Methyl thiazolyl tetrazolium (mtt)

The materials for this work, including CHA, a scaffold of CHA/HCB 40 wt%, CHA/Ti plates, and CHA/HCB/Ti plates to emulate conditions, were adapted from previous studies [7 (link),10 (link),11 (link)]. Penicillin-streptomycin, fungizone, and MEM- $\alpha$ medium were picked up from Gibco (Thermo Fisher Scientific, Carlsbad, CA, USA). Fetal bovine serum (FBS) and phosphate buffered saline (PBS) were bought from Sigma-Aldrich (Sigma-Aldrich Inc., Burlington, MO, USA). MTT was bought from Biobasic (Biobasic Inc., Amherst, NY, USA), and dimethyl sulfoxide (DMSO) was picked up from Merck (Merck KGaA, Darmstadt, Germany). Six-well plates were purchased from Nunc (Nalge Nunc International, Rochester, MA, USA).

The cytotoxicity of silk cocoon and pupa extracts was investigated by MTT assay. In brief, the Vero cells were seeded onto 96-well culture plates approximately 1.5 × 10⁴ cells/well. The two-fold dilutions of the extracts were treated with the cells and incubated for 72 h. Following, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) (Bio Basic, Markham, ON, Canada) was added and incubated for 4 h. The absorbance was measured at a wavelength of 540 nm with a reference wavelength of 630 nm and used to analyze the percentage of cell viability relative to the non-treated control, which was set a 100%. The half maximal cytotoxicity doses, or CD_50, was calculated from a dose–response curve.

Tryptophol (TOH; C10H11NO,), voriconazole (VOZ,), absolute ethanol (EtOH), Tween^™ 20, glycerin, propylene glycol, allantoin, phenoxyethanol, Acridine Orange/Ethidium Bromide (AO/EB), Sodium Lauryl Sulfate (SLS), and croton oil were purchased from Sigma-Aldrich (St. Louis, MO, USA). SEPIGEL 305^™ was purchased from Seppic Inc. (Fairfield, NJ, USA). Triethanolamine was purchased from Dow Chemical (Thailand). Deionized (DI) water used in all experiments was produced from in-house Milli-Q^® IQ 7000 Ultrapure Water System (EMD Millipore, Bedford, MA, USA). Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA) were purchased from Oxoid Ltd. (Hampshire, UK). Roswell Park Memorial Institute 1640 medium (RPMI-1640), Fetal Bovine Serum (FBS), Penicillin-streptomycin (Pen-strep), Phosphate-Buffered Saline (PBS), and Trypsin-EDTA were purchased from Gibco™ (Grand Island, NY, USA). MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was purchased from Bio Basic Inc. (Canada). CytoPainter MitoGreen (#ab176830) was purchased from Abcam. CytoTox 96^® Non-Radioactive Cytotoxicity assay for measuring the release of lactate dehydrogenase (LDH) was purchased from Promega (Madison, WI, USA). All the reagents used in this study were of high purity or HPLC grade.

LA, chloroquine (CQ) and primary antibody for α‐Tubulin were obtained from Sigma‐Aldrich (St. Louis, MO, USA). Rapamycin (Rap) was from LC Laboratories (Woburn, MA, USA). Primary antibodies for Bcl‐2, Bax, phosphor‐Akt (p‐Akt^Ser473), phosphor‐mTOR (p‐mTOR^Ser2448) and total mTOR, phospho‐p70S6K (p‐p70S6K^{Thr421/Ser424}) and p70S6K, VPS34, Beclin‐1, LC3‐II and p62 were obtained from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies for ATG13 were obtained from San Ying Biotechnology (Wuhan, China). A primary antibody for glyceraldehyde‐3 phosphate dehydrogenase was from Bioworld Technology (Louis Park, MN, USA). Complete protease inhibitor cocktail was purchased from Roche (Mannheim, Germany). MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide] reagent was from BioBasic, Inc. (Markham, ON, Canada). Cell‐Light™ EdU Apollo 567 In Vitro Kit was from RiboBio Technology (Guangzhou, China). SuperSignal West Pico chemiluminescent substrate was from Pierce (Rockford, IL, USA). FBS was from Life Technology (Grand Island, NY, USA). The plasmid‐expressed rat LC3 fused to enhanced green fluorescent protein (pEGFP–LC3) was provided by Addgene (Cambridge, MA, USA) [21].

The viability of siRNA-transfected VK2/E6E7 cells was assessed using a 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay [41 (link)]. Cells were seeded at a density of 1 × 10⁴ cells per well in 96-well culture plates and were allowed to adhere overnight. The following day, the culture medium was discarded and replaced with a medium containing 10% MTT (Sigma Aldrich). Cells were then incubated for 3 h at 37 °C. Afterward, the MTT solution was carefully removed from each well, and dimethyl sulfoxide (DMSO; Bio Basic Inc., Markham, ON, Canada) was added to dissolve the formazan crystals. The absorbance of each sample was subsequently measured at 570 nm using a microplate spectrophotometer (Multiskan Go; Thermo Fisher Scientific).

LS 174T cell lines and EA were seeded in 96-well tissue culture plates (2 × 10⁴ cells/well). CM of LS 174T cell lines were collected accordingly and transferred to EA. Following CM treatment, EA cell viability was determined at every 24 h for 72 h using 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Bio Basic Inc., Markham, Canada) assay, as described previously (Chieng & Say, 2015 (link)).