Entranster h4000

Plasmid and shRNA construction were supported by Hitrobio.tech (Beijing, China). Full-length sequences of bovine lncRNA PRANCR were commercially synthesized and cloned into the pcDNA3.1 + vector. pcDNA3.1 + was used as vector control for analysis. shRNA targeting sequences are listed as follows: PRANCR (bovine), 5′-GGTGCTTGTGCACGCACTTCC-3′ and Scramble control sequence, 5′-GTCTCCGAACGTGTCACGT-3′. shRNA targeting sequences were cloned into pLKO.1-Puro. Plasmids and shRNAs were transfected into cells by using the Entranster-H4000 (Engreen Biosystem, Beijing, China) following the manufacturer’s recommended procedures.

The Entranster-H4000 (H4000, Engreen) was diluted from 20 μL to 1 mL. Hydrodynamic dimension and zeta potential were measured. CD25-PE (100 μL, 0.2 mg mL-1) was mixed with EDC (300 μL, 1 mg mL-1) in MES (pH 5.5), and then, H4000 (100 μL) was added overnight in the dark. Finally, H4000-CD25PE was purified using a dialysis bag with a molecular weight of 100 kD and enriched in 100 μL of water. The hydrodynamic force and zeta potential of the product were measured again to check for coupling effect, and 20 μL was used to perform the fluorescence spectrometer test. Activated Cy3-NHS (10 μL, 5 mg mL-1), H4000 (200 μL), and NaHCO3 (200 μL, 0.1 M, pH 7.8) were homogeneously mixed and reacted overnight in the dark at 4°C. Subsequently, the reacted solution was dialyzed (molecular weight: 3500 D) in deionized water for 24 h, and the H4000-cy3 nanoparticles were obtained. Furthermore, H4000 and purified H4000-CY3 were diluted to 0.5 mg mL-1 with deionized water, and the hydration particle size and surface potential of each sample were tested thrice using a laser particle size analyzer (Malvern, zen3690).

Reprogramming plasmids pRK-ATF4 (#26114), plenti-ATF4 (#125238), plenti6-xCT-V5 (#170427), pDONR221-xCT (#132244), and pFETCh_KDM1A (#86260) were obtained from Addgene (USA) as bacterial stabs. These plasmids were transfected using Entranster-H4000 (Engreen Biosystem, China). Proteins were extracted 48 h after transfection to detect the expression levels of related proteins.

The mutant E. coli MscL-G22S cDNA was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and subsequently ligated into a lentiviral vector of pLentai-hEF1a-PuroR-mCherry with a CMV promoter. The plasmid map is shown in Fig. S1. The lentiviral plasmids were co-transfected with helper plasmids into HEK293T cells to package the lentivirus (Taitool Bioscience Co., Ltd., Shanghai, China). For the MscL-G22S expression, SCs were transfected with the lentivirus according to the manufacturer’s instructions. pLentai-hEF1a-PuroR-MCV-mCherry was used as the negative control for the MscL-G22S expression. After transfection, puromycin (2 μg/mL) was added into the culture medium from the second passage to screen the resistant cells. For mitochondrial membrane potential (MMP) measurement, MscL-G22S was amplified using primers containing NheI and EcoRI cut sites and subcloned into a modified pcDNA3.1 vector (Invitrogen, CA, USA) and the MscL-G22S-pcDNA3.1 plasmid were transfected into SCs using EntransterTM-H4000 (Engreen, 4000–5, Beijing, China) according to the manufacturer’s instructions. Expression of MscL-G22S was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blot, and immunostaining.

C48/80, 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide, bovine serum albumin (BSA), Pluronic F-127 and thapsigargin were purchased from Sigma-Aldrich (St Louis, MO, United States). Fluo-3 AM Ester, Calcium Green-5N and Cal-630 AM Ester were from Biotium (San Francisco, CA, United States), Invitrogen (Carlsbad, CA, United States) and MKbio (Shanghai, China), respectively. Plasmid pcDNA3-4mtD3cpv was from Beijing Zoman Biotechnology Co. Ltd. (Beijing, China). The transfection reagent Entranster^TM-H4000 was from Engreen Biosystem (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin were purchased from Gibco BRL (Grand Island, NY). Nycodenz was from Axis-shield (Scotland, United Kingdom). Forsythiaside A (CAS^#79916-77-1), isoforsythiaside (CAS^#1357910-26-9) and phillyrin (CAS^#487-41-2) were from Baoji Herbest Bio-Tech Co. Ltd. (Baoji, Shaanxi, China, purity >98%). All the other reagents were of analytical grade.

Plasmids of pLvx-Flag-ΔNp63α, pLenti6-Flag-ΔNp63α, pLKO.1-shGFP, pLKO.1-shΔNp63α have been described previously [54 (link),79 (link),80 (link)]. We used lentiviral-mediated shRNA interference to achieve CDK1 knockdown in tongue squamous cells. The target sequences of CDK1 shRNA were as follows: shCDK1#1, 5′-ATAGTCCTGTAAAGATTCCAC-3′; shCDK1#2, 5′-AATTAGAAGACGAAGTACAGC-3′. HEK293T cells were transfected with pLvx-Flag-CDK1/pLvx-puro/pLKO.1-shGFP/pLKO.1-shCDK1 along with the lentiviral packaging plasmids psPAX2 and pMD.2G, using Entranster^TM-H4000 (Engreen Biosystem, Beijing, China) as a transfection reagent. The viral particles were collected 48 h post transfection. Then the particles were supplemented into the medium to incubate with UM1 or Cal27 cells overnight in the presence of 10 μg/mL polybrene. Forty-eight hours post infection, the cells were selected in a medium supplemented with 5 μM puromycin (Puro) or (and) blasticidin (BSD) for 72 h.

Dual-luciferase reporter plasmids, consisting of MFSD12 wild-type (WT), mutant (MUT), and control (CON), were constructed in psicheck2.0 vectors (Zolgene, China). 293T cells were co-transfected with reporter plasmids and miR-4732-3p mimics using EntransterTM-H4000 and EntransterTM-R4000 transfection reagents from Engreen. Cell lysates were obtained to perform luciferase reporter assays following transfection for 24 h.