Sc 437275

Prior to transfection, 50,000 PC-12 cells/well were plated on 6-well plates and allowed to grow to 80% confluence. The PC-12 cells were transfected with the desired MOI of Ad-TMBIM6 in 700 µl opti-MEM media and incubated overnight. After 48 h, 700 µl CRISPR plasmid targeting IRE1α (sc-400576-ACT) or control sequence plasmid (sc-437275) (both from Santa Cruz Biotechnology) were added. The plasmids were diluted in 200 µl sterile DNase free water (provided) giving a concentration of 0.1 µg/µl. The stock solution was further diluted with Opti-MEM transfection medium to bring to the desired concentration of 2–8 µg per 0.5–1 million cells. Cells were transfected with CRISPR using Ultracruz transfection reagent. Next, transfected cells were placed in an incubator at 37°C for 5–7 h, after which the medium was removed and replaced with normal medium, and cells prepared for OGD.

To evaluate the transfection efficiency, GFP-encoded plasmids were transfected into the cells. To enhance SIRT1 expression, we used SIRT1 activation CRISPR/dCas9 system (sc-400085-ACT, Santa Cruz Biotechnology, Dallas, TX, USA). Control plasmid (sc-437275, Santa Cruz Biotechnology, Dallas, TX, USA) served as a negative control. Transfection was performed using plasmid transfection reagent (UltraCruz^®, Santa Cruz Biotechnology Dallas, TX, USA). Cells were incubated for 72 h and collected for further experiments. SIRT1 activation was confirmed by qRT-PCR 72 h after transfection. In addition, hCECs were incubated with and without TNF-α (10 ng/mL) or TGF-β1 (10 ng/mL).

Hepa-1c1c7 cells were seeded 24 h prior to transfection. After cells reached 80% confluency, CHOP CRISPR/Cas9 KO plasmid (SC-41970, Santa Cruz Biotechnology) or control CRISPR plasmid (SC-437275, Santa Cruz Biotechnology) were transfected to cells following the manufacturer’s instruction. Transfection efficiency was determined by immunoblot analysis.
For the evaluation of autophagy flux, Hepa1c1c7 cells were transfected with pTF-LC3 (plasmid with Tandem Fluorescent tagged LC3, Plasmid ID # 21074) plasmid (Addgene, Cambridge, MA, USA). This plasmid construct comprises the autophagosome marker LC3 tagged with both red fluorescent and green fluorescent protein in tandem (mRFP-GFP-LC3).

The HECTD1 CRISPR activation plasmid (SC-431500-ACT) and control CRISPR activation plasmid (SC-437275) were purchased from Santa Cruz Biotechnology. Approximately 0.5–1 × 10⁵ cells were seeded into 24-well plates (with unparalleled anti-standard medium), and the medium in the 24-well plates was replaced with 200 μL of unparalleled anti-fresh medium per well until the cells reached 40-80% confluence. First, 1.5 μL of the siRNA reagent were added to 10 μL of the transfection medium to form solution A, and the solution was mixed gently at room temperature for 5 min. Second, 0.3 μg of the plasmid was added to 10 μL of the transfection medium to form solution B, and the solution was mixed gently at room temperature for 5 min. Third, solution B was added dropwise to solution A to form solution C, which was immediately vortexed at room temperature and incubated for ≥ 20 min. Finally, solution C was added dropwise to the wells of the 24-well plate, followed by mixing. 12 h after transfection, 1 mL of medium was added to the medium in each well of the 24-well plate, and the medium was discarded and replaced with 1 mL of fresh medium, if the cells were in good condition. The samples were incubated for 24–72 h in a 37 °C incubator containing 5% CO₂ until use in the western blotting analysis^{16 (link)}.

The CRISPR/dCas9 activation system was used for the overexpression of EPHA3 in AMC HN3 cells. The EPHA3 CRISPR/dCas9 activation plasmid (sc-401565-ACT; Santa Cruz Biotechnology, Dallas, TX, USA) consisted of a pool of three plasmids designed to overexpress the EPHA3 gene. The control CRISPR/dCas9 activation plasmid (sc-437275; Santa Cruz Biotechnology, Dallas, TX, USA) was used as a negative control. The plasmid transfection medium and UltraCruz transfection medium were used following the manufacturers’ protocol. Briefly, cells (1 × 10⁵ cells per well) were seeded on six-well culture plates in 3 mL antibiotic-free DMEM 24 h before transfection and grown to 70% confluence. Cells were transfected with 1 μg of the EphA3 CRISPR/dCas9 activation system using the UltraCruz transfection reagent (Santa Cruz Biotechnology, Dallas, TX, USA) and incubated at 37 °C with 5% CO₂. Three days after transfection, cells were used for evaluation. EPHA3 expression was evaluated at 3 and 7 days, and at every experiment (supplementary Figure S1).