniques: Activation Assay, Multiplex Assay, Western Blot, Expressing, Marker, Control, Activity Assay, Luciferase, Solvent, Immunostaining

Journal: Journal of cell science

Article Title: The laminin-binding integrins regulate nuclear factor κB-dependent epithelial cell polarity and inflammation.

doi: 10.1242/jcs.259161

Figure Lengend Snippet: Fig. 8. The mesenchymal phenotype of α3−/−α6−/−collecting duct cells is mediated by NF-κB-dependent Snail and Slug induction. (A-C) α3f/fα6f/f and α3−/−α6−/−collecting duct cells grown to 80% confluence were lysed and subjected to separation of nuclear and cytosolic fractions. Western blot analysis of the fractions (20 µg total protein/lane) showed increased nuclear Snail and Slug in α3−/−α6−/−collecting duct cells compared to α3f/f/α6f/f collecting duct cells. Lamin A/C was used as a nuclear marker; β-actin was used as a loading control (A). The expression of Snail (B) or Slug (C) was normalized to β-actin and quantified as a ratio of α3f/fα6f/f cells. **P<0.01 between α3−/−α6−/−and α3f/fα6f/f collecting duct cells. (D-H) α3−/−α6−/−collecting duct cells grown to 80% confluence were transfected with non-silencing (n) or NF-κB p65-silencing (p65) siRNA for 48 h. Collecting duct cells were lysed and subjected to separation of nuclear and cytosolic fractions. Western blot analysis of the fractions (20 µg total protein/lane) showed decreased cytosolic and nuclear phospho-p65S536 (p-p65), total p65 and Slug, and decreased nuclear Snail in cells transfected with p65 siRNA compared to non-silencing siRNA. Lamin A/C was used as a nuclear marker; β-actin was used as a loading control (D). The expression of p-p65 (E), p65 (F), Snail (G) or Slug (H) was normalized to β-actin and quantified as a ratio of cells transfected with non-silencing siRNA. **P<0.01 between cells transfected with non-silencing and p65 siRNA. (I-M) α3f/f/α6f/f and α3−/−α6−/−collecting duct cells grown to 80% confluence were transfected with non-silencing or NF-κB p65-silencing (p65) siRNA, and lysed 48 h later. Total cell lysates (40 µg total protein/lane) were analyzed by western blotting for levels of p65, E-cadherin, N-cadherin and collagen I; β-actin was used as a loading control (I). The expression of p65 (J), E-cadherin (K), N-cadherin (L) or collagen I (M) proteins was normalized to actin and quantified as ratio of α3f/fα6f/f cells transfected with non-silencing siRNA. Data are mean±s.e.m. of at least three experiments. **P<0.01 between non-silencing and p65 siRNA-transfected α3f/f/α6f/f or α3−/−/α6−/−collecting duct cells. Statistical significance was determined using two-tailed, unpaired t-tests.

Article Snippet: In some experiments, the collecting duct cells were treated with IκB kinase inhibitor BMS-34554 (100 μM in water/3% Tween 80) or the vehicle alone for 1 h. To silence NFκB p65, collecting duct cells were transfected with non-silencing siRNA (20 nM, transfection control) or p65-silencing siRNA [NFκB p65 siRNA (m), 29411, Santa Cruz Biotechnology] using Lipofectamine RNAiMAX according to manufacturer’s instructions.

Techniques: Western Blot, Marker, Control, Expressing, Transfection, Two Tailed Test

Journal: The EMBO Journal

Article Title: Control of NAD + homeostasis by autophagic flux modulates mitochondrial and cardiac function

doi: 10.1038/s44318-023-00009-w

Figure Lengend Snippet: ( A ) NF-κB luciferase activity in H9c2 cells following treatment with PBS (Veh), CQ, or CQ + Bay11-7085 (Bay). H9c2 cells were first transfected with an NF-κB luciferase reporter vector or non-luciferase vector as a control for 24 h, and then were treated with PBS (Veh), CQ, or CQ + Bay for an additional 24 h in a nutrient-replete medium. n = 3 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. ( B ) RELA (NF-κB p65) phosphorylation at S536 in H9c2 cells following treatment with Veh or CQ for 24 h, n = 5–7 per group. Data are mean ± SEM. An unpaired t test was used to determine statistical significance between two groups. * P < 0.05. ( C ) RELA phosphorylation at S536 in H9c2 cells following Atg3 knockdown, n = 3–4 per group. Data are mean ± SEM. An unpaired t test was used to determine statistical significance between two groups. * P < 0.05. ( D ) RELA phosphorylation at S536 in cAtg3-KO mouse hearts. Mice were randomly fed or fasted for 48 h, n = 6 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. ( E ) Chromatin immunoprecipitation (CHIP) assay of RELA binding to the NNMT promoter region. Chromatin was extracted from WT mouse hearts, and protein-DNA immunoprecipitates were analyzed by RT-PCR, performed using primers specific to the NNMT promoter region. Histone H3 was used as positive control and IgG was used as a negative control. Representative images are shown. ( F ) Fold enrichment of RELA binding to the NNMT promoter in PBS or CQ-treated mouse hearts. n = 3 per group. Data are mean ± SEM. An unpaired t test was used to determine statistical significance between two groups. * P < 0.05. ( G ) NNMT mRNA expression in H9c2 cells. Cells were transfected with either Con si or RELA siRNA (RELA si), and then CQ was added to cells, n = 6–10 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. ( H ) NNMT mRNA expression in H9c2 cells following treatment with Veh, CQ, Bay, or CQ + Bay for 24 h, n = 3 to 6 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. ( I ) NAD + in H9c2 cells following treatment with Veh, CQ, Bay, or CQ + Bay for 24 h, n = 3–5 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. ( J ) Oxygen consumption rate (OCR) in H9c2 cells following treatment with Veh, CQ, Bay, or CQ + Bay for 24 h, n = 6–13 per group. Data are mean ± SEM. Unpaired t tests were used to determine statistical significance between two groups at corresponding time points. * P < 0.05 vs. Veh, # P < 0.05 vs. CQ. ( K ) Protein levels of GAPDH, LC3 (MAP1LC3A), SQSTM1, phosphorylation of RELA at S536, and NNMT; ( L ) NF-κB luciferase activity; ( M ) NNMT mRNA levels; and ( N ) NAD + levels in H9c2 cells transfected with a vector encoding SQSTM1 or HA as control, n = 4–6 per group. Data are mean ± SEM. Unpaired t tests were used to determine statistical significance between two groups. * P < 0.05, ** P < 0.01. ( O ) Oxygen consumption rate (OCR) in H9c2 cells transfected with a vector encoding SQSTM1 or HA as control, n = 18–22 per group. Data are mean ± SEM. Unpaired t tests were used to determine statistical significance between two groups at corresponding time points. * P < 0.05 vs. HA. .

Article Snippet: For siRNA knockdown of Atg3 , SQSTM1 , NNMT , and RELA (NF-κB p65), H9c2 cells were infected with siRNAs against Atg3 , SQSTM1 (6399, Cell Signaling Technology, Danvers, MA; or paired sequences as indicated), NNMT (L-101014-02-0005, GE Dharmacon), and RELA (6261S, Cell Signaling Technology), or scramble siRNA at 80–100 nM for 48–72 h together with Lipofectamine 2000 (11668500, Thermofisher Scientific) under nutrient-replete conditions.

Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Control, Comparison, Phospho-proteomics, Knockdown, Chromatin Immunoprecipitation, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Expressing

Journal: The EMBO Journal

Article Title: Control of NAD + homeostasis by autophagic flux modulates mitochondrial and cardiac function

doi: 10.1038/s44318-023-00009-w

Figure Lengend Snippet: ( A ) Protein levels of ATG3, SQSTM1, NNMT, RELA (NF-κB p65) and RELA phosphorylation at S536, and GAPDH in H9c2 cells. ATG3 and SQSTM1 were silenced as indicated, n = 4–8 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. ( B ) NAD + levels in H9c2 cells. Atg3 and SQSTM1 were silenced as indicated, n = 3–4 to 8 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. ( C ) Oxygen consumption rate (OCR) in H9c2. ATG3 and SQSTM1 were silenced as indicated, n = 9–30 per group. Data are mean ± SEM. Unpaired t tests were used to determine statistical significance between two groups at corresponding time points. * P < 0.05 vs. Con si, # P < 0.05 vs. Atg3 si. ( D ) Protein levels of GAPDH, SQSTM1, NNMT, RELA phosphorylation at S536, and RELA in cardiac-specific SQSTM1 KO mouse hearts, n = 3–6 per group. Data are Mean ± SEM. Unpaired t tests were used to determine statistical significance between two groups. * P < 0.05. ( E ) NAD + levels in cardiac-specific SQSTM1 KO mouse hearts. n = 3–6 per group. Data are mean ± SEM. An unpaired t test was used to determine statistical significance between two groups. * P < 0.05. ( F ) Protein levels of SQSTM1, LC3 (MAP1LC3A), and GAPDH in WT and cAtg3-KO mouse hearts. Mice, at ~30 weeks of age, were injected with 5-Amino-1-methylquinolinium iodide (NNMTi) or Veh as indicated. n = 3 per group. Typical blots are shown. ( G ) NAD + levels in WT and cAtg3-KO mouse hearts. Mice, at ~30 weeks of age, was injected with NNMTi or Veh as indicated. n = 3–8 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. ( H ) Heart rates (HR) of WT and cAtg3-KO mice under isoflurane anesthesia. Mice, at ~30 weeks of age, were injected with NNMTi or Veh as indicated. n = 5–8 per group. ( I ) Fractional shortening (FS) in WT and cAtg3-KO mouse hearts under isoflurane anesthesia. Mice, at ~30 weeks of age, were injected with NNMTi or Veh as indicated. n = 5–8 per group. Data are mean ± SEM. One-way ANOVA followed by Bonferroni’s multiple comparison tests was used to determine statistical significance. * P < 0.05. .

Article Snippet: For siRNA knockdown of Atg3 , SQSTM1 , NNMT , and RELA (NF-κB p65), H9c2 cells were infected with siRNAs against Atg3 , SQSTM1 (6399, Cell Signaling Technology, Danvers, MA; or paired sequences as indicated), NNMT (L-101014-02-0005, GE Dharmacon), and RELA (6261S, Cell Signaling Technology), or scramble siRNA at 80–100 nM for 48–72 h together with Lipofectamine 2000 (11668500, Thermofisher Scientific) under nutrient-replete conditions.

Techniques: Phospho-proteomics, Comparison, Injection

Journal: Cellular signalling

Article Title: Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression.

doi: 10.1016/j.cellsig.2019.01.014

Figure Lengend Snippet: Fig. 4. Minocycline inhibits PDGF-BB-induced NF-κB and AP-1 activation. A, PDGF-BB induces time-dependent NF-κB activation. Quiescent SMC were incubated with PDGF-BB (10 ng/ml). At the indicated time periods, activation of NF-κB was analyzed by immunoblotting using antibodies that specifically detect phos- phorylated p65 at Ser536. B, PDGF-BB induces time-dependent AP-1 activation. Quiescent SMC incubated as in A were analyzed for AP-1 activation by im- munoblotting using antibodies that specifically detect phosphorylated c-Jun at Ser73. C, Silencing IKKβ or pre-treatment with minocycline inhibit PDGF-BB-induced NF-κB activation. SMC incubated with lentiviral IKKβ shRNA (moi0.5 for 48 h) were made quiescent and treated with PDGF-BB (10 ng/ml for 30 min). In a subset of experiments, quiescent SMC were incubated with minocycline (10 μM for 15 min) and then treated with PDGF-BB (10 ng/ml for 30 min). Activation of NF-κB was analyzed as in A. D, Silencing JNK2 or pre-treatment with minocycline inhibits PDGF-BB-induced AP-1 activation. SMC incubated with lentiviral JNK2 shRNA (moi 0.5 for 48 h) were made quiescent and treated with PDGF-BB (10 ng/ml for 30 min). In a subset of experiments, quiescent SMC were incubated with minocycline (10 mM for 15 min) and then treated with PDGF-BB (10 ng/ml for 30 min). Activation of AP-1 was analyzed as in B. Silencing IKKβ and JNK2 was confirmed by immunoblotting. JNK2 and IKKβ served as off-targets in IKKβ and JNK2 silenced cells, respectively (right hand panels in C and D). Tubulin served as a loading control. Bar graphs at the bottom of panels in A-D represent densitometric analyses from three independent experiments. ⁎P < .05 control, †P < .05 versus PDGF- BB (n = 3).

Article Snippet: The following lentiviral shRNA vectors against IKKβ (#sc-35645-V), p65 (#sc-29410-V), JNK2 (#sc-39101-V), and c-Jun (#sc-29223-V) were purchased from Santa Cruz Biotechnology, Inc. Lentiviral shRNA against RECK (SHCLNV-NM_021111; TRCN0000376461) and eGFP (SHC005V) were purchased from Sigma-Aldrich.

Techniques: Activation Assay, Incubation, Western Blot, shRNA, Control

Journal: Cellular signalling

Article Title: Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression.

doi: 10.1016/j.cellsig.2019.01.014

Figure Lengend Snippet: Fig. 5. PDGF-BB induces SMC proliferation in part via miR-221 and miR-222. A, B, PDGF-BB induces miR-221 (A) and miR-222 (B) expression via IKKβ, NF-κB, JNK and AP-1. Quiescent SMC incubated with PDGF-BB (10 ng/ml) for 1 h were analyzed for miR- 221 (A) and miR-222 (B) expression by TaqMan® Advanced miRNA assays. The results were normal- ized to corresponding U6 expression. In a subset of experiments, SMC were incubated with lentiviral IKKβ, p65, JNK2 or c-Jun shRNA (moi0.5 for 48 h), made quiescent and then treated with PDGF-BB. C, D, miR-221 and miR-222 mediate PDGF-BB-induced SMC migration (C) and proliferation (D), without affecting cell viability (E). SMC were transduced with miR-221 or miR-222 inhibitor prior to the ad- dition of PDGF-BB (10 ng/ml). Cell migration was analyzed after 18 h using transwell migration assays (C). Cell proliferation was analyzed after 48 h by CyQUANT® Cell Proliferation Assay (D). Cleaved caspase-3 levels, as an indicator of reduced cell via- bility, was analyzed after 8 h by immunoblotting using antibodies that detect both total and cleaved caspase-3 levels (E). Hydrogen peroxide (H2O2, 100 μM) served as a positive control. ⁎P < at least 0.05 versus control, †P < .05 versus PDGF-BB (n = 6).

Article Snippet: The following lentiviral shRNA vectors against IKKβ (#sc-35645-V), p65 (#sc-29410-V), JNK2 (#sc-39101-V), and c-Jun (#sc-29223-V) were purchased from Santa Cruz Biotechnology, Inc. Lentiviral shRNA against RECK (SHCLNV-NM_021111; TRCN0000376461) and eGFP (SHC005V) were purchased from Sigma-Aldrich.

Techniques: Expressing, Incubation, shRNA, Migration, Transduction, CyQUANT Assay, Proliferation Assay, Western Blot, Positive Control, Control

Journal: Oncotarget

Article Title: HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling

doi: 10.18632/oncotarget.17699

Figure Lengend Snippet: ChIP assay was performed to determine which EGR1 promotor segments bind to p65 in TaxP cells ( A ). PCR products were subjected to electrophoresis on a 1.2% agarose gel. Results are averaged from three independent ChIP assays ( B ). Error bars show standard deviations. TaxP or MT2 cells were transfected with pNFκB-luc and p65 shRNA, and luciferase activity was measured after 48 h ( C ). TaxP or MT2 cells were transfected with EGR1-luc (E1) and p65 shRNA, and luciferase activity was measured after 48 h ( D ). TaxP or MT2 cells were transfected with pNFκB-luc ( E ) or EGR1-luck ( F ) and treated with Bay 11-7082. After 48 h, luciferase activity was measured. Results are shown as relative expression normalized to the control. * p < 0.01.

Article Snippet: To construct a plasmid expressing full-length Tax with an RFP tag, the Tax CDS was subcloned from pCMV-Bam-Tax into the Bgl II and Hind III sites of the pmCherry-C1 vector. pCMV-M22 Tax and pCMV-M47 Tax plasmids were kindly provided by Edward Harhaj (The University of Miami). p65 shRNA (sc-29410-SH) was purchased from Santa Cruz.

Techniques: Electrophoresis, Agarose Gel Electrophoresis, Transfection, shRNA, Luciferase, Activity Assay, Expressing, Control

Journal: Oncotarget

Article Title: HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling

doi: 10.18632/oncotarget.17699

Figure Lengend Snippet: MT2 or TaxP cells were transfected with p65 shRNA for 48 h, and ACTB, EGR1, and p65 were detected by western blot ( A ). MT2 or TaxP cells were treated with or without 5 mM BAY 11-7082 for 48 h, and ACTB, EGR1, and p65, Tax, p-IκB, and IκB were detected by western blot ( B ). Jurkat cells were transfected with pNF-κB-luc and plasmids expressing wild-type Tax, M22 Tax, or M47 Tax for 48 h, and luciferase activity was measured ( C ). Jurkat cells were transfected with the indicated EGR1 promoter-luc plasmids and plasmids expressing wild-type Tax, M22 Tax, or M47 Tax for 48h, and luciferase activity was measured ( D ). Jurkat cells were transfected with plasmids expressing wild-type Tax, M22 Tax, or M47 Tax for 48 h and EGR1, CREB, p65, Tax, and ACTB were detected by western blot E. Jurkat cells were transfected with EGR1-luc plasmids and plasmids expressing wild-type Tax, M22 Tax, or M47 Tax for 48 h, and luciferase activity was measured F. * p < 0.01.

Article Snippet: To construct a plasmid expressing full-length Tax with an RFP tag, the Tax CDS was subcloned from pCMV-Bam-Tax into the Bgl II and Hind III sites of the pmCherry-C1 vector. pCMV-M22 Tax and pCMV-M47 Tax plasmids were kindly provided by Edward Harhaj (The University of Miami). p65 shRNA (sc-29410-SH) was purchased from Santa Cruz.

Techniques: Transfection, shRNA, Western Blot, Expressing, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling

doi: 10.18632/oncotarget.17699

Figure Lengend Snippet: TaxP ( A ) or MT2 cells ( B ) were transfected with pNF-κB-luc and different quantities of pcDNA3.0-EGR1 for 48 h, and luciferase activity was measured. TaxP ( C ) or MT2 ( D ) cells were transfected with different quantities of pcDNA3.0-EGR1 for 48 h, and EGR1, p65, Tax, and ACTB were detected by western blot. TaxP cells were transfected with pcDNA3.0-EGR1 for 24 h, and p65, Lamin B, and EGR1 in the nuclei were analyzed by western blot ( E ) Relative levels of nuclear p65 were analyzed from three individual experiments. TaxP or MT2 cells were transfected with EGR1 siRNA for 48 h and EGR1 was detected by western blot ( F ) MT2 or TaxP cells were transfected with EGR1 siRNA and pNF-κB-luc for 48 h, and luciferase activity was detected ( G ) Schematic showing the positive feedback loop between EGR1 and NF-κB activation in HTLV-1-infected cells ( H ) * p < 0.01.

Article Snippet: To construct a plasmid expressing full-length Tax with an RFP tag, the Tax CDS was subcloned from pCMV-Bam-Tax into the Bgl II and Hind III sites of the pmCherry-C1 vector. pCMV-M22 Tax and pCMV-M47 Tax plasmids were kindly provided by Edward Harhaj (The University of Miami). p65 shRNA (sc-29410-SH) was purchased from Santa Cruz.

Techniques: Transfection, Luciferase, Activity Assay, Western Blot, Activation Assay, Infection

Journal: Journal of Bio-X Research

Article Title: GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

doi: 10.1097/jbr.0000000000000010

Figure Lengend Snippet: Figure 3. GPER1, but not ERa/b, is responsible for FAK phosphorylation in breast cancer cells. MCF-7 cells were exposed to either 10nM 17b-estradiol (E2) or 1m MGPER1 agonist G1 for 20minutes in the presence or absence of the pure ER antagonist ICI 182780 (ICI; 1mM) or GPER1 antagonist G15 (1mM). Top, Western blot of FAK and phosphorylated FAK; bottom, quantification of p-FAK. p-FAK densitometry values were adjusted to FAK intensity and then normalized to expression from the control sample. ∗P<.05, ∗∗P<.01, versus control; ###P<.001, versus E2 or G1. (B) MDA-MB-231 cells were transfected with empty vector or GPER1 plasmid for 48hours and then exposed to 10nM E2 or 1mM G1 for 20minutes. Western blot for p-FAK, FAK, GPER1and b-actin was performed. (C) MCF-7 cells were transfected with scrambled siRNA, ERa, ERb, or GPER1 targeted siRNA for 48hours. ERa, ERb, or GPER1protein expressions were detected by western blot; b-actin intensity was used as loading control. (D–F) MCF-7 cells were transfected with 100nM scrambled siRNA or target siRNAs for ERa, ERb, or GPER1 for 48hours and then treated with E2 (10nM) or G1 (1mM) for 20minutes. Top, Western blot of FAK or phosphorylated FAK; bottom, densitometric analysis of p-FAK. ∗P<.05, ∗∗P<.01, versus control; ##P<.01, versus E2. All the experiments were repeated at least three times with consistent results and representative images are shown. ER=estrogen receptor, GPER1=G-protein-coupled ER, FAK=focal adhesion kinase.

Article Snippet: The siRNAs used for experiments included ERa siRNA (#sc29305, Santa Cruz, CA), ERb siRNA (#SR301462, OriGene, Rockville, MD, USA), GPER1 siRNA (#SR301912, OriGene), cSrc siRNA (#SR304574, OriGene, Rockville, MD, USA) and p65 siRNA (#6534, Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Transfection, Plasmid Preparation

Journal: Journal of Bio-X Research

Article Title: GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

doi: 10.1097/jbr.0000000000000010

Figure Lengend Snippet: Figure 5. c-Src/NF-kB-p65 cascade is involved in the phosphorylation of FAK induced by E2. (A) SK-BR-3 cells were exposed to 10nM E2 for 20minutes, in presence or absenceofthePI3KinhibitorLY294002(LY;50mM),theMEKinhibitorPD98059(PD;5mM),thec-SrcinhibitorPP2(10mM),theGproteininhibitorPTX(100ng/mL)ortheNF-kB inhibitor PDTC (10mM). Top, Western blot of FAK and p-FAK; bottom, p-FAK densitometry values were adjusted to FAK intensity and then normalized to expression from the control sample. ∗P<.05, ∗∗P<.01, versus control; ##P<.01, versus E2. (B, C) SK-BR-3 breast cancer cells were treated with E2 (B) or G1 (C) at the indicated concentrations. Top, Western blot of c-Src, Tyr416-phosphorylated c-Src (p-c-Src), p65 or Ser536-phophor-p65; bottom, densitometry values of p-c-Src and p-p65 were adjusted to c-Src and p65 intensities, respectively, andthen normalized to expression from the controlsample. ∗P<.05, ∗∗P<.01, versus control. (D) SK-BR-3 cells were treated with 10nM E2 for 20 minuteswithorwithoutc-SrcinhibitorPP2(10mM)orNF-kBinhibitor PDTC (10mM).Top:Westernblotanalysis;bottom,p-c-Srcandp-p65densitometryvalues wereadjusted to c-Src and p65 intensities, respectively, then normalized to expression from the control sample. ∗P<.05, ∗∗P<.01, versus control; ##P<.01, versus E2. (E, F) SK-BR-3 cells were transfected with 100nM scrambled siRNA or target siRNAs for c-Src (E) or p65 (F) for 48hours, followed by treatment with E2 (10nM) or G1 (1mM) for 20minutes. Top, WesternblotforphosphorylationofFAK,c-Srcandp65;bottom,densitometricanalysisofphosphorylatedFAK,c-Srcandp65normalizedbyb-actin. ∗P<.05, ∗∗P<.01,versus control; ##P<.01,versuscorrespondingE2orG1inscrambledsiRNAgroup.Alltheexperimentswererepeatedatleast3timeswithconsistentresultsandrepresentativeimages were shown. NF-kB=nuclear factor kappa B, FAK: focal adhesion kinase, E2=17b-estradiol, GPER1=G-protein-coupled estrogen receptor.

Article Snippet: The siRNAs used for experiments included ERa siRNA (#sc29305, Santa Cruz, CA), ERb siRNA (#SR301462, OriGene, Rockville, MD, USA), GPER1 siRNA (#SR301912, OriGene), cSrc siRNA (#SR304574, OriGene, Rockville, MD, USA) and p65 siRNA (#6534, Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Transfection

Journal: Journal of Bio-X Research

Article Title: GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

doi: 10.1097/jbr.0000000000000010

Figure Lengend Snippet: Figure 6. GPER1 interacts with c-Src to promote cell migration and invasion. SK-BR-3 cells were transfected with 100nM GPER1 siRNAs or scrambled siRNAs for 48hours, followed by treatment with E2 (10nM) or GPER1 agonist G1 (1mM) for 20minutes. Left, Western blot; right, densitometry of phosphorylated FAK, c-Src and p65. ∗P<.05, ∗∗P<.01, versus control; #P<.05, ##P<.01, versus corresponding E2 or G1 in scrambled siRNA group. (B) SK-BR-3 cells were treated with vehicle, E2 or G1 for 20minutes and lysates were subjected to immunoprecipitation using anti-GPER1 antibody followed by Western blot with anti-c-Src antibodies. (C) Cells were transfected with 100nM GPER1 siRNAs or scrambled siRNAs for 48hours and then treated with E2 (10nM) or G1 (1mM) for an additional 48hours. Cell migration distances are shown below images as mean migration distance (mm)±SD. ∗∗∗P<.001, versus control; ###P<.001, versus E2 or G1 in scrambled siRNA group. (D) Cell invasion was assayed using invasion chambers. Representative images and invasion indexes are shown. ∗∗∗P<.001, vs. control; ###P<.001, versus E2 or G1 in scrambled siRNA group. All the experiments were repeated at least 3 times with consistent results. GPER1=G-protein-coupled estrogen receptor; FAK=focal adhesion kinase.

Article Snippet: The siRNAs used for experiments included ERa siRNA (#sc29305, Santa Cruz, CA), ERb siRNA (#SR301462, OriGene, Rockville, MD, USA), GPER1 siRNA (#SR301912, OriGene), cSrc siRNA (#SR304574, OriGene, Rockville, MD, USA) and p65 siRNA (#6534, Cell Signaling Technology).

Techniques: Migration, Transfection, Western Blot, Control, Immunoprecipitation