Dimethyl sulfoxide (dmso)

MDA-MB-231 cells were cultured in a 96-well plate for 24 h at an initial density of 3 × 10³ cells/well, before treatment with different concentrations (1.0, 2.5, and 5.0 mM) of CaLac for 24, 48, or 72 h at 37 °C. After CaLac treatment, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to each well, and then the cells were incubated for 1 h at 37 °C in a humidified environment of 5% CO₂. After discarding the media, 200 μL of dimethyl sulfoxide (Cell Signaling Technology, Danvers, MA, USA) was added to each well. The absorbance was then read at 570 nm using a microplate reader (iMark Microplate Absorbance Reader, Bio-Rad, Hercules, CA, USA).

Saleable pasteurized homogenized whole milk (3.25% fat content) was spiked using stock solutions of ceftiofur, as previously described (6 (link)). Briefly, 60 mg of ceftiofur sodium powder (93.6% purity, Sigma-Aldrich, St. Louis, MO) was diluted in 93.6 ml of distilled water (Millipore Corp., Bedford, MA) with 0.96% of dimethyl sulfoxide (Cell Signaling Technology, Danvers, MA, USA) added to increase the solubility of ceftiofur, to a stock concentration of 600 μg/ml, which was used to spike a volume of 3 l of milk, to a final concentration of 200 ppb for heat treatment trials and 400 ppb for pH trials. Stock solutions were stored in individual vials at −80°C until used. Concentrations of ceftiofur targeted in milk batches were based on previously reported concentrations of ceftiofur in waste milk on dairy farms in the US (7 (link), 12 (link)).
A total of four repetitions with new milk batches were conducted for each heat treatment and pH assay. This number of repetitions was based on reported references for heat and pH stability of antimicrobials, where we estimate an 80.5% statistical power to identify a significant difference between samples after treatment when compared to the control group (α = 0.05, standard deviation = 0.22, difference to detect = 0.18) (8 (link), 11 (link), 13 (link)).

Colorectal cancer cells were cultured in a 96-well plate (3 × 10³ cells/well) for 24 h, and then treated with different concentrations of CaLac (0.5, 1, and 2.5 mM) for 24 h at 37 °C. Then, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to each well, and the cells were incubated at 37 °C for 1 h in a humidified environment containing 5% CO₂. After the media was discarded, 200 μL of dimethyl sulfoxide (Cell Signaling Technology, Danvers, MA, USA) was added to each well. The absorbance was read at 570 nm using a microplate reader (iMark Microplate Absorbance Reader, Bio-Rad, Hercules, CA, USA).

Chemical inhibitors of MEK, U0126 and PD98059 (Cell Signaling Technology), were used at concentrations of 10 and 50 μM, respectively. Bay43-9006 (Cayman Chemical), a chemical inhibitor of Raf, was used at 15 μM. Retro2 (Sigma–Aldrich), a retrograde trafficking inhibitor, was used at a concentration of 100 μM (Nelson et al., 2013 (link)). All chemical inhibitors were reconstituted in DMSO (Cell Signaling Technology), which served as a volume-specific vehicle control. EGFR and ERK1/2 siRNAs (Cell Signaling Technology) were transfected into SVG-A cells with RNAiMax (Thermo Fisher) at 10 pmol per well per manufacturer’s instructions. Successful transfections of the siRNAs were confirmed using BLOCK-iT Red (Thermo Fisher) (DuShane et al., 2018 (link)).

DMSO was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Dasatinib was purchased from Cell Signaling Technology (CST, Danvers, MA, USA) and dissolved in DMSO. ATO was purchased from the First Affiliated Hospital of Harbin Medical University. c‐ABL1, Bcl‐2, Mcl‐1, Noxa and p53 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); β‐actin antibody was bought from Sigma‐Aldrich; ERK, p‐ERK, STAT5, p‐STAT5, PI3K, p‐PI3K, AKT, p‐AKT, caspase‐9, caspase‐3, PARP, XIAP, Survivin, Bcl‐w, Bik, Bak, Bad, PUMA, Bid, p21, JNK, p‐JNK, p‐ATF‐2, p‐JUN, IRE1, ASK1 and ATF4 antibodies were purchased from CST; TRAF2 antibody was purchased from ABclonal Biotechnology (Wuhan, Hubei, China); PML and ATF6 were purchased from Abcam (Cambridge, MA, USA).