Journal: Biomaterials science

Article Title: ZBTB20-mediated titanium particle-induced peri-implant osteolysis by promoting macrophage inflammatory responses.

doi: 10.1039/d0bm00147c

Figure Lengend Snippet: Fig. 2 TiPs induce the activation of NF-κB signaling pathway and phosphorylation of IRF-3 in BMDMs. (A) Protein concentrations of p-IKKβ, p-p65, and p-IκBα increased in the BMDMs after Ti0.2 or LPS (1 μg ml−1) stimulation for 1 h (p < 0.05). (B) Nuclear protein p65 of BMDMs increased after Ti0.2 stimulation for 30, 60, or 90 min (p < 0.05). (C) p65 protein (red) translocated from the cytoplasm into the nucleus (blue) after Ti0.2 stimulation for 1 h in BMDMs. (D) Ti0.2 induced the phosphorylation of IRF-3 at the indicated time points (p < 0.05). (E) After pretreatment with Ti0.2 for 8 h, IRF3 (yellow) aggregated into the nucleus (blue) in BMDMs. (F) Secreted IFN-β was significantly increased after Ti0.2 stimulation for 4 or 8 h (p < 0.05). *p < 0.05. Statistical significance was determined by one-way ANOVA with Fisher’s LSD test. All the data are shown as mean ± SD. The assays were performed at least three times for each sample.

Article Snippet: The antibodies used were p-IKKβ (rabbit, 2697, Cell Signaling Technology), IKKβ (rabbit, 8943, Cell Signaling Technology), p-p65 (rabbit, 3033, Cell Signaling Technology), p65 (rabbit, 8242, Cell Signaling Technology), p-IκBα (rabbit, 2859, Cell Signaling Technology), IκBα (mouse, 4814, Cell Signaling Technology), GAPDH (rabbit, 5174, Cell Signaling Technology), p-IRF3 (rabbit, 29047, Cell Signaling Technology), IRF3 (rabbit, ab68481, Abcam), ZBTB20 (rabbit, NBP2-20936, Novus Biologicals), p-JNK (rabbit, 4668, Cell Signaling Technology), p-ERK (rabbit, 4370, Cell Signaling Technology), p-p38 (rabbit, 4511, Cell Signaling Technology), and histone H3 (rabbit, This journal is © The Royal Society of Chemistry 2020 Biomater.

Techniques: Activation Assay, Phospho-proteomics

Journal: Biomaterials science

Article Title: ZBTB20-mediated titanium particle-induced peri-implant osteolysis by promoting macrophage inflammatory responses.

doi: 10.1039/d0bm00147c

Figure Lengend Snippet: Fig. 5 ZBTB20 positively regulates TiPs-induced NF-κB activation by repressing IκBα transcription. (A) TiPs- or LPS-induced p-IKKβ and p-p65 decreased in ZBTB20-knockdown BMDMs, and the level of IκBα increased in ZBTB20-knockdown BMDMs (p < 0.05). (B) TiPs-induced nuclear trans- location of p65 was suppressed in the ZBTB20-siRNA group when compared with the NC-siRNA group. (C) mRNA level of IκBα in the ZBTB20-siRNA group was significantly higher than that in the NC-siRNA group at each time point. (D) ZBTB20-expressing plasmid repressed the relative luciferase activity of IκBα-promoter-luciferase plasmid in a dose-dependent manner. (E) IκBα-siRNA rescued the impaired cytokines production of TNF and IL-6 caused by ZBTB20 knockdown. (F) IκBα-siRNA rescued the impaired TiPs-induced cellular TNF (red) and IL-6 (green) caused by ZBTB20 knock- down. *p < 0.05. Two-sided Student’s t-test was utilized in (C). One-way ANOVA with Fisher’s LSD test was utilized in (D) and (E). Statistical signifi- cance was determined by a two-sided Student’s t-test. All the data are shown as mean ± SD. The assays were performed at least three times for each sample.

Article Snippet: The antibodies used were p-IKKβ (rabbit, 2697, Cell Signaling Technology), IKKβ (rabbit, 8943, Cell Signaling Technology), p-p65 (rabbit, 3033, Cell Signaling Technology), p65 (rabbit, 8242, Cell Signaling Technology), p-IκBα (rabbit, 2859, Cell Signaling Technology), IκBα (mouse, 4814, Cell Signaling Technology), GAPDH (rabbit, 5174, Cell Signaling Technology), p-IRF3 (rabbit, 29047, Cell Signaling Technology), IRF3 (rabbit, ab68481, Abcam), ZBTB20 (rabbit, NBP2-20936, Novus Biologicals), p-JNK (rabbit, 4668, Cell Signaling Technology), p-ERK (rabbit, 4370, Cell Signaling Technology), p-p38 (rabbit, 4511, Cell Signaling Technology), and histone H3 (rabbit, This journal is © The Royal Society of Chemistry 2020 Biomater.

Techniques: Activation Assay, Knockdown, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal: Cell Death and Differentiation

Article Title: EBV infection-induced GPX4 promotes chemoresistance and tumor progression in nasopharyngeal carcinoma

doi: 10.1038/s41418-022-00939-8

Figure Lengend Snippet: mRNA and protein expression levels of GPX4 and SLC7A11 in EBV-negative and EBV-positive NPC cells were determined by RT–qPCR ( A ) ( n = 3) and immunoblotting ( B ) ( n = 3). C The p62-Keap1-NRF2 pathway was examined in EBV-negative and EBV-positive NPC cells by immunoblotting. D Cytoplasmic and nuclear proteins from EBV-negative and EBV-positive NPC cells were fractionated and detected by immunoblotting. Lamin B1 and GAPDH were used as controls for the nuclear and cytoplasmic fractions, respectively. E Immunofluorescence staining showing the localization of NRF2 in EBV-negative and EBV-positive NPC cells. mRNA and protein levels of NRF2 and GPX4 in EBV-negative and EBV-positive NPC cells were determined by RT–qPCR ( F ) ( n = 3) and immunoblotting ( G ) ( n = 3) after siRNA knockdown of endogenous NRF2. H GPX4 was highly expressed in CNE2 EBV + xenografts. I Representative images of immunohistochemistry staining showing GPX4 expression in paraffin-embedded tumor sections from NPC patients. J GPX4 expression in different groups according to EBV copy number in 181 NPC patients. Data are shown as the mean ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant. A , F Two-tailed unpaired t test ( F , compared to si-NC). J two-tailed Mann–Whitney test. S cale bars: 20 µm ( E ) and 100 µm ( H , I ).

Article Snippet: The following antibodies were used in this study: anti-4-hydroxynonenal (4-HNE), Abcam (ab46545), for immunoblotting and IHC; anti-p62, Abcam (ab109012), for immunoblotting; anti-NRF2, Abcam (ab62352), for immunoblotting and Cell Signaling Technology (#12721), for immunofluorescence staining; anti-Keap1, Abcam (ab227828), for immunoblotting; anti-SLC7A11, Abcam (ab175186), for immunoblotting; anti-GPX4, Abcam (ab125066), for immunoblotting and IHC; anti-GAPDH, Cell Signaling Technology, #5174, for immunoblotting; anti-β-actin, Abcam (ab8226), for immunoblotting; anti-TAK1, Abcam (ab109526), for immunoblotting; anti-p-TAK1(T187), Cell Signaling Technology, #4536, for immunoblotting; anti-TAB1, Abcam (ab76412), for immunoblotting and immunofluorescence staining; anti-TAB3, Abcam (ab124723), for immunoblotting and immunofluorescence staining; anti-Flag, Sigma (F1804), for immunoblotting and immunofluorescence staining; anti-IKKα, Cell Signaling Technology, #11930, for immunoblotting; anti-IKKβ, Cell Signaling Technology, #8943, for immunoblotting; anti-p-IKKα/β(Ser176/180), Cell Signaling Technology, #2697, for immunoblotting; anti-p-NFκB (Ser536), Cell Signaling Technology, #3033, for immunoblotting; anti-NFκB, Cell Signaling Technology, #8242, for immunoblotting; anti-p-IκBα (Ser32), Cell Signaling Technology, #2859, for immunoblotting; anti-IκBα (Ser32), Cell Signaling Technology, #4814, for immunoblotting; anti-p-JNK(Thr183/Tyr185), Cell Signaling Technology, #4668, for immunoblotting; anti-JNK, Cell Signaling Technology, #9252, for immunoblotting; anti-p-p38(Thr180/Tyr182), Cell Signaling Technology, #4511, for immunoblotting; and anti-p38, Cell Signaling Technology, #8690, for immunoblotting.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Knockdown, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: Cell Death and Differentiation

Article Title: EBV infection-induced GPX4 promotes chemoresistance and tumor progression in nasopharyngeal carcinoma

doi: 10.1038/s41418-022-00939-8

Figure Lengend Snippet: A . Two gRNA sequences targeting the EBNA1 gene of EBV. B EBNA1 protein expression was examined in EBV-positive NPC cells transduced with control or EBNA1-targeting gRNAs. C CNE2 cells carrying recombinant EBV-GFP virions were transduced with control or EBNA1-targeting gRNAs and assayed for GFP expression by fluorescence microscopy and flow cytometry ( n = 3). D NRF2 and GPX4 signaling in CRISPR/Cas9-mediated EBV-negative and CRISPR/Cas9-positive NPC cells was examined by immunoblotting. E Twenty-four hours after 5 µM RSL3 treatment, lipid ROS production was determined by C11-BODIPY staining and flow cytometry ( n = 3). F Cell death of CNE2 sgVector, CNE2-EBV sgEBNA1 or HK1 sgVector, HK1-EBV sgEBNA1 was measured and quantified after 5 µM RSL3 treatment for 24 h by flow cytometry ( n = 3). Data are shown as the mean ± SD. *** p < 0.001; **** p < 0.0001. C , E , and F , two-tailed unpaired t test. C , scale bar, 100 µm.

Article Snippet: The following antibodies were used in this study: anti-4-hydroxynonenal (4-HNE), Abcam (ab46545), for immunoblotting and IHC; anti-p62, Abcam (ab109012), for immunoblotting; anti-NRF2, Abcam (ab62352), for immunoblotting and Cell Signaling Technology (#12721), for immunofluorescence staining; anti-Keap1, Abcam (ab227828), for immunoblotting; anti-SLC7A11, Abcam (ab175186), for immunoblotting; anti-GPX4, Abcam (ab125066), for immunoblotting and IHC; anti-GAPDH, Cell Signaling Technology, #5174, for immunoblotting; anti-β-actin, Abcam (ab8226), for immunoblotting; anti-TAK1, Abcam (ab109526), for immunoblotting; anti-p-TAK1(T187), Cell Signaling Technology, #4536, for immunoblotting; anti-TAB1, Abcam (ab76412), for immunoblotting and immunofluorescence staining; anti-TAB3, Abcam (ab124723), for immunoblotting and immunofluorescence staining; anti-Flag, Sigma (F1804), for immunoblotting and immunofluorescence staining; anti-IKKα, Cell Signaling Technology, #11930, for immunoblotting; anti-IKKβ, Cell Signaling Technology, #8943, for immunoblotting; anti-p-IKKα/β(Ser176/180), Cell Signaling Technology, #2697, for immunoblotting; anti-p-NFκB (Ser536), Cell Signaling Technology, #3033, for immunoblotting; anti-NFκB, Cell Signaling Technology, #8242, for immunoblotting; anti-p-IκBα (Ser32), Cell Signaling Technology, #2859, for immunoblotting; anti-IκBα (Ser32), Cell Signaling Technology, #4814, for immunoblotting; anti-p-JNK(Thr183/Tyr185), Cell Signaling Technology, #4668, for immunoblotting; anti-JNK, Cell Signaling Technology, #9252, for immunoblotting; anti-p-p38(Thr180/Tyr182), Cell Signaling Technology, #4511, for immunoblotting; and anti-p38, Cell Signaling Technology, #8690, for immunoblotting.

Techniques: Expressing, Transduction, Control, Recombinant, Fluorescence, Microscopy, Flow Cytometry, CRISPR, Western Blot, Staining, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: EBV infection-induced GPX4 promotes chemoresistance and tumor progression in nasopharyngeal carcinoma

doi: 10.1038/s41418-022-00939-8

Figure Lengend Snippet: A A partial list of interacting proteins identified by mass spectrometry using cells stably expressing GPX4. The unique and total peptide numbers for the indicated proteins are shown. B Representative peptides of TAK1, TAB1, and TAB3. C 293 T cells transfected with empty vector control or Flag-GPX4 for 48 h were subjected to the co-IP assay. D Representative immunofluorescence images showing the colocalization of GPX4 and the indicated genes in CNE2 cells. E Schematic diagram showing the structure of the TAK1 protein and the designs of different truncations for domain mapping. F 293 T cells transfected with Flag-GPX4 and full-length or truncated myc-TAK1 for 48 h were subjected to a co-IP assay. G Purified GPX4 proteins were precipitated with GST-vector, GST-TAK1 1-606aa, or GST-TAK11-305aa proteins and detected by immunoblotting using anti-GPX4 antibody. GST-fusion proteins were detected by Coomassie blue staining. H 293 T cells transfected with the vector control or Flag-GPX4 and myc-TAK1 expression plasmids for 48 h were subjected to co-IP assay. I Analysis of the TAK1-NFκB/MAPK signaling pathway in the indicated stable cell lines by immunoblotting. mock, no shRNA; Ctrl, negative control shRNA. J Representative immunofluorescence images of NFκB (p65) and p38 in GPX4 knockdown or control CNE2 EBV-positive cells. shCtrl, negative control shRNA. Data are representative of three biologically independent experiments. D and J Scale bars: 20 µm.

Article Snippet: The following antibodies were used in this study: anti-4-hydroxynonenal (4-HNE), Abcam (ab46545), for immunoblotting and IHC; anti-p62, Abcam (ab109012), for immunoblotting; anti-NRF2, Abcam (ab62352), for immunoblotting and Cell Signaling Technology (#12721), for immunofluorescence staining; anti-Keap1, Abcam (ab227828), for immunoblotting; anti-SLC7A11, Abcam (ab175186), for immunoblotting; anti-GPX4, Abcam (ab125066), for immunoblotting and IHC; anti-GAPDH, Cell Signaling Technology, #5174, for immunoblotting; anti-β-actin, Abcam (ab8226), for immunoblotting; anti-TAK1, Abcam (ab109526), for immunoblotting; anti-p-TAK1(T187), Cell Signaling Technology, #4536, for immunoblotting; anti-TAB1, Abcam (ab76412), for immunoblotting and immunofluorescence staining; anti-TAB3, Abcam (ab124723), for immunoblotting and immunofluorescence staining; anti-Flag, Sigma (F1804), for immunoblotting and immunofluorescence staining; anti-IKKα, Cell Signaling Technology, #11930, for immunoblotting; anti-IKKβ, Cell Signaling Technology, #8943, for immunoblotting; anti-p-IKKα/β(Ser176/180), Cell Signaling Technology, #2697, for immunoblotting; anti-p-NFκB (Ser536), Cell Signaling Technology, #3033, for immunoblotting; anti-NFκB, Cell Signaling Technology, #8242, for immunoblotting; anti-p-IκBα (Ser32), Cell Signaling Technology, #2859, for immunoblotting; anti-IκBα (Ser32), Cell Signaling Technology, #4814, for immunoblotting; anti-p-JNK(Thr183/Tyr185), Cell Signaling Technology, #4668, for immunoblotting; anti-JNK, Cell Signaling Technology, #9252, for immunoblotting; anti-p-p38(Thr180/Tyr182), Cell Signaling Technology, #4511, for immunoblotting; and anti-p38, Cell Signaling Technology, #8690, for immunoblotting.

Techniques: Mass Spectrometry, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Purification, Western Blot, Staining, shRNA, Negative Control, Knockdown

Journal: Cell Death and Differentiation

Article Title: EBV infection-induced GPX4 promotes chemoresistance and tumor progression in nasopharyngeal carcinoma

doi: 10.1038/s41418-022-00939-8

Figure Lengend Snippet: A . The TAK1-NFκB/MAPK signaling pathway was examined in EBV-negative and EBV-positive NPC cells by immunoblotting . B Protein expression of TAK1 in EBV-negative and EBV-positive CNE2 cells transduced with siRNAs against endogenous TAK1. CCK-8 assay ( C ) and colony formation assay ( D ) in the indicated cells ( n = 3). E . Dose–response curve for DPP, 5-FU, and TAX treatment in the indicated cells. F The TAK1-NFκB/MAPK signaling pathway was examined in the indicated stable cell lines treated with TAK1 siRNA by immunoblotting. G CCK-8 assay of CNE2 EBV-negative cells with stable overexpression of GPX4 treated with TAK1 siRNA ( n = 4). H, I . Cell cycle analysis of the indicated cells by flow cytometry. J Colony formation of the indicated cells ( n = 3). si Ctrl, negative control. Data are shown as the mean ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001. C , D and G , two-tailed unpaired t test.

Article Snippet: The following antibodies were used in this study: anti-4-hydroxynonenal (4-HNE), Abcam (ab46545), for immunoblotting and IHC; anti-p62, Abcam (ab109012), for immunoblotting; anti-NRF2, Abcam (ab62352), for immunoblotting and Cell Signaling Technology (#12721), for immunofluorescence staining; anti-Keap1, Abcam (ab227828), for immunoblotting; anti-SLC7A11, Abcam (ab175186), for immunoblotting; anti-GPX4, Abcam (ab125066), for immunoblotting and IHC; anti-GAPDH, Cell Signaling Technology, #5174, for immunoblotting; anti-β-actin, Abcam (ab8226), for immunoblotting; anti-TAK1, Abcam (ab109526), for immunoblotting; anti-p-TAK1(T187), Cell Signaling Technology, #4536, for immunoblotting; anti-TAB1, Abcam (ab76412), for immunoblotting and immunofluorescence staining; anti-TAB3, Abcam (ab124723), for immunoblotting and immunofluorescence staining; anti-Flag, Sigma (F1804), for immunoblotting and immunofluorescence staining; anti-IKKα, Cell Signaling Technology, #11930, for immunoblotting; anti-IKKβ, Cell Signaling Technology, #8943, for immunoblotting; anti-p-IKKα/β(Ser176/180), Cell Signaling Technology, #2697, for immunoblotting; anti-p-NFκB (Ser536), Cell Signaling Technology, #3033, for immunoblotting; anti-NFκB, Cell Signaling Technology, #8242, for immunoblotting; anti-p-IκBα (Ser32), Cell Signaling Technology, #2859, for immunoblotting; anti-IκBα (Ser32), Cell Signaling Technology, #4814, for immunoblotting; anti-p-JNK(Thr183/Tyr185), Cell Signaling Technology, #4668, for immunoblotting; anti-JNK, Cell Signaling Technology, #9252, for immunoblotting; anti-p-p38(Thr180/Tyr182), Cell Signaling Technology, #4511, for immunoblotting; and anti-p38, Cell Signaling Technology, #8690, for immunoblotting.

Techniques: Western Blot, Expressing, Transduction, CCK-8 Assay, Colony Assay, Stable Transfection, Over Expression, Cell Cycle Assay, Flow Cytometry, Negative Control, Two Tailed Test