Cell culture
Human cervix adenocarcinoma HeLa (ATCC® CCL-2™) and human breast adenocarcinoma MCF-7 (ATCC® HTB-22™) cells were obtained from the American Type Culture Collection (2015), mycoplasma tested (last test August 2019), and maintained in a humidified incubator containing 5% CO2 at 37 °C. Cells were cultured in DMEM-F12 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies, Grand Island, NY), and were routinely grown in plastic tissue-culture dishes (Life Sciences, Corning, NY).
Peripheral blood mononuclear cells (PBMC) extraction.
After obtaining written informed consent, PBMC were isolated from healthy donors by density gradient centrifugation with Ficoll-Paque™ PLUS (GE Healthcare, Chicago, Ilinois, USA) and maintained at 4X106 cells/mL in cell culture plates at 37 °C in 5% CO2 atmosphere, using RPMI 1640 medium (GIBCO Thermofisher, Waltham, Massachusetts, USA) supplemented with 1 μg/mL amphotericin B, 1 μg/mL penicillin and 2.5X10− 3 μg/mL streptomycin (GIBCO Thermofisher, Waltham, Massachusetts, USA) and 10% of FBS (GIBCO Thermofisher, Waltham, Massachusetts, USA). The study was approved by the Institutional Ethics Committee at the Universidad Autonoma de Nuevo León, College of Biological Sciences.
Cell death analysis
Cell death was determined by staining cells with 2.5 μg/mL APC Annexin V (BD Pharmingen, San Jose, CA) and 0.5 μg/mL propidium iodide (PI) (Sigma-Aldrich, ST. Louis, MO). In brief, 5 × 104 cells were seeded in 24-well plates and were incubated with IMMUNEPOTENT CRP for 24 h, with or without pre-incubation with QVD.oph (10 μM), N-acetyl-L-cysteine (NAC) (5 mM) or Spautin-1 (Sp-1) (15 μM). Cells were then recuperated, washed with PBS (Phosphate-buffered saline) and then resuspended in 100 μl of binding buffer (10 mM HEPES/ NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Finally, cells were stained, incubated at 4 °C for 20 min and assessed with BD Accury C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The results were analyzed using FlowJo Software (LLC, Ashland, OR).
Mitochondrial membrane potential analysis
Mitochondrial membrane potential was measured using 500 nM TMRE (Sigma-Aldrich, ST. Louis, MO). In brief, 5 × 104 cells in 24-well dishes were incubated with or without IMMUNEPOTENT CRP (CC50) for 24 h. Cells were then recuperated, washed with PBS, stained, incubated at 37 °C for 30 min, and measured by flow cytometry as described above.
ROS production analysis
ROS generation was measured using 2.5 μM DCFDA (Thermo Fisher Scientific, Waltham, MA). In brief, 5 × 104 cells in 24-well dishes were incubated with IMMUNEPOTENT CRP (CC50) for 24 h, with or without pre-incubation with NAC. Cells were then recuperated, washed with PBS, stained, incubated at 37 °C for 30 min, and measured by flow cytometry as mention.
Cell cycle analysis
Cell cycle distributions were determined by PI (Sigma-Aldrich, ST. Louis, MO) staining. 2 × 105 cells in 6-well dishes were incubated with ICRP for 24 h. Cells were then washed with PBS and fixed in 70% ethanol. Cells were washed with PBS, then incubated with 50 μg/mL PI and simultaneous 50 μg/mL RNase (Sigma-Aldrich, ST. Louis, MO) treatment at 37 °C for 30 min. Cell DNA contents were measured by flow cytometry as explained above.
eIF2α phosphorylation analysis
For this assay, 2 × 105 cells were plated in 12-well dishes and were incubated with ICRP for 18 h. Cell were collected and fixed (eBioscience™ Foxp3 / Transcription Factor Fixation/Permeabilization) for 1 h at 4 °C, washed with 2%-FACS Buffer (PBS 1x and 2% FBS), centrifuged twice at 2000 rpm during 20 min, suspended in 10%-FACS Buffer (PBS 1x and 10% FBS) and incubated for 30 min in shaken. Then, anti-EIF2S1 (phospho S51) antibody [E90] (Abcam, ab32157) (1:100) was added and incubated for 2 h and washed twice. Next, goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, ab150077) (1:200) was added and incubated for 1 h in darkness. Finally, cells were washed and eIF2α phosphorylation was measured by flow cytometry as described above.
For confocal microscopy 1.5 × 105 cells were planted on cover slides into 12-well dishes, treated with ICRP (CC50) and incubated for 24 h. Then, cells were washed and fixed with 4% PFA, washed and permeabilized with 0.1% Triton Buffer, washed twice with 2% FACS buffer, and 10%-FACS buffer was added and incubated during 45 min. Next, recombinant anti-EIF2S1 (phospho S51) antibody [E90] (Abcam, ab32157) (1:250) was added, incubated for 2 h and washed thrice. Finally, goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, ab150077) (1:100) was added, incubated for 30 min in darkness, washed twice and assessed by confocal microscopy (Olympus X70).
Calreticulin exposure
For this evaluation, 1 × 106 cells were plated, left untreated or treated with ICRP, and incubated for 24 h. Cells were harvested, washed, and stained with Calreticulin-Phycoerythrin (Calreticulin-PE, FMC-75; Enzo Life Science, Farmingdale, NY) antibody (1:1000) in 2%-FACS buffer. After 1 h in darkness at room temperature (RT), cells were washed and suspended in 100 microliters uL of 2%-FACS buffer to be assessed by flow cytometry as mention before.
For confocal microscopy, 2.5 × 105 cells were plated, and then left untreated (control) or treated with ICRP (CC50) and incubated for 24 h. Then, cells were washed with PBS, stained with Calreticulin-PE antibody (2 μg/mL) (Enzo Life Science, Farmingdale, NY) and incubated for 1 h in FACS buffer. Finally, cells were washed twice with PBS and assessed by confocal microscopy (Olympus X70).
ATP release assay
For this, 1 × 106 cells/mL were treated with ICRP for 24 h. Supernatants were used to assess extracellular ATP by a luciferase assay (ENLITEN kit, Promega, Madison, WI) following the manufacturer’s instructions. Bioluminescence was assessed in the Synergy HT microplate reader using the Software Gen5 (BioTek, Winooski, VT) at 560 nm.
High-mobility group box 1 release assay
Supernatants of untreated and ICRP-treated cells (1 × 106 cells/mL) were used to measure extracellular HMGB1 using an HMGB1 ELISA kit (BioAssay ELISA kit human; US Biological Life Science, Salem, MA), following the manufacturer’s instructions. Absorbance was assessed in the Synergy HT microplate reader using the Software Gen5 (BioTek, Winooski, VT) at 450 nm.
Cell morphology assessment
HeLa and MCF-7 cells were cultured in 24-well plates and left untreated or incubated for 24 h with ICRP, with or without pre-incubation with NAC (20 min). After the incubation time, cells were observed in an inverted microscope (NIKON TS100) and pictures were obtained with an Infinity1 (Lumera) camera (10X).
Autophagosome formation analysis
For this assay, 5 × 104 cells were cultured in 24-well plates (Life Sciences) and left untreated or incubated for 24 h with ICRP, with or without pre-incubation with Sp-1 or NAC. Then, cells were recuperated, washed with PBS, stained with CYTO-ID Autophagy Detection Kit (Enzo Life Science, Farmingdale, NY) and measured by flow cytometry as explained above.
Statistical analysis
The results presented here represent the mean of at least three independent experiments done in triplicate (mean ± SD). Statistical analysis was done using paired student T-test, and the statistical significance was defined as p < 0.05. The data was analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA).