β actin

Antibodies against PKC-α, JNK, VCAM-1, CD11b, and β-actin were purchased from GeneTex (Hsinchu, Taiwan). Antibodies against p-PKCα and p-JNK were purchased from Cell Signaling Technology (Danvers, MA, USA). 2′,7′-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester (BCECF-AM) and GF109203X, Gö6976 and SP600125 inhibitors were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Membrane proteins were extracted from LS174T cells using an s compartmental protein extraction kit (Chemicon Inc, Temecula, CA, USA). After the various treatments, LS174T cells were lysed with RIPA lysis buffer supplemented with protease and inhibitor for 30 min on ice followed by centrifugation at 4 °C. The amount of protein was measured by BCA assay (Thermo Fisher Scientific, Waltham, MA, USA) and an equal amount of protein lysates was electrophoresed on an 8–15% polyacrylamide gel at 100 volts for 2 h. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (0.2 µm pore size) through wet blotting at 250 mAmp for 2 h (in cold room) (Biorad, Billerica, MA, USA). Blocking was performed using 5% BSA in 1x PBS-T (1X PBS 0.1% TWEEN-20) for 1 h at RT, and then the membrane was incubated with primary antibodies at 4 °C overnight. After that, a washing process was performed four times with 1x PBS-T, and the samples were incubated with secondary antibody HRP–goat–anti-rabbit/mouse (1:10,000) for 1 h at RT. Then, membranes were washed with 1X PBS-T and developed using a Femto chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA) and the image analysis was carried out using ImageJ software. Antibodies for Western blotting, including GPX4 (GTX54095, 1:1000), β actin (GTX109639, 1:5000), were purchased from GeneTex, Irvine, CA, USA.

Oxaliplatin was purchased from Sanofi Pharmaceutical Company (NY, USA). The procedure for synthesizing the CTSS inhibitor RJW-58 was described previously 14 (link). The following primary antibodies were purchased: Cathepsin S (Thermo Fisher catalog: #PA5-81369), EBF (Santa Cruz, #sc-137065), ATF-3 (Biossua, #BS-0519R), α-tubulin (Sigma, MO, USA), IRF-1 (Bio-Rad, #VPA00801), CBP (GeneTex, #GTX101249), Iba1 (Abcam, #ab-5076), NeuN (Novus, #NBP2-10491), IL-10 (Abcam, #ab189392), STIM1 (Cell signaling (D88E10), #5668S), CD11b (Abcam, #ab52478), CD45 (BD Biosciences, #553079), STAT3 (BD, #50402), P-STAT3 (Cell signaling, #9145S), NFκB (Cell signaling, #4727S), OR5B3 (Abcam, #ab186624), OR5M3 (LSBio, #LS-C200406), and β-actin (GeneTex, #GTX109639). Horseradish-peroxidase-conjugated secondary antibodies were purchased from GeneTex (#GTX213110, #GTX213111, #GTX224125, and #GTX232040).

WISP-3, VEGF-C, ERK, p38, JNK, and β-actin antibodies were obtained from GeneTex (Hsinchu, Taiwan). The phosphorylated forms of ERK, p38, and JNK antibodies were bought from Cell Signaling (Danvers, MA, USA). ERK, p38, JNK, and control ON-TARGETplus siRNAs were purchased from Dharmacon (Lafayette, CO, USA). Taqman^® One-Step PCR Master Mix and qPCR primers and probes were bought from Applied Biosystems (Foster City, CA, USA). Recombinant human WISP-3 was acquired from PeproTech (Rocky Hill, NJ, USA). All other chemicals not already mentioned were acquired from Sigma-Aldrich (St. Louis, MO, USA).