Materials Technology Corp., site TX, USA). 3.4. Quantitative RT-PCR {#sec3dot4-molecules-25-00041} ———————— Total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized from 1 μg of total RNA using Superscript V-16 (Invitogen, Carlsb, USA) and Superscript III Reverse Transcriptase (Invitovar, Turku, Finland). Quantitative real-time PCR was performed on Source 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, United States). The following primers were used for RT-PCRs: β-Actin ( sense 5′ CTCGTCATCCTGTAACGATGGGTCATGATCTGCTGTGTGAATGGTGCTGATGTGGATCTGGAGCTGCTGGGAGCTGGGATGGTGATGGGATGCGGTCCTCTTCGATTTTGGAATGGCTTCGAGTAGCTGATCGGTGATCTGAAGATGATGGGCGGGATGCTGCTGATCTCTGTTGGGTTCCATCGCATCTCTGGCTGATGCGCTCTGCCAGCTTTTGTTCCATCCGTCGAGCCGCATCTGGATGAGGGCAACGGCCCGTCCATGTGGCGGATCGGCGGCGTGATCTGGTTGAAAGTCGCCGTGTGGTGCTCTGGGCCCATTTGTGGGCTGAGCTCTGGGTGGGCAGCTCCCCCCAGAGCCGACATCGGCTGGTCGCTGCCGTCTGTTCGGCTGTCGCTCTCCCCATCATCCCTCCCTCCTTTCGGAGGCTCCGTGCCATTTCCTTCCATGCTGGCAATGACCTGGGCTGGCCGCTGTCCGCTTCGGGCTGAACTGGTGATGCAACCTGGTGAAGGTGCTTCGCTCCGATGAGCCATGAGGCTGTGGCCAAGCCTTGTGGCGGGGTCTGCCTGCTCTGCTTGCCCTTTGTCTCTGAGAGCTGGTGCCATCTGGCCACTGCCTTGCTCGTAGCCTTGCCATGCCCCATGATGCCTCCGCCCTTTCCATATGGGAGCGAGATGGGTCCAAGGGATAGGGCTCTGGTAGCGGGCGCTGGATACGGCCAAAGTCGGATAGACCTCCAGGGCGCCCCATTTCTCGATTTGCTGTTCCAAGGAAGAGACGCCGGTGATCGGGATCAGAAGGTTGGCTGGAGACCCAAGGCGGGGGTGGCGTCTGAGCGGGACTCGGAAAGCGCTCCTTTTGGTGCTGGATCTCTCTGGTCTGAGCCCGCTGGGTCTGCTCACCCTTGGGCGCCATCTGCCATCGGGTGCTTTGGGCTTCGCCGGGATCCGGAGCTCCTTGGTGCCACCGCACATGCCCTGGATCAGAGGGGCCTTCTTGGCGGAAATGGAGAAGGCAGAGACCCGGGCGTCAGTCCACCCTCTGGAGCGGCAGCTGGCCAGCTCCAGCTGATCGCCATGGTCTGCCCCTCTTCCGCTGGGTCCGTCGGGATCGATGCTGTGCCTTTGCTGAGGGCAGGGCGGACCCAGCTAGCGGAGGATGGCTCGTTGCTTTGCTGGCTGGGCCAAAGCCACCAGGCGCCTTCCGATCTGTGCTCCTTCCCCGTCAGGCTGGTCGGACCCTCGTTCGGCCGTTGGCTGAGATCGGGGCTGTCCGGGCTGTCTGGCTCTGTCGGCCGGCCGAAGCCAGCGGCCCTGTGGGCCGGCMaterials Technology Corp. provided the facilities and materials for the experiments.

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Additional technical assistance was provided by F.R.T. (Dr. Timothy J. Nelson) and M.C. (Dr Michael M.

Case Study click for source [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [https://doi.org/10.1371/journal.pone.0129091](10.13 71/journal.

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psone.0127091) Materials Technology Corp., Changsha, China) for the protease treatment. For cDNA synthesis, mRNA extraction and cDNA synthesis were performed with the Ambion ExoScript kit (Life Technologies, Grand Island, NY, USA) according to the manufacturer\’s instructions. The expression of *let-7* mRNA was detected by quantitative reverse transcription PCR. Total RNA was Read Full Report using TRIzol Reagent (Life Technologies). The cDNA was synthesized from 1.5 μg protein using the PrimeScript RT reagent kit (Life technologies).

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The RT-PCR reaction was performed using the TaqMan Universal PCR Master Mix (Life technologies) and a QuantiTect Reverse Transcription kit (Qiagen, Valencia, CA, USA) with the following conditions: 95°C for 5 min, 94°C for 30 s, 55°C for 35 s, 72°C for 40 s, and then at 72°C until the temperature reached 65°C. The primer sequences are as follows: *let-6* (5′-GTCGAGAAAGGAAGGACAA-3′), *let-16* (5′)-GTCAGGCGCTCCACATCC-3′ and *let-17* (5′). Antibodies {#sec2-7} ———- The primary antibodies used were: mouse anti-IKV-1 (1:1000), goat anti-β-actin (1:500) and rabbit polyclonal anti-IT-7 (1:200). The secondary antibody was goat anti-rabbit IgG (1:2000), goat anti–mouse IgG (2:1000). The secondary antibodies were goat anti-goat IgG (3:1000), rabbit anti-mouse IgG-HRP (1:100), and donkey anti-goats IgG (4:1000). Western Blotting {#sec3-5} —————- HEK 293T cells were seeded in a 96-well plate and cultured for 48 h. The cells were harvested by centrifugation and lysed by RIPA lysis buffer. The cells then were lysed in RIPA lysate Buffer supplemented with protease and phosphatase inhibitors (Roche).

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The proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride membranes, and analyzed by Western blotting with antibodies against IT-7 (ab132593), IKV-1, and β-actin. Immunofluorescence {#sec1-4} —————— The cells were fixed with methanol and permeabilized by incubation with 2% formaldehyde for 1 h at room temperature. After blocking with 5% normal donkey serum, the cells were incubated with primary antibodies at 4°C overnight, followed by incubation for 1 h with a goat anti-mouse antibody at 1:500 dilution. The secondary antibody conjugated with Alexa Fluor 488 was applied at a 1:500 concentration. The cells incubated with secondary antibodies were visualized with a fluorescence microscope (Leica, Wetzlar, Germany). Fluorescence images were captured on a Delta FLS-S microscope (Leucher, Germany). Cell Death Assay {#sec4} ————— HEKA-293T cells were cultured in DMEM supplemented with 10% fetal bovine serum and 50 U/ml penicillin-streptomycin for 24 h. The medium was collected, and the cells were lysened by lysis buffer (15 mM Tris-HCl, pH 7.

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5, 150 mM NaCl, and visit this page glycerol). The lysate was made by centrifuging, and the supernatant was incubated with 0.5 ml of a Protein G-Sepharose resin (GE Healthcare Life Sciences), which was then incubated with the cell lysate for 2 h at 4°. The resin was washed three times with 100% methanol, and the beads were eluted with 5 mM EDTA and stored at −70°C. After precipitation with 50% methanolic acetic acid, the beads were washed three times, and the lysate

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