Fig 2: Silencing LINC00665 decreased TUBB expression through upregulating miR-339-3p in CM cells. (A) TUBB expression in CM (M21, MEL-RM, A375, and A2058) cells and HEMn cells was analyzed by qRT-PCR. (B–E) TUBB expression in A375 cells and A2058 cells, both of which underwent transfection with miR-339-3p mimic or miR-339-3p inhibitor, or co-transfection with siLINC00665 and miR-339-3p inhibitor, was analyzed by qRT-PCR (B, D) and western blot (C, E), with GAPDH serving as the reference gene. +++ p or *** p or ### p or ^^^p < 0.001; + vs. HEMn; * vs. MC; # vs. IC; ^ vs. I (CM, Cutaneous Melanoma; I, miR-339-3p inhibitor; IC, inhibitor control; M: miR-339-3p mimic; MC, mimic control; siLINC00665, siRNA-LINC00665; siNC, siRNA-negative control; TUBB, Tubulin beta chain).

Fig 3: The comparison between the Western blot analysis of an individual protein and the total protein staining(A) Western blot (WB) analyses of ACTB, GAPDH, TUBB, and the total protein staining with SYPRO Ruby with serial gradient amounts of total protein loaded on the gels. (B) The relationship between the quantified band intensities and the actual amounts of the total protein loaded on the gels. The band intensities were quantified using ImageJ software.

Fig 4: Expression of Cytochrome c, Smac/Diablo and HtrA2/Omi in cancer cells.MCF-7 breast cancer (A) and DU145 prostate cancer (B) cells were treated for 48 h with NC, NutramilTM Complex, at 4% concentration; NC-CC, NutramilTM Complex without calcium caseinate, at 4% concentration; or ST, staurosporine positive control, at 1.5 µM concentration. Cell extracts were prepared using Cell Lysis Buffer (Cell Signaling Technology, MA, USA) with the addition of Protease Inhibitor Cocktail (BioShop, Canada). Protein extracts were then separated on a polyacrylamide gel and transferred to a nitrocellulose filter (Bio-Rad, CA, USA) by wet-electroblotting. The immobilized proteins were incubated with Cytochrome c (#11940), Smac/Diablo (#2954), and HtrA2/Omi (#9745) primary antibody (Cell Signaling Technology, MA, USA). ß-Tubulin (#2128, Cell Signaling Technology, MA, USA) was used as a reference protein. Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA).

Fig 5: BCAT1 silencing leads to reduced IRG1 and itaconate levels in activated human macrophages.(a) Western Blot following BCAT1 (Si-BCAT1) and non-targeting (Si-Control) siRNA treatment in hMDMs stimulated with LPS (100 ng ml-1, 8 h). BCAT1, IRG1, TUBB and ACTB blots are shown on three independent donor hMDMs. (b) BCAT1 and IRG1 Western Blot on Si-BCAT1 and Si-Control hMDMs from donor four and corresponding itaconate levels measured by GC/MS are shown in the right panel. Itaconate levels measured by GC/MS were further confirmed in si-BCAT1 and si-Control hMDMs from independent donors (n=4, lower panel). Error bars are s.e.m. Significance was tested using one sample t-test (two-tailed). *P<0.05 by one-sample-t-test.