Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . Analysis of BTZ effects on NRF1 by two different antibodies. The c-19 antibody was used for further experiments in this study because it readily revealed all three forms of NRF1 (glycosylated, full length, and processed). B . A phosphorylated peptide that included the predicted ERK1/2 motif PYSP was identified by mass spectrometry at the C terminal of NRF1 (see text). C . In HEK293-NRF1 3×FLAG cells, inhibition of P97/VCP ATPase by the P97 inhibitors DBeQ and NMS-873 results in accumulation of the glycosylated but not further processed forms of NRF1. By contrast, ERK1/2 inhibition with SCH772984 does not disrupt NRF1 processing. D . Western blot detection of FLAG-tagged NRF1 by NRF1 antibody in HEK293-NRF1 3×FLAG cells. E . Colloidal blue staining of the co-immunoprecipitation sample that was analyzed by mass spectrometry. F . Quantification of NRF1 nuclear localization in the experiments shown in and , *p<0.05.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Mass Spectrometry, Inhibition, Western Blot, Staining, Immunoprecipitation

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . Interaction between NRF1 and ERK1/2. Co-immunoprecipitation from HEK293 cells revealed a physical interaction between ERK1/2 and NRF1 3XFLAG that is enhanced by BTZ and inhibited by SCH772984. B . Dependence of NRF1 serine phosphorylation on ERK1/2. Immunoprecipitation from HEK293 cells showing that phosphorylation of NRF1 3XFLAG was blocked by SCH772984. C . BTZ-induced nuclear localization of NRF1 is dependent upon ERK1/2. A western blot shows that treatment decreases representation of NRF1 in the nuclear fraction. D . Immunofluorescence staining showing that SCH772984 inhibits BTZ-induced accumulation of NRF1 3XFLAG protein in HEK293 cell nuclei, which are labeled with DAPI. Quantification is shown in Figure S4F. Scale bar, 10 µm. E . The phosphosite S734A is required for NRF1 nuclear localization. Immunofluorescence staining and analysis was performed as in D. Scale bar, 10 µm. F . Diagram indicating phosphosite S734 within NRF1.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Immunoprecipitation, Phospho-proteomics, Western Blot, Immunofluorescence, Staining, Labeling

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . SCH772984 treatment increases BTZ-induced cell death in the Melanoma cell line A2058, detected by CAS3 cleavage. B . Flow cytometry detection of A2058 cell apoptosis (monitored by propidium iodide and Annexin V staining) after treatment with BTZ or/and SCH772984. Quantification of the Annexin V (+) PI (-) fractions are shown in . C . SCH772984 treatment increases BTZ-induced apoptosis in MM.1s cells, detected by western blotting for CAS3 cleavage. D . Flow cytometry analysis of apoptosis (assayed by propidium iodide and Annexin V staining) in MM.1s cells that were treated with BTZ or/and SCH772984. Quantification of the Annexin V (+) PI (-) fraction is shown in . E . ERK1/2 phosphorylation, an indicator of activity, is diminished by treatment with the ERK1/2 inhibitors SCH772984 and SCH772984-HCL. F . MM.1s cell growth and viability is impaired by combined BTZ and SCH772984 treatment, indicated by the Cell Counting Kit-8 colorimetric assay of cell proliferation, ****p<0.0001.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Flow Cytometry, Staining, Western Blot, Phospho-proteomics, Activity Assay, Cell Counting, Colorimetric Assay

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . Proteasome activity was reduced in SH-SY5Y Nrf1 shRNA cells compared to SH-SY5Y shRNA control cells, ****p<0.0001. B . SCH772984 treatment does not significantly reduce the background level of proteasome activity in HepG2 cells, ns, p>0.05. C . Screening of a commercial MAPK inhibitor library for small molecules that could increase the level of BTZ-induced apoptosis in HepG2 cells, as shown in D. Red and yellow indicate strong (robust CAS3 increase over control) and moderate (detectable CAS3 increase over control) hits, respectively. D . Induction of apoptosis in HepG2 cells that were treated with BTZ along with the indicated MAPK inhibitors from the library in C, with cell death detected by western blotting for cleavage of CAS3. E . Repeat analysis of “strong” hits from the screen shown in C and D.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Activity Assay, shRNA, Control, Western Blot

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . NRF1 knockdown sensitizes tumor cells to BTZ-induced cell death. Western blotting shows that NRF1 knockdown increases BTZ-induced cell death in HepG2 cells, indicated by CAS3 cleavage. B . NRF1 but not NRF2 knockdown sensitizes HepG2 cells to BTZ-induced apoptosis. C . Inhibition of ERK1/2 by SCH772984 sensitizes HepG2 cells to BTZ-induced apoptosis. D . Inhibition of ERK1/2 by SCH772984 sensitizes MM.1s multiple myeloma cells to BTZ-induced apoptosis. Propidium iodide and Annexin V staining were monitored by flow cytometry. *p<0.05. E . SCH772984 sensitizes A2058 melanoma cells to BTZ-induced apoptosis, detected by flow cytometry, *p<0.05, **p<0.01. F . Result of a small inhibitor screen to identify drugs that increase HepG2 cell death synergistically with BTZ, detected by CAS3 western blot. 16 of 18 MEK inhibitors and 4 of 6 ERK inhibitors showed a positive synergistic effect. G, H . BTZ-induced expression of psma7 ( G ) and pcmd1 ( H ) proteasome subunit genes is decreased by SCH772894 treatment, assayed by RT-PCR in HepG2 cells, *p<0.05.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Knockdown, Western Blot, Inhibition, Staining, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . SCH772984 enhances BTZ-induced inhibition of A2058 tumor cell growth. Mice were treated with Control, SCH772984 (20 mg/kg, i.p. daily), BTZ (0.5 mg/kg, i.p. every other day), or a combination of SCH772984 and BTZ for 10 days, n=14 for each cohort. Tumor size was measured at day 0, 4, 6, 8 and 10, *p<0.05. B . Quantification of the tumor growth inhibitory (TGI) effect of BTZ alone (Cont) or BTZ combined with SCH772984, *p<0.05. C, D . Inhibition of tumor cell proliferation by combined BTZ and SCH772894 treatment. Representative images (C) and quantification (D) of Ki67 staining are shown for tumors collected from mice described in A (n=20 per cohort), Scale bar, 100 µm, ns, p>0.05, **p<0.01. E . Combined BTZ and SCH772894 treatment reduced tumor cell weight. A2058 tumors were weighed after a 10 day treatment with solvent control, SCH772984 (20 mg/kg, i.p. daily), BTZ (0.5 mg/kg, i.p. every other day), or a combination of SCH772984 and BTZ, ns, p>0.05, *p<0.05. F . Inhibition of the proteasome recovery response in vivo by ERK1/2 inhibition. An RT-PCR analysis shows the expression of 6 proteasome subunit genes in tumors collected in (A), *p<0.05, **p<0.01. G . Schematic depiction of the ERK1/2-NRF1 pathway and the proteasome recovery response. Inhibition of the ERK1/2-NRF1 pathway represents a promising strategy for pharmacologically enhancing the efficacy of proteasome inhibition in cancer therapy.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Inhibition, Control, Staining, Solvent, In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . Inhibition of A2058 tumor growth by SCH772984 and BTZ treatment in vivo . A2058 cells were injected into nude mice, and treated with solvent control, SCH772984 210 mg/kg, i.p. daily), BTZ (0.5 mg/kg, i.p. every other day), or a combination of SCH772984 and BTZ for 10 days. All tumors of each cohort were collected and shown. B . Western blot showing the relative levels of MEK, pMEK, ERK and pERK in tumor samples from (A). SCH772984 inhibits ERK but not MEK activity.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Inhibition, In Vivo, Injection, Solvent, Control, Western Blot, Activity Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: RPS15 interacted with IGF2BP1 to promote esophageal squamous cell carcinoma development via recognizing m 6 A modification

doi: 10.1038/s41392-023-01428-1

Figure Lengend Snippet: RPS15 overexpression promotes the translation of core E2F and p38 MAPK pathway proteins. a Schematic illustrating the ribosome profiling of KYSE30 cells (Con) and RPS15-overexpressing KYSE30 cells (RPS15). b GO analysis (upper) and KEGG-pathway analysis (lower) of transcripts preferentially bound by ribosomes in the RPS15 group. The most enriched ribosomal/translational GO gene sets and associated FDR values are shown, which were mainly enriched in translation and MAPK signaling pathways. c Heat map of the log2 fold-change of the RPS15 group relative to that of the control group for ribosome protein genes for mRNA expression, ribosome-protected fragments (RPF), and translation efficiency (TE). d Heat map of the log2 fold-change of the RPS15 group relative to that of the control for each gene of the E2F pathway and p38 MAPK pathway target-gene set for mRNA expression, RPF, and TE. Genes were categorized according to their GO biological process functions in the relative order of the TE fold-change. e Scatter plot of the translational efficiency of individual RP genes (right panel) and the hallmark p38 MAPK-target gene set transcripts (left panel). The x-axis represents the log2 fold-change in RNA-seq and the y-axis represents the log2 fold-change in ribosome profiling. f Expression of the hallmark p38 MAPK-target gene set in RPS15-overexpressing KYSE30 cells (left) and KYSE450 cells (right) determined by Western blot. g Western blot analysis results of expression of the hallmark p38 MAPK-target gene set in KYSE150 cells (left) and KYSE510 cells (right) with or without RPS15 knockout. h , i Statistical analyses of Boyden Chamber migration and invasion assays for KYSE30 cells ( h ) and KYSE450 cells ( i ) with stably transfected control vector or RPS15-overexpressing vector with or without SB203580 treated for 24 h. The statistical analysis results are shown. j Growth curves measured using Incucyte live-cell analyses of KYSE30 cells stably transfected with control vector (blue) or RPS15-overexpressing vector (red) and treated with SB203580 or SB203580 combined DDP for 72 h. k Growth curves measured using Incucyte live-cell analyses of KYSE450 cells stably transfected with control vector (blue) or RPS15-overexpressing vector (red) and treated with SB203580 or SB203580 combined DDP for 72 h. Data were analyzed using unpaired t -tests. * P < 0.05, ** P < 0.01, and *** P < 0.001. l Western blot analysis to detect expression of the hallmark MAPK-target gene set in KYSE30 cells (upper) and KYSE450 cells (lower) stably transfected with control vector or RPS15-overexpressing vector and treated with SB203580 or DDP for 2 days. Con control, Veh vehicle

Article Snippet: Approximately 24 h later, 10 μM folic acid (T0062, TargetMol), 8 μM p38 MAPK inhibitor SB203580 (T1746, TargetMol), and/or cisplatin were added at the indicated concentrations.

Techniques: Over Expression, Control, Expressing, RNA Sequencing Assay, Western Blot, Knock-Out, Migration, Stable Transfection, Transfection, Plasmid Preparation

Journal: Signal Transduction and Targeted Therapy

Article Title: RPS15 interacted with IGF2BP1 to promote esophageal squamous cell carcinoma development via recognizing m 6 A modification

doi: 10.1038/s41392-023-01428-1

Figure Lengend Snippet: IGF2BP1 binds the 3′-UTR of MKK6 and MAPK14 in an m6A-dependent manner. a Distribution of m 6 A peaks of IGF2BP1 RIP-seq data across MAPK14 and MKK6 mRNA transcripts. b Enrichment of m6A modification in 3′-UTR region of MKK6 with Flag-tagged IGF2BP1 in KYSE30 cells (left); RIP-qPCR showing the binding of IGF2BP1 to the 3′-UTR region of MKK6 (right). c Enrichment of m6A modification in 3′-UTR region of MAPK14 with Flag-tagged IGF2BP1 in KYSE30 cells (left); RIP-qPCR showing the binding of IGF2BP1 to the 3′-UTR region of MAPK14 (right). d Agarose gel electrophoresis showing the binding of IGF2BP1 to the 3′-UTR region of MKK6 and MAPK14 in KYSE30 cells (left) and KYSE450 cells (right). e Western blot detected protein expression of the hallmark p38 MAPK-target gene set in RPS15-overexpressing KYSE30 cells (left) and KYSE450 cells (right) with or without IGF2BP1 knockdown. f Growth curves measured using Incucyte live-cell analyses of KYSE30 cells (upper) and KYSE450 cells (lower) stably transfected with control vector (blue) or RPS15-overexpressing vector (purple) and treated with IGF2BP1 knockdown or without IGF2BP1 knockdown for 72 h. Data were analyzed using unpaired t-tests. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Approximately 24 h later, 10 μM folic acid (T0062, TargetMol), 8 μM p38 MAPK inhibitor SB203580 (T1746, TargetMol), and/or cisplatin were added at the indicated concentrations.

Techniques: Modification, Binding Assay, Agarose Gel Electrophoresis, Western Blot, Expressing, Knockdown, Stable Transfection, Transfection, Control, Plasmid Preparation

Journal: Signal Transduction and Targeted Therapy

Article Title: RPS15 interacted with IGF2BP1 to promote esophageal squamous cell carcinoma development via recognizing m 6 A modification

doi: 10.1038/s41392-023-01428-1

Figure Lengend Snippet: Schematic depicting the contribution of RPS15 and IGF2BP1 to ESCC progression via the p38 MAPK pathway. a RPS15 overexpression promotes the proliferation and motility of ESCC cells. Mechanistic investigation revealed that RPS15 interacts with the KH domain of IGF2BP1, which directly binds and recognizes the MAPK14 and MKK6 mRNA 3′-UTR, and promotes translation of core p38 MAPK pathway proteins. b , RPS15 inhibition with folic acid treatment, IGF2BP1 ablation, or treatment with SB203580 suppresses ESCC proliferation and metastasis via the p38 MAPK signaling pathway

Article Snippet: Approximately 24 h later, 10 μM folic acid (T0062, TargetMol), 8 μM p38 MAPK inhibitor SB203580 (T1746, TargetMol), and/or cisplatin were added at the indicated concentrations.

Techniques: Over Expression, Inhibition

Journal: Bioengineered

Article Title: Coordination of PRKCA/PRKCA-AS1 interplay facilitates DNA methyltransferase 1 recruitment on DNA methylation to affect protein kinase C alpha transcription in mitral valve of rheumatic heart disease

doi: 10.1080/21655979.2021.1971482

Figure Lengend Snippet: The role of p38/MAPK in regulating PRKCA-AS1 expression in RHD. (a) the RNA levels of PRKCA-AS1 and PRKCA in TNF-α-induced AC16 cells treated with inhibition of different signaling pathways. (b) Smad2 occupancy on PRKCA-AS1 in TNF-α-induced AC16 cells treated with inhibition of different signaling pathways. (c) Smad2 occupancy on PRKCA-AS1 in mitral valve of RHD. the given data from triplicate experiments was processed as mean ± standard error and compared by student’s t-test. ‘FC’: fold change; ‘BK’: blocking

Article Snippet: The conditions of 100 ng/mL tumor necrosis factor-α (TNF-α) (APExBIO, Houston, TX, USA) for 16 h [ ], 20 ng/mL 5-Azacytidine (APExBIO) for 24 h [ ], 1 μM p38/MAPK inhibitor (2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-(pyridin-4-yl)-1H-pyrazol-3(2 H)-one) (APExBIO) for 4 h, 50 mM transforming growth factor β1 (TGFB1) inhibitor (4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4 H-yrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide) (APExBIO) for 4 h, 0.1 μM Rho associated coiled-coil containing protein kinase 1/2 (ROCK1/2) (4-[(1 R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide) (APExBIO) for 6 h, 10 μM peroxisome proliferator activated receptor gamma/delta (PPARγ/δ) (2,5-dichloro-N-(2-methyl-4-nitrophenyl) benzenesulfonamide) (APExBIO) for 4 h, and 100 μM signal transducer and activator of transcription 3 (STAT3) (5,15-diphenyl-21 H,23 H-porphine) (APExBIO) for 6 h were adopted.

Techniques: Expressing, Inhibition, Blocking Assay

Journal: Annals of the rheumatic diseases

Article Title: MicroRNA-199a* regulates the expression of cyclooxygenase-2 in human chondrocytes

doi: 10.1136/annrheumdis-2011-200519

Figure Lengend Snippet: miR-199a* directly inhibits the expression of cyclooxygenase-2 (COX-2) in osteoarthritis chondrocytes. (A) Luciferase activity in chondrocytes transfected with the reporter vector and premiR-199a* (*p<0.05). (B, C) Expression of COX-2 protein in premiR-199a* or antimiR-199a* transfected chondrocytes after 72 h of stimulation with interleukin-1β (IL-1β). (D, E) Expression of COX-2 protein in negative control premiRNA or negative control-antimiRNA-transfected chondrocytes after 72 h of stimulation with IL-1β. (F, G) Expression levels of miR-199a* and COX-2 in chondrocytes treated with the p38-MAPK inhibitor (SB202190) and stimulated with IL-1β were determined by TaqMan assays. Gene expression in unstimulated chondrocytes was used as a control and expression of RNU6B/glyceraldehyde 3-phosphate dehydrogenase was used as an endogenous control. COX-2 protein expression was determined by western immunoblotting. Each experiment was performed in duplicate with samples from the indicated number of patients. *p<0.05 vs control; #p<0.05 vs IL-1β-stimulated chondrocytes.

Article Snippet: OA chondrocytes were serum-starved overnight and then stimulated with IL-1β (5 ng/ml; R&D Systems, St Paul, Minnesota, USA) or p38-MAPK inhibitor (SB202190 100 μM; A G Scientific Inc, San Diego, California, USA) for the indicated time and total RNA was prepared using the TRIZOL reagent (Invitrogen, Carlsbad, California, USA).

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Negative Control, Gene Expression, Control, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Excessive Neutrophil Extracellular Trap Formation Aggravates Acute Myocardial Infarction Injury in Apolipoprotein E Deficiency Mice via the ROS-Dependent Pathway

doi: 10.1155/2019/1209307

Figure Lengend Snippet: APOE3 affects NET formation through the MAPK-MSK1 pathway. (a) Quantitation of ex vivo NET formation of WT- and ApoE −/− -derived neutrophils stimulated with PMA and different concentrations of the P38 MAPK inhibitor (SB239063 or losmapimod) or the ERK inhibitor (FR180204) for 4 hours. (b, c) Western blots and quantitation of the ERK1/2 (ERK), phosphorylated ERK1/2 (p-ERK), JNK, phosphorylated JNK (p-JNK), P38 MAPK, and phosphorylated P38 MAPK (p-P38 MAPK) expressions in WT- and ApoE −/− -derived neutrophils stimulated with PBS, PMA, or PMA plus APOE3 for 1 hour. (d, e) Western blots and quantitation of the phosphorylated MSK1 (p-MSK1) and the phosphorylated ATF2 (p-ATF2) expression in WT- and ApoE −/− -derived neutrophils stimulated with PBS, PMA, or PMA plus APOE3 for 1 hour. Columns filled with black: WT mouse-derived neutrophils. Columns filled with white: ApoE −/− mouse-derived neutrophils. Data are shown as mean ± SEM. Statistical tests include the one-way analysis of variance followed by Dunnett's multiple comparison test (a) and the Kruskal-Wallis test followed by Dunn's multiple comparison test (c, e). ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: A NADPH oxidase inhibitor (apocynin, Selleck Chemicals), recombinant human APOE3 (PeproTech), P38 MAPK inhibitor (SB239063 or losmapimod, Beyotime Institute of Biotechnology), or ERK1/2 inhibitor (FR 180204, Beyotime Institute of Biotechnology) was pretreated 30 min before phorbol-12-myristate-13-acetate (PMA, Beyotime Institute of Biotechnology), Triton X-100 (Invitrogen), or H 2 O 2 was added into wells according to protocol or demand.

Techniques: Quantitation Assay, Ex Vivo, Derivative Assay, Western Blot, Expressing, Comparison

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Excessive Neutrophil Extracellular Trap Formation Aggravates Acute Myocardial Infarction Injury in Apolipoprotein E Deficiency Mice via the ROS-Dependent Pathway

doi: 10.1155/2019/1209307

Figure Lengend Snippet: Schematic model of the role of ApoE/APOE3 in regulating neutrophil function, especially NET formation, in the context of acute myocardial infarction. After acute myocardial infarction, neutrophils are mainly mobilized from the bone marrow, infiltrate into the infarcted heart, and form NETs via the NADPH oxidase-ROS-MAPK-MSK1 signal pathway. In this process, apolipoprotein E regulates neutrophil infiltration and NET formation. As for NET formation, apolipoprotein E fails to reduce ROS generation but can inhibit MSK1 phosphorylation. Additionally, P38 MAPK is sensitive to apolipoprotein E deficiency. The APOE3 supplement inhibits P38 MAPK phosphorylation under the conditions of apolipoprotein E deficiency. Moreover, ApoE deficiency promotes PMA-induced ERK1/2 phosphorylation, but APOE3 fails to inhibit ERK1/2 phosphorylation after PMA treatment.

Article Snippet: A NADPH oxidase inhibitor (apocynin, Selleck Chemicals), recombinant human APOE3 (PeproTech), P38 MAPK inhibitor (SB239063 or losmapimod, Beyotime Institute of Biotechnology), or ERK1/2 inhibitor (FR 180204, Beyotime Institute of Biotechnology) was pretreated 30 min before phorbol-12-myristate-13-acetate (PMA, Beyotime Institute of Biotechnology), Triton X-100 (Invitrogen), or H 2 O 2 was added into wells according to protocol or demand.

Techniques: Phospho-proteomics