Journal: bioRxiv

Article Title: FUS controls muscle differentiation and structure through LLPS mediated recruitment of MEF2 and ETV5

doi: 10.1101/2024.09.18.613669

Figure Lengend Snippet: FUS is a transcriptional co-activator of PEA3 transcription factors a: Pie chart showing the distribution of FUS binding sites identified by ChIP-seq analysis in C2C12 cells. FUS binds mostly to promoters, close to the transcription start sites (TSS). b-c: Mean profiles established for FUS binding at TSS using SeqMiner in 3 independent replicates. d: Genome browser visualization of FUS binding on the promoter region of the Mrps18c and Helq genes. e: Gene ontology analysis of genes with FUS binding sites identified by ChIP-seq in C2C12 cells. f-g: MEME motif enriched in ChIP-seq peaks bound by FUS (F) and the predicted motif bound by PEA3 transcription factor ETV4 (G). h-j: Light units relative to empty control plasmid in C2C12 cells 24 h after transfection of an Mrps18c- luciferase plasmid and an expression plasmid for either hFUS WT, hFUS R495X or empty control. Cells were simultaneously transfected with si Ctr (25 nM) or si ETV1 (25 nM, H), si ETV4 (25 nM, I) and si ETV5 (25nM, J). Nested one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001, **P<0.01, *P < 0.05. Each dot represents the mean of an individual experiment (n=3), each consisting of 4 technical replicates. Data represent the mean ± SEM. k: Light units relative to si Ctr in C2C12 cells 24 h after transfection of Mrps18c- luciferase plasmid and si Ctr (25 nM) or si FUS and an expression plasmid for either ETV5 or empty control. Nested one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001. Each dot represents the mean of an individual experiment (n=3), each consisting of 4 technical replicates. Data represent the mean ± SEM.

Article Snippet: ETV1 (# TP310533), ETV4 (# TP760035), ETV5 (# TP761784) and MEF2A (# TP312830) were purchased from Origen.

Techniques: Binding Assay, ChIP-sequencing, Control, Plasmid Preparation, Transfection, Luciferase, Expressing

Journal: bioRxiv

Article Title: FUS controls muscle differentiation and structure through LLPS mediated recruitment of MEF2 and ETV5

doi: 10.1101/2024.09.18.613669

Figure Lengend Snippet: a: Genome browser snapshot of MEF2A, ETV5 and FUS ChIP-seq binding sites at the MRPS18C/HELQ locus. b: Venn diagram showing the overlap between FUS, MEF2A and ETV5 binding sites. Hypergeometric p-values: Fus vs Etv5: 3.66×10 -2 ; Fus vs Mef2a: <5×10 -324 ; Etv5 vs Mef2a: <5×10 -324 . c-e: Light units relative to Empty vector + si Ctr in C2C12 cells 24 h after transfection of a 3xMEF2 luciferase plasmid and an expression plasmid for either hFUS WT, hFUS R495X or empty control. Cells were simultaneously transfected with si Ctr (25 nM) or si ETV1 (25 nM, C), si ETV4 (25 nM, D), and si ETV5 (25nM, E). ETV5, but not ETV1 or ETV4, is required for co-activation of the MEF2 reporter by FUS. Nested one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001, **P<0.01, *P < 0.05. Each dot represents the mean of an individual experiment (n=4), each consisting of 4 technical replicates. Data represent the mean ± SEM.

Article Snippet: ETV1 (# TP310533), ETV4 (# TP760035), ETV5 (# TP761784) and MEF2A (# TP312830) were purchased from Origen.

Techniques: ChIP-sequencing, Binding Assay, Plasmid Preparation, Transfection, Luciferase, Expressing, Control, Activation Assay

Journal: eLife

Article Title: Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer

doi: 10.7554/eLife.66524

Figure Lengend Snippet: ( a ) Upregulated genes by KRAS G12V overexpression for 1 day and 7 days in comparison with a GFP overexpression control in H2107 and H82 cells. The numbers of genes upregulated (>1.5 fold) are indicated. ETV1 , ETV4 , and ETV5 are shown in red. ( b ) Heat map of the PEA3 family ETS transcription factors ( ETV1 , ETV4 , and ETV5 ) and neuroendocrine transcription factors ( ASCL1 , NEUROD1 , INSM1 , and POU3F2 [ BRN2 ]) in small cell lung cancer and lung adenocarcinoma cell lines. Red and blue denote high and low expression, respectively. ( c ) Western blot showing the effects of ERK inhibition using 1 μM SCH772984 on the expression of ETV1, ETV4, and ETV5 with or without KRAS G12V transduction for 72 hr. Lysates from HA-tagged ETV1-overexpressing H524 cells were used as a positive control for ETV1. GAPDH was used as a loading control. ( d ) Effects of HA-tagged ETV1 induction as assessed by western blot in H2107, H82, and H524 cells, upon treatment with 100 ng/mL doxycycline (dox) for 3 and 7 days. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated control values (set to two in each panel) after normalization to GAPDH. ( e ) Effects of HA-tagged ETV5 induction as assessed by western blot in H82 and H524 cells, upon treatment with 100 ng/mL dox for 3 and 7 days. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated control values (set to one in each cell line panel) after normalization to GAPDH. ( f ) Western blot showing the effects of KRAS G12V induction and treatment with siRNA pools targeting ETV4 , ETV5 , or both on expression of neuroendocrine transcription factors in H82 cells. Cells were treated with 100 ng/mL dox and indicated siRNAs for 72 hr. Scrambled siRNA (siScr) was used as a negative control. GAPDH was used as a loading control. Numbers below blots show the amounts of each band relative to the corresponding non-dox-treated and siScr-treated control values (set to 1) after normalization to GAPDH. ( g ) Western blot showing the effects of MSK/RSK and/or CBP/p300 inhibition on KRAS G12V -mediated expression of ETV4 and ETV5 in H82 cells. Cells were treated with 5 μM SB-747651A (a MSK-RSK inhibitor) and/or 400 nM A-485 (a CBP/p300 inhibitor) as well as 100 ng/mL dox for 72 hr. GAPDH was used as a loading control. Numbers below the ETV5 blots indicate the amounts of ETV5 relative to the dox-treated and non-drug-treated (DMSO-treated) control values (set to lane 5) after normalization to GAPDH. Immunoblots are representative of at least two biological replicates.

Article Snippet: Human ETV1 (Addgene, Cambridge, MA, USA; plasmid #82209) and ETV5 (Horizon Discovery, Cambridge, UK; clone 100008315) were transferred to pInducer20 using Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific).

Techniques: Over Expression, Comparison, Control, Expressing, Western Blot, Inhibition, Transduction, Positive Control, Negative Control

Journal: eLife

Article Title: Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer

doi: 10.7554/eLife.66524

Figure Lengend Snippet: ( a ) Western blot of RB in RB1 -proficient parental PC9 and H1975 cells as well as RB1 -knockout polyclonal and clonal cells. GAPDH was used as a loading control. ( b ) Western blot showing effects of 100 nM osimertinib treatment for up to 5 days on protein expression of neuroendocrine transcription factors and SOX2 in RB1 -proficient and -deficient PC9 and H1975 cells. Lysates from the small cell lung cancer cell line, H2107 or H82, were used as a positive control for neuroendocrine markers. Lysates from H2107 were used as a positive control for SOX2. GAPDH was used as a loading control. ( c ) Mean relative proliferation of parental PC9 and H1975 cells with or without RB1 knockout treated with osimertinib. Cells were treated with osimertinib or DMSO for 72 hr. Bars represent mean (three biological replicates) ± SD. The IC 50 values for each clone are as follows: PC9-sgControl, 7.0 nM; PC9-sg RB1 #1, 5.9 nM; PC9-sg RB1 #15, 4.8 nM; H1975-sgControl, 19 nM; H1975-sg RB1 #9, 145 nM; and H1975-sg RB1 #22, 57 nM. IC 50 analysis of dose-response curves were compared by the extra sum-of-squares F test. ( d ) Mean relative proliferation of PC9 and H1975 cells with acquired resistance to osimertinib are plotted. Osimertinib-resistant RB1 -proficient cells as well as RB1 -knockout clonal cells were treated with osimertinib for 72 hr. Control cells were treated with DMSO as a vehicle control. Osimertinib-resistant cells were generated by either a stepwise dose-escalation or initial high-dose method. Bars represent mean (three biological replicates) ± SD. ( e ) Profiling of acquired genetic alterations through osimertinib treatment in PC9 and H1975 cells with or without RB1 knockout assessed by MSK-IMPACT. Abbreviations: stepwise, stepwise dose-escalation method; initial, initial high-dose method. ( f ) Western blot for profiling expression of EGFR, ERK, RAS, and neuroendocrine factors in parental and osimertinib-resistant PC9 and H1975 cells with or without RB1 knockout. Parental and resistant cells were harvested under treatment with 0.1% DMSO or osimertinib (2 μM for H1975 [stepwise dose-escalation method] and 1 μM for the others), respectively. Lysates from the small cell lung cancer cell line, H2107 or H82, were used as a positive control for neuroendocrine markers. GAPDH was used as a loading control. ( g ) Western blot for profiling expression of CIC as well as its downstream targets ETV1, ETV4, and ETV5 in parental and osimertinib-resistant H1975 cells. Parental cells were treated with 100 nM osimertinib or DMSO for 3 days. Osimertinib-resistant cells were cultured with 2 μM (stepwise dose-escalation method) or 1 μM (initial high-dose method) osimertinib. GAPDH was used as a loading control.

Article Snippet: Human ETV1 (Addgene, Cambridge, MA, USA; plasmid #82209) and ETV5 (Horizon Discovery, Cambridge, UK; clone 100008315) were transferred to pInducer20 using Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific).

Techniques: Western Blot, Knock-Out, Control, Expressing, Positive Control, Generated, Cell Culture

Journal: eLife

Article Title: Extracellular signal-regulated kinase mediates chromatin rewiring and lineage transformation in lung cancer

doi: 10.7554/eLife.66524

Figure Lengend Snippet:

Article Snippet: Human ETV1 (Addgene, Cambridge, MA, USA; plasmid #82209) and ETV5 (Horizon Discovery, Cambridge, UK; clone 100008315) were transferred to pInducer20 using Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific).

Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Mutagenesis, Recombinant, Sequencing, Control, Gene Expression, Extraction, Software

Journal: The Oncologist

Article Title: Combined RAF and MEK Inhibition to Treat Activated Non-V600 BRAF-Altered Advanced Cancers

doi: 10.1093/oncolo/oyad247

Figure Lengend Snippet: Correlative studies of pharmacodynamic markers of pathway inhibition in patient tumors. ( A ) Reverse transcription-polymerase chain reaction (RT-PCR) measurements of transcript levels of the ERK output genes, DUSP6, SPRY2, and ETV1, in pretreatment and on-treatment (day 7) biopsies. ( B ) Summary of immunohistochemical analysis of pretreatment and on-treatment samples. ( C ) Representative images of immunohistochemical staining.

Article Snippet: TaqMan primers and control reagents were purchased from Applied Biosystems (GAPDH- HS02758991_g1, SPRY2- HS00183386_m1, ACTB- HS01060665_g1, ETV1- HS00951941_m1, DUSP6- HS04329637_m1).

Techniques: Inhibition, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining