Journal: Oncogenesis

Article Title: LIN28B inhibition sensitizes cells to p53-restoring PPI therapy through unleashed translational suppression

doi: 10.1038/s41389-022-00412-8

Figure Lengend Snippet: A Schematic diagram of RNA immunoprecipitation (RNA-IP) and qPCR detection of TP53 mRNA enrichment in A2780 and TOV-112D cells using isotype control IgG or anti-LIN28B antibody. Representative data of three independent experiments (mean ± s.e.m.). * p < 0.05, ** p < 0.01. B Schematic of RNA-pulldown and representative western blots of LIN28B and nucleolin in input and RNA-pulldown samples. CDS coding sequence. C Representative interaction heatmap between TP53 mRNA (5′UTR) and LIN28B protein by catRAPID prediction. D Secondary structure of TP53 mRNA (5′UTR) by RNAfold prediction. E Schematic diagram of truncation mutations of TP53 5′UTR and representative western blots of LIN28B, nucleolin and β-actin in input and RNA-pulldown samples. Exp. = exposure time. * p < 0.05.

Article Snippet: The primary antibodies are as follows: β-Actin (1:10,000; Sigma); α-Tubulin (1:5000; Sigma); LIN28B (1:1000; Cell Signaling Technology, #11965); TP53 (1:1000; Cell Signaling Technology, #2527); RPL26 (1:1000; Abcam, ab59567); cleaved caspase-3 (1:1000; Cell Signaling Technology, #9664); Nucleolin (1:1000; Cell Signaling Technology, #14574); MDM2 (1:1000; Cell Signaling Technology, #51541); MDM4 (1:1000; Sigma-Aldrich, 04-1556); and LaminB1 (1:1000; Abcam, ab16048).

Techniques: RNA Immunoprecipitation, Control, Western Blot, Sequencing

Journal: Molecular and Cellular Biology

Article Title: ARID1a-DNA Interactions Are Required for Promoter Occupancy by SWI/SNF

doi: 10.1128/MCB.01008-12

Figure Lengend Snippet: The ARID domain of ARID1a is required for BAF-A occupancy at THBS1. (A) Mouse/human VISTA alignment of the THBS1 promoter and 5′ transcribed region. Salmon-colored peaks denote evolutionarily conserved regions, whereas lavender peaks denote exons. The locations of ChIP amplicons within this interval are plotted. (B to H) Formaldehyde-cross-linked chromatin from wild-type and Arid1aV1068G/V1068G MEFs was immunoprecipitated with ARID1a, ARID1b, ARID2, BRG1, BRM, INI1/SNF5, or Pol II antibodies. DNA was amplified by quantitative PCR to determine if loss of ARID-DNA binding leads to changes in BAF-A occupancy across THBS1. An intergenic, nonconserved downstream region (located at approximately kb +20) and two unlinked promoter control elements (GAPDH and INS-1) were used as negative genomic controls. Data were plotted as the percentage of total input or chromatin bound. (I) Whole-embryo protein lysates from triplicate pooled samples were used to examine THBS1 protein (reduced, monomeric form) expression differences by Western blotting. An overexposed image of the Western blot was also included to further emphasize these expression differences. The constitutive nuclear matrix protein, nucleolin, was used as a loading control. (J) cDNA synthesized from RNA isolated from wild-type or Arid1aV1068G/V1068G MEFs was used in a quantitative PCR to examine THBS1 expression differences following transfection with mock (nontargeting control), BRG1, or BRM siRNA. (K) Normalized luciferase activity of the THBS1 −2.8 kb promoter-luciferase fragment cotransfected with 0.05 to 0.5 μg of wild-type or mutant HA-mARID1a expression plasmids into NIH 3T3 cells. Cells cotransfected with the empty luciferase vector (−luc) or THBS1 −2.8 kb promoter-luciferase fragment and with empty pcDNA expression plasmids served as negative and positive controls, respectively. (L) Normalized luciferase activity of THBS1 −2.8 kb and −0.48 kb promoter-luciferase reporter plasmids cotransfected with 0.25 μg of pcDNA only (−) or wild-type or mutant HA-mARID1a expression plasmids. Empty luciferase reporter plasmid was used as a negative control. (M) Summary model of ChIP and expression data. Error bars in panels B to H and in panel J represent the SEMs. Error bars in panels K and L represent the standard deviations. Significant differences were calculated using a two-tailed Student t test (*, P < 0.05).

Article Snippet: Western blotting was performed using standard procedures and the following antibodies: ARID1a (A301-040A or A301-041A; Bethyl Labs), ARID1b (sc-32762; Santa Cruz), ARID2 (A302-230A; Bethyl Labs), BRG1 (sc-17796; Santa Cruz), BRM (sc-6450; Santa Cruz), INI1/SNF5 (A301-087A; Bethyl Labs), BAF60a (611728; BD Biosciences), cullin-2 (ab1870; Abcam), THBS-1 (MS-421-P0; Thermo Scientific), hemagglutinin (HA) tag (A190-108A; Bethyl Labs), β-actin (ab8226; Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma), and nucleolin (A300-711A; Bethyl Labs).

Techniques: Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Binding Assay, Control, Expressing, Western Blot, Synthesized, Isolation, Transfection, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Negative Control, Two Tailed Test

Journal: Cell reports

Article Title: mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching

doi: 10.1016/j.celrep.2023.112868

Figure Lengend Snippet: (A) MEFs stably expressing HA-eIF3D proteins were treated with/without Torin1 (250 nM) for 24 h, and RNA immunoprecipitation and qRT-PCR were performed. Data are the mean ± SD of three technical replicates. Statistical significance (*p < 0.05) was assessed by t test. (B) eIF3D interacting RBPs identified by mass spectrometry. (C–E) MEFs stably expressing control, HA-hnRNPF (C), HA-hnRNPK (D), and HA-SSB (E) were treated with Torin1 for 24 h, and their interacting proteins were identified and analyzed. (F–K) Stably knocked down MEFs with indicated shRNAs were treated with/without Torin1 for 24 h, and immunoblot blot analysis was performed. (L) RBP knockdown MEFs expressing HA-eIF3D were treated with Torin1 for 24 h, and RNA immunoprecipitation and qRT-PCR were performed. Data are the mean ± SD of three technical replicates. Statistical significance (*p < 0.05) was assessed by t test. (M) Stably knocked-down MEFs with indicated shRNAs were treated with/without Torin1 for 24 h, and qRT-PCR was performed. Data are the mean ± SD of three technical replicates. Statistical significance (*p < 0.05) was assessed by t test. See also .

Article Snippet: Anti-Stat3, anti-hnRNPK, and anti-hnRNPF antibodies were obtained from Proteintech.

Techniques: Stable Transfection, Expressing, RNA Immunoprecipitation, Quantitative RT-PCR, Mass Spectrometry, Control, Western Blot, Knockdown

Journal: Cell reports

Article Title: mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching

doi: 10.1016/j.celrep.2023.112868

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Stat3, anti-hnRNPK, and anti-hnRNPF antibodies were obtained from Proteintech.

Techniques: Recombinant, Software, Mass Spectrometry