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

Journal: RNA biology

Article Title: hnRNPH1-MTR4 complex-mediated regulation of NEAT1v2 stability is critical for IL8 expression.

doi: 10.1080/15476286.2021.1971439

Figure Lengend Snippet: Figure 1. Depletion of hnRNPH1 stabilizes NEAT1v2 in HeLa TO cells. (a) Venn diagram showing the overlap between proteins that interact with MTR4 and those that co-localize with NEAT1. Four proteins (hnRNPH1, hnRNPF, AKAP8L and RBM7) were determined to be MTR4-interacting factors. (b) qRT-PCR analysis of hnRNPH1, hnRNPF, AKAP8L and RBM7 expression in HeLa TO cells. Results are expressed as the mean ± s.d. (n = 3). *P < 0.05. (c) qRT-PCR analysis of NEAT1v2 expression in HeLa TO cells transfected with siRNA targeting indicated genes. Results are expressed as the mean ± s.d, (n = 3), *P < 0.05. (d) Degradation of NEAT1v2 and GAPDH mRNA was determined by BRIC followed by qRT-PCR analysis. Dot and solid line indicate control siRNA or siRNA targeting hnRNPH1 condition in HeLa TO cells, respectively. GAPDH mRNA was used as a negative control. In each degradation curve, data are shown as an exponential plot. Results are expressed as the mean ± s.d, (n = 2). (e) Schematic representation of the NEAT1 promoter luciferase reporter construct. (f) HeLa TO cells that had been transfected with an siRNA targeting hnRNPH1 or a control siRNA were transfected with the NEAT1 promoter reporter construct and subjected to luciferase assays. Bars represent the luciferase activity divided by the activity in cells transfected with siControl. The GFP expressing vector was cotransfected to normalize transfection efficiency. Results are expressed as the mean ± s.d. (n = 3). (g) Luciferase assays were performed with HeLa TO cells that had been transfected with NEAT1 promoter reporter construct in the presence of poly I or poly I:C. Results are expressed as the mean ± s.d. (n = 3). *P < 0.05.

Article Snippet: Anti-HNRNPF (67,701-1-Ig) and AKAP8L (10,103-1-AP) were purchased from Proteintech.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Negative Control, Luciferase, Construct, Activity Assay, Plasmid Preparation