Journal: Nature Communications

Article Title: ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

doi: 10.1038/s41467-023-44089-y

Figure Lengend Snippet: a A schematic representation of in vitro biochemical analyses used in this study. Gray and white circles without labels, proteins; a black curved line, a nascent RNA molecule. b Left, silver-stained recombinant ERK1 (K1) and ERK2 (K2) proteins used in in vitro analyses. SM, standard protein size marker. Right, the result of immunoblotting confirming ERK1 and ERK2. c Immobilized template assay results showing the increased occupancies of Pol II, CDK9, and MED23 by ERK2 on the EGR1 TSS. CTRL, ERK-free, buffer only control throughout the figures. Data are presented as mean values and standard deviation (SD) ( n = 3 independent experiments ) . P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. Not significant (ns). d SDS-PAGE and silver-staining of the constitutively active ERK1m and ERK2m proteins used in this study. e Immobilized template assay showing ERK1 and ERK2 proteins associated with the template. K1, ERK1; K1 m , ERK1m; K2, ERK2; K2 m , ERK2m. INPUT, 20% NE used for the assay. f Immobilized template assay results of CDK9, and MED23 ( n = 3 independent experiments). Data are presented as mean values and SD. P -values for the bar graphs were calculated with the unpaired, one sided Student’s t test. g in vitro transcription assay results showing EGR1 gene activation by ERK2 and ERK2m ( n = 3 independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. Source data are provided as a Source Data file.

Article Snippet: The medium was replaced with Opti-MEM (Gibco, 31985) before transfecting with scrambled siRNA (Santa Cruz Biotechnology, sc-37007) and ERK1 siRNA (Cell Signaling Technology, #6436), ERK2 siRNA (Santa Cruz Biotechnology, sc-35335), or both ERK siRNA species.

Techniques: In Vitro, Staining, Recombinant, Marker, Western Blot, Control, Standard Deviation, SDS Page, Silver Staining, Transcription Assay, Activation Assay

Journal: Nature Communications

Article Title: ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

doi: 10.1038/s41467-023-44089-y

Figure Lengend Snippet: a Immobilized template assay results presenting TOP2B enrichment by ERK1m and ERK2 on the WT template but not on the ELK1 mut template ( n = 4 independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. b Deubiquitinated TOP2B used for the in vitro kinase assays, silver-stained. c Silver-stained reactions following the in vitro kinase assay. d Autoradiograms of the in vitro kinase assay with TOP2B and ERKs. ELK1, a positive control. e In vitro kinase assay followed by SDS-PAGE for the preparation of mass spectrometry analyses. Red boxes showing sliced gel pieces used for the analyses. CTRL, ERK storage buffer only; SM, size marker (KDa). f A flow chart showing the steps of mass spectrometry analyses performed in this study. g A schematic representation of the TOP2B residues phosphorylated by ERK2/ERK2m (red), or both ERK1/ERK1m and ERK2/ERK2m kinases (blue). Source data are provided as a Source Data file.

Article Snippet: The medium was replaced with Opti-MEM (Gibco, 31985) before transfecting with scrambled siRNA (Santa Cruz Biotechnology, sc-37007) and ERK1 siRNA (Cell Signaling Technology, #6436), ERK2 siRNA (Santa Cruz Biotechnology, sc-35335), or both ERK siRNA species.

Techniques: In Vitro, Staining, Kinase Assay, Positive Control, SDS Page, Mass Spectrometry, Marker

Journal: Nature Communications

Article Title: ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

doi: 10.1038/s41467-023-44089-y

Figure Lengend Snippet: a qRT-PCR results presenting the effects of ERK2 catalytic inhibition on EGR1 and HSP70 transcription. HEK293 cells were not synchronized ( n = 3). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. b qRT-PCR results presenting the effects of ERK2 catalytic inhibition on the EGR 1 and FOS transcription ( n = 3). HEK293 cells were synchronized at G 0 (S0) before they were serum-induced to early G 1 (S15). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. c ChIP-qPCR results showing S2 Pol II occupancy changes in the EGR1 gene with or without functional ERK2 ( n = 3 independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. d ChIP-qPCR results showing a dramatic increase of TOP2B at EGR1 upon the catalytic inhibition of ERK2 ( n = 3 independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. e Immunoblots of ERK1 (K1 KD ), ERK2 (K2 KD ), and ERK1/ERK2 double KD (Double KD ) HEK293 cells. SCR, scrambled siRNA; Tubulin, a reference. f qRT-PCR and ChIP-qPCR results presenting the effects of ERK KDs on EGR1 transcription (left) and TOP2B occupancies at the EGR1 TSS (right), respectively ( n = 3 independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. g qRT-PCR and ChIP-qPCR results presenting the effects of ERK KDs on FOS transcription (left) and TOP2B occupancies at the FOS TSS (right), respectively ( n = independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. Source data are provided as a Source Data file.

Article Snippet: The medium was replaced with Opti-MEM (Gibco, 31985) before transfecting with scrambled siRNA (Santa Cruz Biotechnology, sc-37007) and ERK1 siRNA (Cell Signaling Technology, #6436), ERK2 siRNA (Santa Cruz Biotechnology, sc-35335), or both ERK siRNA species.

Techniques: Quantitative RT-PCR, Inhibition, ChIP-qPCR, Functional Assay, Western Blot

Journal: Nature Communications

Article Title: ERK2-topoisomerase II regulatory axis is important for gene activation in immediate early genes

doi: 10.1038/s41467-023-44089-y

Figure Lengend Snippet: During Pol II pausing at EGR1 , ELK1 and ERK2 are inactive. TOP2B is ubiquitinated by the BRCA1/BARD1 complex (purple/pink circles) . Upon transcriptional activation, ERK2 is activated to phosphorylate ELK1 and TOP2B (phosphorylation marked with a black star and a letter “P”). TOP2B generates DNA bending, relaxes DNA supercoiling, mediates DNA strand break (DSB), triggering DNA damage response signaling that phosphorylates and activates multiple enzymes (tan circles) including PI3K, TRIM28, and BRCA1 , , . If ERK2 is catalytically null or absent, gene activation is hindered and TOP2B is abnormally increased in the EGR1 TSS. Nascent RNA, an orange curved line.

Article Snippet: The medium was replaced with Opti-MEM (Gibco, 31985) before transfecting with scrambled siRNA (Santa Cruz Biotechnology, sc-37007) and ERK1 siRNA (Cell Signaling Technology, #6436), ERK2 siRNA (Santa Cruz Biotechnology, sc-35335), or both ERK siRNA species.

Techniques: Activation Assay, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: ERK1/2-Nanog signaling pathway enhances CD44(+) cancer stem-like cell phenotypes and epithelial-to-mesenchymal transition in head and neck squamous cell carcinomas

doi: 10.1038/s41419-020-2448-6

Figure Lengend Snippet: a , b Graphs of the positive area (%) per field for the CSC marker proteins CD24, CD44, CD133, and Musashi-1 and the self-renewal proteins Sox2, Oct-4, Nanog, and c-Myc. c Immunofluorescence (IF) staining of commercially available tissue array slides containing 45 HNSCC and 5 corresponding normal tissues with DAPI (blue), CD44 (green), and Nanog (red). Scale bar = 20 µm. d , e Western blotting of CSC marker proteins and self-renewal proteins in three HNSCC cell lines grown as monolayers or as spheroids. f Western blotting of CD44, Sox2, Oct-4, Nanog, and c-Myc following FACS for CD44(−) and CD44(+) cells. g IF of CD44(+) SCC-15 and QLL-1 cells transduced with sh.NANOG or sh.Scr. Scale bar = 50 µm. h GFP-expressing cell in a single cell assay using CD44(+) SCC-15 and QLL-1 cells following transduction with GFP, or NANOG shRNA (sh.Nanog), or scrambled shRNA (sh.Scr). Error bars represent standard deviation. * p < 0.05.

Article Snippet: ERK1/2 and β-catenin were silenced via lentiviral transduction of human ERK1 shRNA, ERK2 shRNA and β-catenin shRNA (SC-44252-V, SC-29307-V, and SC-35335-V; Santa Cruz Biotechnology).

Techniques: Marker, Immunofluorescence, Staining, Western Blot, Transduction, Expressing, shRNA, Standard Deviation

Journal: Cell Death & Disease

Article Title: ERK1/2-Nanog signaling pathway enhances CD44(+) cancer stem-like cell phenotypes and epithelial-to-mesenchymal transition in head and neck squamous cell carcinomas

doi: 10.1038/s41419-020-2448-6

Figure Lengend Snippet: a Phospho-RTK protein array analysis using CD44(+) and CD44(−) cells. b Western blotting of CD44, Nanog, phosphorylated ERK1/2, total ERK2, phosphorylated JNK1/2, total JNK2, phosphorylated p38, total p38, and β-actin in CD44(+) and CD44(−) cells. c Western blotting of total ERK1/2, total ERK1, total ERK2, CD44, and Nanog in CD44(+) cells transduced with sh.ERK1/2 or sh.Scr. d IF of CD44(+) cells transduced with sh.ERK1/2 or sh.Scr for CD44 and Nanog. e Single cell assay and f colony formation assay of CD44(+) SCC-15 and QLL-1 cells following transduction with sh.ERK1/2 or sh.Scr. g Migration and Invasion assay of CD44(+) SCC-15, SCC-25, and QLL-1 cells following transduction with sh.ERK1/2 or sh.Scr. h Western blotting of ERK1/2, N-cadherin, Snail, Slug, Zeb1, and β-actin and immunofluorescent images of N-cadherin and Snail staining in CD44(+) cells transduced with sh.ERK1/2 or sh.Scr. Scale bar = 20 µm. Error bars represent standard deviation. * p < 0.05.

Article Snippet: ERK1/2 and β-catenin were silenced via lentiviral transduction of human ERK1 shRNA, ERK2 shRNA and β-catenin shRNA (SC-44252-V, SC-29307-V, and SC-35335-V; Santa Cruz Biotechnology).

Techniques: Protein Array, Western Blot, Transduction, Colony Assay, Migration, Invasion Assay, Staining, Standard Deviation

Journal: Cell Death & Disease

Article Title: ERK1/2-Nanog signaling pathway enhances CD44(+) cancer stem-like cell phenotypes and epithelial-to-mesenchymal transition in head and neck squamous cell carcinomas

doi: 10.1038/s41419-020-2448-6

Figure Lengend Snippet: a Western blotting of ERK1/2, ERK1, and ERK2 in CD44(+) cells transduced with sh.ERK1/2 or sh.Scr. b Tumor growth curves of CD44(+) SCC-15 cell xenografts after treatment with sh.ERK1/2, sh.Scr, or 8 Gy. c Representative images of tumors from each treatment group on the 20th day. d IF analysis of treated tumors for CD44 (green), Nanog (red), and Snail (white) in SCC-15 xenografts treated with sh.ERK1/2, sh.Scr, or 8 Gy. Scale bar = 50 µm. Error bars represent standard deviation. * p < 0.05.

Article Snippet: ERK1/2 and β-catenin were silenced via lentiviral transduction of human ERK1 shRNA, ERK2 shRNA and β-catenin shRNA (SC-44252-V, SC-29307-V, and SC-35335-V; Santa Cruz Biotechnology).

Techniques: Western Blot, Transduction, Standard Deviation