Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 1. JNK1 directly binds to TAp63c at its transactivation domain. (A), Hela cells transfected with Flag-JNK1 were lysed and subjected to GST pull-down assay with GST alone or GST-TAD. 10 lg of total protein from the same cell lysates were directly loaded as an input control. The adsorbates were analyzed by immunoblotting analysis (IB) using anti-Flag (top panel). Comparable amounts of GST and GST-TAD fusion proteins were shown by staining the membrane with Coomassie blue R-250 (bottom panel). (B and C) H1299 cells were co-transfected with HA-TAp63c and Flag-JNK1. Whole-cell lysates were subjected to immunoprecipitation (IP) with normal mouse IgG, anti-HA or anti-Flag. IP products were subjected to IB analysis with indicated primary antibodies and trueblot secondary antibody.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Transfection, Pull Down Assay, Control, Western Blot, Staining, Membrane, Immunoprecipitation

Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 2. Overexpression of JNK1 inhibits TAp63c transactivity. (A) H1299 cells were transfected with a mixture of Bax-Luc and TK-Renilla plus HA-TAp63c or (and) Flag-JNK1 plasmid; (B) H1299 cells were transfected with a mixture of Bax-Luc and TK-Renilla plus HA-TAp63c and increasing amounts of Flag-JNK1 plasmid. Firefly and Renilla luciferase activities were measured. The Bax-Luc activity was normalized to Renilla activity and presented as percentage of Bax-Luc expression with standard deviation (n = 3). Bax-Luc expression in cells transfected with TAp63c alone with Bax-Luc/TK-Renilla mixture was set as 100%. IB analysis was performed to detect the expression of TAp63c and JNK1 using anti-HA and anti-Flag respectively.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Over Expression, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Standard Deviation

Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 3. Ser12 in TAp63c is critical for JNK1-mediated trans-inhibition. H1299 cells were transfected with Bax-Luc/TK-Renilla mixture plus HA-TAp63c (wild-type or S12A mutant) or (and) Flag-JNK1 plasmid. Firefly and Renilla luciferase activities were measured. The Bax-Luc activity was normalized to Renilla activity and presented as percentage of Bax-Luc expression with standard deviation (n = 3). Bax-Luc expression in cells transfected with wild-type TAp63c alone with Bax-Luc/ TK-Renilla mixture was set as 100 percent. IB analysis was performed to detect the expression of TAp63c and JNK1 using anti-HA and anti-Flag respectively.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Inhibition, Transfection, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Standard Deviation

Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 4. Inhibition or knockdown of JNK1 increases TAp63c transactivity. (A) H1299 cells were transfected with Bax-Luc/TK-Renilla mixture with or without HA-TAp63c, and treated with JNK inhibitor SP600125 (JNKin); (B) H1299 cells were transfected with Bax-Luc/TK-Renilla mixture plus HA-TAp63c or (and) siJNK1. Firefly and Renilla luciferase activities were measured. The Bax-Luc activity was normalized to Renilla activity and presented as percentage of Bax-Luc expression with standard deviation (n = 3). Bax-Luc expression in cells transfected with TAp63c alone with Bax-Luc/TK-Renilla mixture was set as 100%. IB analysis was performed to detect the expression of TAp63c, total or phosphorylated (p-JNK1) JNK1 using anti-HA, antibody specific to total or T183-phosphorylated JNK1, respectively.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Inhibition, Knockdown, Transfection, Luciferase, Activity Assay, Expressing, Standard Deviation

Journal: FEBS letters

Article Title: JNK1 inhibits transcriptional and pro-apoptotic activity of TAp63γ.

doi: 10.1016/j.febslet.2015.10.028

Figure Lengend Snippet: Fig. 5. JNK1 inhibits TAp63c-mediated cell apoptosis. H1299 cells were transfected with HA-TAp63c and (or) Flag-JNK1. 48 h post transfection, cells were trypsinized and subjected to IB (A) or flow cytometry analysis (B) of apoptosis. Cleaved PARP1 (CL-PARP), JNK1 and TAp63c were detected with antibody to N-terminal cleaved PARP1, Flag, or HA respectively.

Article Snippet: JNK1 siRNA or scrambled control (Santa Cruz) was transfected with Lipofectamin 2000 to knock down endogenous JNK1.

Techniques: Transfection, Cytometry

Journal: Scientific Reports

Article Title: JNK confers 5-fluorouracil resistance in p53-deficient and mutant p53-expressing colon cancer cells by inducing survival autophagy

doi: 10.1038/srep04694

Figure Lengend Snippet: (A) Pharmacological inhibition of autophagy with 3-MA or CQ. Representative cell morphological changes are detected by light microscopy; characteristic morphological features of apoptosis were observed, including detachment and cell shrinkage. (B) The expression of ATG5 or Beclin1 in HCT116 p53 −/− and HT29 cells transiently transfected with negative control siRNA, siAtg5 or siBeclin1 were determined by western blotting. (C) Cell viability was quantified by the MTT assay in HCT116 p53 −/− and HT29 cells transiently transfected with negative control siRNA, siAtg5 or siBeclin1, *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (D) Clonogenic survival assay, *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (E) Representative results of annexin V-FITC/PI staining and quantitative analysis; values are expressed as the mean ± SD of three independent experiments; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (F) HCT116 p53 −/− and HT29 cells were treated by 20 µM 5-FU with or without 3-MA for 24 h. Cells were fluorescently labeled and imaged using a confocal microscope. Green, FITC-labeled LC3; Blue, DAPI-labeled nucleus; quantitative analyses of punctate fluorescence numbers are shown; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (G) The expression of LC3B II and active caspase-3 was examined by western blotting.

Article Snippet: Cells were transfected with either nonspecific siRNA (Qiagen,1027280), Atg5 siRNA (Qiagen, SI02655310), Beclin1 siRNA (Cell Signaling Technology, #6222) or JNK siRNA (Cell Signaling Technology, #6233) via LipofectAMINE RNAi max (Invitrogen, 13778150) according to the manufacturer's instructions.

Techniques: Inhibition, Light Microscopy, Expressing, Transfection, Negative Control, Western Blot, MTT Assay, Clonogenic Cell Survival Assay, Staining, Labeling, Microscopy, Fluorescence

Journal: Scientific Reports

Article Title: JNK confers 5-fluorouracil resistance in p53-deficient and mutant p53-expressing colon cancer cells by inducing survival autophagy

doi: 10.1038/srep04694

Figure Lengend Snippet: (A) HCT116 p53 −/− and HT29 cells were treated with 5-FU with or without SP600125 for 24 h. Cells were fluorescently labeled and imaged using a confocal microscope. Green, FITC-labeled LC3; Blue, DAPI-labeled nucleus; quantitative analyses of punctate fluorescence numbers are shown; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (B) JNK, c-Jun and Bcl-2 phosphorylation, as well as the expression of active caspase-3 and several key autophagic regulators, were analyzed in HCT116 p53 −/− and HT29 cells treated with the combination of 5-FU and SP600125 (the blots were cropped, and the full-length blots are included in the ). (C) Cell viability was quantified by the MTT assay in these resistant cells treated with the combination of 5-FU and SP600125; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (D) Clonogenic survival assay; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (E) The expression of JNK in HCT116 p53 −/− and HT29 cells was analyzed by RT-PCR when these cells were transiently transfected with the negative control siRNA siJNK (F) JNK, c-Jun and Bcl-2 phosphorylation, as well as the expression of active caspase-3 and several key autophagic regulators, were analyzed in HCT116 p53 −/− and HT29 cells transiently transfected with the negative control siRNA siJNK (the blots were cropped, and the full-length blots are included in the ). (G) Cell viability was quantified by the MTT assay in these resistant cells transiently transfected with the negative control siRNA siJNK; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate.

Article Snippet: Cells were transfected with either nonspecific siRNA (Qiagen,1027280), Atg5 siRNA (Qiagen, SI02655310), Beclin1 siRNA (Cell Signaling Technology, #6222) or JNK siRNA (Cell Signaling Technology, #6233) via LipofectAMINE RNAi max (Invitrogen, 13778150) according to the manufacturer's instructions.

Techniques: Labeling, Microscopy, Fluorescence, Phospho-proteomics, Expressing, MTT Assay, Clonogenic Cell Survival Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control

Journal: Journal of translational medicine

Article Title: c-Jun N-terminal kinase activation by nitrobenzoxadiazoles leads to late-stage autophagy inhibition.

doi: 10.1186/s12967-016-0796-x

Figure Lengend Snippet: Fig. 1 Effects of MC3181 on TRAF2/JNK signaling in U-2OS cells. a Confocal microscopy for the in situ detection of the TRAF2-GSTP1-1 complex in cells treated with vehicle (CTRL) or 5 μM MC3181 (i.e. a concentration about five times the IC50 value) for the indicated times. The TRAF2-GSTP1-1 complex was visualized by PLA, which generates fluorescent dots (red) when the two proteins are in close proximity. DAPI (blue) was used to counterstain cell nuclei. Scale bar 10 μM. b Amount per cell of TRAF2-GSTP1-1 complex as calculated from the analysis of PLA images (mean ± SEM, n ≥ 3); *p < 0.05. c Western blot analysis of JNK phospho-activation in cells treated with vehicle (CTRL) or 5 μM MC3181 for the indicated times. The filter was probed with anti-phospho-JNK (p-JNK) and anti-JNK1 antibodies; β-actin was used to ensure equal loading and transfer of samples

Article Snippet: Vectors for JNK1 RNA interference were purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Confocal Microscopy, In Situ, Concentration Assay, Western Blot, Activation Assay

Journal: Journal of translational medicine

Article Title: c-Jun N-terminal kinase activation by nitrobenzoxadiazoles leads to late-stage autophagy inhibition.

doi: 10.1186/s12967-016-0796-x

Figure Lengend Snippet: Fig. 4 Autophagic flux studies in JNK1-silenced cells. a Western blot for JNK1 in untransfected, scrambled shRNA-transfected and JNK1 shRNA- transfected cells. β-Actin was used to ensure equal loading and transfer of samples. b Western blot analysis of JNK phospho-activation in cells treated with vehicle (CTRL) or 5 μM NBDHEX for the indicated times. The filter was probed with anti-phospho-JNK (p-JNK) and anti-JNK1 antibodies; β-actin was used to ensure equal loading and transfer of samples. c Lysates from scrambled shRNA- and JNK1 shRNA-transfected cells treated with vehicle (CTRL) or 5 μM NBDHEX for 24 h, incubated or not with 100 nmol/l BAF for 3 h before lysis, were subjected to western blot for LC3-II and β-actin. d LC3-II/β-actin ratios as determined from the densitometric analysis of autoradiograms obtained in replicates of the experiment shown in c, expressed in arbitrary units (mean ± SEM, n ≥ 3; *p < 0.05 vs. CTRL; #p < 0.05 vs. minus BAF). e Autophagic flux indexes calculated from the data illustrated in d as the difference in LC3-II/actin ratios between samples plus and minus BAF (*p < 0.05 vs. CTRL), expressed in arbitrary units (a.u.)

Article Snippet: Vectors for JNK1 RNA interference were purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Western Blot, shRNA, Transfection, Activation Assay, Incubation, Lysis

Journal: Journal of translational medicine

Article Title: c-Jun N-terminal kinase activation by nitrobenzoxadiazoles leads to late-stage autophagy inhibition.

doi: 10.1186/s12967-016-0796-x

Figure Lengend Snippet: Fig. 5 Cell death studies in JNK1-silenced cells. a Caspase-3 activity assay in scrambled shRNA- and JNK1 shRNA-transfected cells treated with vehicle (CTRL), 5 μM MC3181 or 5 μM NBDHEX for 24–48 h (mean ± SEM, n ≥ 3; *p < 0.05 vs. CTRL). b, c Analysis of DNA content by flow cytometry on scrambled shRNA- and JNK1 shRNA-transfected cells treated with vehicle (CTRL), 5 μM MC3181 or 5 μM NBDHEX for 48 h. b A representative experiment with the percentage of sub-G1 apoptotic cells is shown. c Histograms illustrating the percentage of cells in each phase of cell cycle, resulting from four independent experiments

Article Snippet: Vectors for JNK1 RNA interference were purchased from OriGene Technologies, Rockville, MD, USA.

Techniques: Caspase-3 Activity Assay, shRNA, Transfection, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Antrodia cinnamomea Galactomannan Elicits Immuno-stimulatory Activity Through Toll-like Receptor 4

doi: 10.7150/ijbs.24564

Figure Lengend Snippet: ACP induces cytokine expression through MAPK. ( A ) J774A.1 macrophages were incubated for 0-60 min with or without ACP (100 µg/mL). The phosphorylation levels of ERK1/2, JNK1/2 and p38 were assayed by Western blot. ( B, C ) J774A.1 macrophages stably transfected with a control shRNA plasmid (sh-SC), a ERK1 shRNA plasmid (sh-ERK1), a JNK1 shRNA plasmid (sh-TLR2), a p38α shRNA plasmid (sh-p38α), and a p38β shRNA plasmid (sh-p38β) were incubated for 24 h with or without ACP (100 μg/mL). The levels of TNF-α ( B ) and IL-6 ( C ) in the culture medium were measured by ELISA. The Western blot results are representative of those obtained in three different experiments. The ELISA data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001.

Article Snippet: All plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): ERK1 shRNA plasmid (sh-ERK1, sc-29308-SH), JNK1 shRNA plasmid (sh-JNK1, sc-29381-SH), p38α shRNA plasmid (sh-p38α, sc-29434-SH), p38β shRNA plasmid (sh-p38β, sc-39117-SH), TLR2 shRNA plasmid (sh-TLR2, sc-40257-SH) and TLR4 shRNA plasmid (sh-TLR4, sc-40261-SH).

Techniques: Expressing, Incubation, Western Blot, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Biological Sciences

Article Title: Antrodia cinnamomea Galactomannan Elicits Immuno-stimulatory Activity Through Toll-like Receptor 4

doi: 10.7150/ijbs.24564

Figure Lengend Snippet: ACP pretreatment induces LPS tolerance. ( A, B ) J774A.1 macrophages ( A ) and mouse peritoneal macrophages ( B ) were incubated for 24 h with or without ACP (100 μg/mL) or LPS (0.1 μg/mL)(1 st stimulus). After washing, cell cultures were changed for fresh medium and then were incubated for 24 h with or without LPS (1 μg/mL)(2 nd stimulus). The levels of TNF-α and IL-6 in the culture medium were measured by ELISA. ( C ) J-Blue cells were incubated for 24 h with or without ACP (100 μg/mL) or LPS (0.1 μg/mL)(1 st stimulus). After washing, cell cultures were changed for fresh medium and then were incubated for 24 h with or without LPS (1 μg/mL)(2 nd stimulus). The activation levels of NF-κB were measured by an NF-κB reporter assay. (D) J774A.1 macrophages were incubated for 24 h with or without ACP (100 μg/mL). After washing, cell cultures were changed for fresh medium and then were incubated for 0-30 min with or without LPS (1 µg/mL). The phosphorylation levels of ERK1/2, JNK1/2 and p38 were assayed by Western blot. The Western blot results are representative of those obtained in three different experiments. The data are expressed as the means ± SD of three separate experiments. *** indicates a significant difference at the level of p < 0.001, compared to 1 st P/2 nd L group. P: PBS; L: LPS.

Article Snippet: All plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA): ERK1 shRNA plasmid (sh-ERK1, sc-29308-SH), JNK1 shRNA plasmid (sh-JNK1, sc-29381-SH), p38α shRNA plasmid (sh-p38α, sc-29434-SH), p38β shRNA plasmid (sh-p38β, sc-39117-SH), TLR2 shRNA plasmid (sh-TLR2, sc-40257-SH) and TLR4 shRNA plasmid (sh-TLR4, sc-40261-SH).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Reporter Assay, Western Blot

Journal: Aging (Albany NY)

Article Title: MUC1 promotes RIF by regulating macrophage ROS-SHP2 signaling pathway to up-regulate inflammatory response and inhibit angiogenesis

doi: 10.18632/aging.205560

Figure Lengend Snippet: Diagnostic efficiency of hub genes and validation. ( A ) ROC analysis of each hub gene using dataset 111974. Only the hub genes whose AUC was higher than 0.75 were selected. The expression levels of selected hub in dataset 111974 ( B ), dataset 92324 ( B ), and local plasma samples ( C , D ). ( E ) ROC analysis of each hub gene using local plasma samples. ( F ) ROC analysis of gene set using local plasma samples, dataset111974 and dateset92324. Gene set means a 4-gene (MUC1-PTGS1-PTGS2-CXCL11) signature. * p < 0.05, *** p < 0.001. Abbreviations: ROC: receiver operating characteristic; AUC: area under the curve.

Article Snippet: RAW264.7 and THP-1 cells of the four groups are stimulated with LPS (20 ng/ml) for 24 h. The small interfering RNA (siRNA) of MUC1 was synthesized by RiboBio (China).

Techniques: Diagnostic Assay, Biomarker Discovery, Expressing, Clinical Proteomics

Journal: Aging (Albany NY)

Article Title: MUC1 promotes RIF by regulating macrophage ROS-SHP2 signaling pathway to up-regulate inflammatory response and inhibit angiogenesis

doi: 10.18632/aging.205560

Figure Lengend Snippet: MUC1 can promote oxidative stress levels in macrophages. ( A ) Protein bands of MUC1, P22, P47 and gp91. ( B ) Statistics of relative protein expression levels of MUC1, P22, P47 and gp91. N = 3; ** P < 0.01; ns P > 0.05.

Article Snippet: RAW264.7 and THP-1 cells of the four groups are stimulated with LPS (20 ng/ml) for 24 h. The small interfering RNA (siRNA) of MUC1 was synthesized by RiboBio (China).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: MUC1 promotes RIF by regulating macrophage ROS-SHP2 signaling pathway to up-regulate inflammatory response and inhibit angiogenesis

doi: 10.18632/aging.205560

Figure Lengend Snippet: MUC1 can promote inflammatory responses in macrophages. ( A ) Protein bands of p-SHP2, p-TBK1, IFN-γ and IL-1β. ( B ) Statistics of relative protein expression levels of p-SHP2, p-TBK1, IFN-γ and IL-1β. N = 3; ** P < 0.01; ns P > 0.05.

Article Snippet: RAW264.7 and THP-1 cells of the four groups are stimulated with LPS (20 ng/ml) for 24 h. The small interfering RNA (siRNA) of MUC1 was synthesized by RiboBio (China).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: MUC1 promotes RIF by regulating macrophage ROS-SHP2 signaling pathway to up-regulate inflammatory response and inhibit angiogenesis

doi: 10.18632/aging.205560

Figure Lengend Snippet: MUC1 can inhibit angiogenesis. ( A ) Statistics of protein bands and relative protein expression levels of p-PI3K, p-mTOR, HIF1α and VEGF. ( B ) Diagram of experimental results of angiogenesis and statistics of the number of angiogenesis. N = 3; ** P < 0.01; ns P > 0.05.

Article Snippet: RAW264.7 and THP-1 cells of the four groups are stimulated with LPS (20 ng/ml) for 24 h. The small interfering RNA (siRNA) of MUC1 was synthesized by RiboBio (China).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: MUC1 promotes RIF by regulating macrophage ROS-SHP2 signaling pathway to up-regulate inflammatory response and inhibit angiogenesis

doi: 10.18632/aging.205560

Figure Lengend Snippet: Inhibition of MUC1 can promote angiogenesis. ( A ) Protein bands of MUC1, p-PI3K and VEGF. ( B ) Statistics of relative protein expression levels of MUC1, p-PI3K and VEGF. ( C ) Diagram of experimental results of angiogenesis and statistics of the number of angiogenesis. N = 3; ** P < 0.01; ns P > 0.05.

Article Snippet: RAW264.7 and THP-1 cells of the four groups are stimulated with LPS (20 ng/ml) for 24 h. The small interfering RNA (siRNA) of MUC1 was synthesized by RiboBio (China).

Techniques: Inhibition, Expressing

Journal: Aging (Albany NY)

Article Title: MUC1 promotes RIF by regulating macrophage ROS-SHP2 signaling pathway to up-regulate inflammatory response and inhibit angiogenesis

doi: 10.18632/aging.205560

Figure Lengend Snippet: MUC1 promotes RIF by regulating macrophage ROS-SHP2 signaling pathway to up-regulate inflammatory response and inhibit angiogenesis.

Article Snippet: RAW264.7 and THP-1 cells of the four groups are stimulated with LPS (20 ng/ml) for 24 h. The small interfering RNA (siRNA) of MUC1 was synthesized by RiboBio (China).

Techniques:

Journal: Oncotarget

Article Title: Anti-proliferation of triple-negative breast cancer cells with physagulide P: ROS/JNK signaling pathway induces apoptosis and autophagic cell death

doi: 10.18632/oncotarget.19299

Figure Lengend Snippet: (A) Representative images of MDA-MB-231 and MDA-MB-468 cells transfected with Ad-mCherry-GFP-LC3B adenovirus after PP treatment (6 μM, 18 h). Scale bars = 5 μm. (B) MDA-MB-231 cells were transfected with 100 nM of LC3B siRNA for 24 h and then treated with different concentrations of PP for 24 h. The viability of MDA-MB-231 cells was then assessed with an MTT assay. (C) Apoptotic MDA-MB-231 cells were detected by flow cytometry after treatment with 6 μM PP for 24 h in the presence or absence of LC3B siRNA. (D) The protein expression levels of cleaved caspase-9, cleaved caspase-3 and LC3BII in MDA-MB-231 cells transfected with LC3B siRNA in the presence or absence of 6 μM PP. The results were similar in at least three independent experiments. * p < 0.05, ** p < 0.01 vs. si NC group.

Article Snippet: JNK siRNA [ ], LC3B siRNA [ ], p21 siRNA pools (siRNA1: CAGGCGGUUAUGAAAUUCAdTdT, siRNA2: GAUGGAACUUCGACUUUGUdTdT and siRNA3: CCUCUGGCAUUAGAAUUAUdTdT) and non-targeting siRNA were synthesized by Biomics (Nantong, China).

Techniques: Transfection, MTT Assay, Flow Cytometry, Expressing

Journal: Oncotarget

Article Title: Anti-proliferation of triple-negative breast cancer cells with physagulide P: ROS/JNK signaling pathway induces apoptosis and autophagic cell death

doi: 10.18632/oncotarget.19299

Figure Lengend Snippet: (A) The ROS levels in MDA-MB-231 and MDA-MB-468 cells incubated with various concentrations of PP for 24 h were measured by flow cytometry. (B) The cells were treated with 6 μM PP alone or in combination with NAC (5 mM) and then subjected to flow cytometry to measure ROS levels. (C) The expression levels of cell cycle-regulated proteins in cells treated with 6 μM PP alone or in combination with NAC were measured by Western blotting. (D) The cell cycle was evaluated by flow cytometry in cells treated with 6 μM PP alone or in combination with NAC. (E) The ROS levels and the protein expression level of p21 in MDA-MB-231 cells transfected with p21 siRNA in the presence or absence of 6 μM PP. The results were similar in at least three independent experiments. * p < 0.05, ** p < 0.01, vs. control group or si NC group; # p < 0.05, ## p < 0.01 vs. PP group.

Article Snippet: JNK siRNA [ ], LC3B siRNA [ ], p21 siRNA pools (siRNA1: CAGGCGGUUAUGAAAUUCAdTdT, siRNA2: GAUGGAACUUCGACUUUGUdTdT and siRNA3: CCUCUGGCAUUAGAAUUAUdTdT) and non-targeting siRNA were synthesized by Biomics (Nantong, China).

Techniques: Incubation, Flow Cytometry, Expressing, Western Blot, Transfection, Control

Journal: Oncotarget

Article Title: Anti-proliferation of triple-negative breast cancer cells with physagulide P: ROS/JNK signaling pathway induces apoptosis and autophagic cell death

doi: 10.18632/oncotarget.19299

Figure Lengend Snippet: (A) MDA-MB-231 and MDA-MB-468 cells were treated with different concentrations of PP for 24 h and 6 μM PP for different periods, and the expression levels of JNK and phospho-JNK were then measured by Western blotting. (B) The protein expression levels of JNK and phospho-JNK in MDA-MB-231 cells transfected with JNK siRNA in the presence or absence of 6 μM PP. (C) Apoptotic MDA-MB-231 cells were measured by flow cytometry after treatment with 6 μM PP for 24 h in the presence or absence of JNK siRNA. (D) The protein levels of cleaved caspase-9, cleaved caspase-7, cleaved caspase-3 and cleaved PARP in MDA-MB-231 cells transfected with JNK siRNA in the presence or absence of 6 μM PP. (E) The protein expression levels of Bax and Bcl-2 in MDA-MB-231 cells transfected with JNK siRNA in the presence or absence of 6 μM PP. (F) The changes in mitochondrial membrane potential in MDA-MB-231 cells were assessed by flow cytometry after treatment with 6 μM PP for 24 h in the presence or absence of JNK siRNA. (G) The protein expression levels of LC3BII in MDA-MB-231 cells transfected with JNK siRNA in the presence or absence of 6 μM PP. (H) The cell cycle was assessed by flow cytometry in MDA-MB-231 cells after treatment with 6 μM PP for 24 h in the presence or absence of JNK siRNA. (I) The protein levels of JNK and phospho-JNK in MDA-MB-231 and MDA-MB-468 cells treated with PP alone or in combination with NAC. (J) The ROS levels in MDA-MB-231 cells were detected by flow cytometry after treatment with 6 μM PP for 24 h in the presence or absence of JNK siRNA. The results were similar in at least three independent experiments. * p < 0.05, ** p < 0.01, vs. control group or si NC group; # p < 0.05, ## p < 0.01 vs. PP/si NC+PP group.

Article Snippet: JNK siRNA [ ], LC3B siRNA [ ], p21 siRNA pools (siRNA1: CAGGCGGUUAUGAAAUUCAdTdT, siRNA2: GAUGGAACUUCGACUUUGUdTdT and siRNA3: CCUCUGGCAUUAGAAUUAUdTdT) and non-targeting siRNA were synthesized by Biomics (Nantong, China).

Techniques: Expressing, Western Blot, Transfection, Flow Cytometry, Membrane, Control