Journal: Cancer cell

Article Title: Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation

doi: 10.1016/j.ccell.2019.07.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After lyophilisation, each concatenated fraction was dissolved in 250 μl of 1x immuno-Affinity Purification Buffer (IAP buffer, #9993, Cell Signaling Technologies, CST) and subjected to two consecutive steps of methyl-R-peptides enrichment using the SDMA antibody-conjugated beads (PTMScan [sdme-R] Kit #13563, Cell Signaling Technologies) and the MMA antibody-conjugated beads (PTMScan Mono-Methyl Arginine Motif [mme-RG] Kit #12235, Cell Signaling Technologies), following the manufacturer’s instructions.

Techniques: Recombinant, Luminescence Assay, MTS Assay, Knock-In, Transgenic Assay, Sequencing, Software

Journal: Cell Reports

Article Title: TIAM1-RAC1 promote small-cell lung cancer cell survival through antagonizing Nur77-induced BCL2 conformational change

doi: 10.1016/j.celrep.2021.109979

Figure Lengend Snippet: Nur77 mediates BCL2 BH3 domain exposure and apoptosis upon TIAM1-RAC1 inhibition (A) Representative images showing Nur77 localization in siControl or siTIAM1-treated DMS53 cells. Scale bars, 10 μm. (B) Quantification of the nuclear-to-cytoplasmic ratio for (A). Each data point represents one microscopic field of cells. Error bars indicate ±SEM from n = 2 independent biological experiments. ∗∗∗ p = 0.0006 for siTIAM1 #1, ∗∗ p = 0.0013 for siTIAM1 #3 (unpaired t test, two tailed). (C) Representative images showing Nur77 staining in control or Nur77 KO DMS53 cells. Scale bars, 10 μm. (D) Representative images showing BCL2 BH3 domain exposure in control and Nur77 KO DMS53 cells (same cells as in C) treated with either DMSO or NSC-23766. Scale bars, 10 μm. (E) Quantification of (D). Error bars indicate ±SEM with n > 40 cells for each condition. ∗∗∗ p = 0.0002, ∗∗∗∗ p < 0.0001 (one-way ANOVA, Sidak’s multiple-comparisons test). (F) Fold change in % Annexin V +ve H526 and H2171 cells treated with control or Nur77 siRNAs followed by treatment with either DMSO or NSC-23766 overnight with representative FACS plots and quantification of fold change. Error bars indicate ±SEM from n = 3 independent experiments. For all comparisons, ∗∗∗∗ p < 0.0001 (two-way ANOVA, Sidak’s multiple-comparisons test). See also .

Article Snippet: Rabbit polyclonal anti-Nur77 antibody , Proteintech , Cat# 12235-1-AP; RRID:AB_ 10644125.

Techniques: Inhibition, Two Tailed Test, Staining

Journal: Cell Reports

Article Title: TIAM1-RAC1 promote small-cell lung cancer cell survival through antagonizing Nur77-induced BCL2 conformational change

doi: 10.1016/j.celrep.2021.109979

Figure Lengend Snippet: Molecular characterization of the interaction between Nur77 and TIAM1 (A) Exogenous full-length TIAM1 co-immunoprecipitated with exogenous Myc-Nur77 expressed following doxycycline addition from H446 nuclear extracts. Successful isolation of nuclear and cytosolic fractions is also shown. Blots are representative of 3 independent experiments. (B) Representative immunoblot showing the interaction of NLS-TIAM1 with Myc-Nur77 following transfection in HEK293T cells and treatment with either DMSO or 50 μM NSC-23766 overnight and immunoprecipitation with anti-Myc. (C) Quantification of (B). Error bars indicate ±SEM from n = 4 independent experiments. ∗∗∗ p = 0.0003 (unpaired t test, two tailed). (D) Immunoblot showing the interaction of Myc-tagged full-length or deletion mutants of Nur77 with FLAG-TIAM1-C1199 following their transfection in HEK293T cells and immunoprecipitation with anti-Myc. (E) Quantification of (D). Error bars indicate ±SEM from n = 3 independent experiments. ∗∗ p = 0.0064, ∗∗∗ p = 0.0007 (one-way ANOVA, Sidak’s multiple-comparisons test). (F) Schematic of full-length and C1199 and DH-PH mutants of TIAM1. Alignment of the amino acid sequences of TIAM1 containing the LXXXLLL motif is highlighted orange for human and yellow for other species. Leucine-to-alanine substitutions to create the TIAM1 2A or TIAM1 4A mutants are shown in red. (G) Immunoblot showing the interaction of Myc-Nur77 with either FLAG-TIAM1-C1199 or FLAG-TIAM1-DH-PH following transfection in HEK293T cells and immunoprecipitation with anti-Myc. Blots are representative of 3 independent experiments. (H) Immunoprecipitation of NLS-TIAM1-WT and NLS-TIAM1-4A with Myc-Nur77 from nuclear extracts of HEK293T cells. Successful isolation of nuclear and cytosolic fractions is also shown. (I) Quantification of (H) from n = 2 independent experiments. See also .

Article Snippet: Rabbit polyclonal anti-Nur77 antibody , Proteintech , Cat# 12235-1-AP; RRID:AB_ 10644125.

Techniques: Immunoprecipitation, Isolation, Western Blot, Transfection, Two Tailed Test

Journal: Cell Reports

Article Title: TIAM1-RAC1 promote small-cell lung cancer cell survival through antagonizing Nur77-induced BCL2 conformational change

doi: 10.1016/j.celrep.2021.109979

Figure Lengend Snippet: TIAM1-Nur77 interaction is required to prevent apoptosis in SCLC (A) Fold change in % Annexin V +ve control (NTC1) or TIAM1 KO H526 cells (left panel) or H2171 cells (right panel) with or without doxycycline-induced expression of TIAM1 WT-GFP or TIAM1 4A-GFP. Error bars indicate ±SEM from n = 3 independent experiments. For the H526 experiment: ∗ p = 0.0146 for NTC1 versus TIAM1 KO cells expressing TIAM1 WT-GFP. For all other comparisons: ∗∗∗∗ p < 0.0001 or ns. For the H2171 experiment: ∗ p = 0.0244 for NTC1 versus TIAM1 KO in control (−dox) cells, ∗∗ p = 0.0016 for TIAM1 KO control (−dox) versus TIAM1 KO expressing TIAM1 WT-GFP (all significance tests were two-way ANOVA, Sidak’s multiple-comparisons test). (B) Representative immunoblots of TIAM1 expression in control (NTC1) or TIAM1 KO H526 or H2171 cells with or without expression of TIAM1 WT-GFP or TIAM1 4A-GFP following doxycycline addition (+dox). (C) Model: TIAM1 expression is upregulated in NE SCLC. TIAM1 sequesters Nur77 in the nucleus. Depletion of TIAM1 or inhibition of RAC1 activation by TIAM1 leads to cytoplasmic redistribution of Nur77. In the cytoplasm, Nur77 induces exposure of the BH3 domain of BCL2 promoting apoptosis.

Article Snippet: Rabbit polyclonal anti-Nur77 antibody , Proteintech , Cat# 12235-1-AP; RRID:AB_ 10644125.

Techniques: Expressing, Western Blot, Inhibition, Activation Assay

Journal: Cell Reports

Article Title: TIAM1-RAC1 promote small-cell lung cancer cell survival through antagonizing Nur77-induced BCL2 conformational change

doi: 10.1016/j.celrep.2021.109979

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Nur77 antibody , Proteintech , Cat# 12235-1-AP; RRID:AB_ 10644125.

Techniques: Recombinant, In Vivo, Transfection, Produced, Binding Assay, Purification, Staining, Protease Inhibitor, Cell Viability Assay, Mutagenesis, Generated, Plasmid Preparation, Software