Agnieszka B. Bialkowska

Supplementary Figure S14. TR-107 treatment impacts the transcriptomic profile of DLD-1 cells. (A) and (B) RT-PCR analysis of components of ISR and ferroptosis pathways in DLD-1 after 24 and 72 hours of TR-107 treatment, respectively. The green lane denotes a 2-fold cut-off, and the red lane – is a 0.5-fold cut-off. Data are presented as mean ± SD values (N=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Supplementary Figure S11. TR-107 treatment reduces integrated stress response and antioxidant protein expression in CRC cells. Immunoblotting of ATF3 and ATF4 and densitometry analysis of colorectal cancer cells (A) DLD-1, (B) LS 1034, (C) HT29, and (D) HCT116 cells treated with TR-107 (50 nmol/L) for 24 and 72 hours indicate a reduction in stress response. Data are presented as mean ± SD values (N=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Supplementary Figure S13. TR-107 treatment impacts the transcriptomic profile of HCT116 cells. (A) Downregulated pathway analysis in HCT116 cells identified related transcripts, which were grouped by pathway and filtered by significance and strength of association (Odds ratio) with treatment condition. (B) and (C) RT-PCR analysis of components of ISR and ferroptosis pathways in HCT116 after 24 and 72 hours of TR-107 treatment, respectively. The green lane denotes a 2-fold cut-off, and the red lane – is a 0.5-fold cut-off. Data are presented as mean ± SD values (N=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Supplementary Figure S1. Assessment of IC50 of TR-107 in NIH 3T3 cells. (A) NIH 3T3 cells were treated with vehicle control (0.1% DMSO) or various log-fold concentrations of TR-107 as described in Materials and Methods. CellTiter-Glo cell viability assay was performed 72 hours post-treatment. IC50 values (nmol/L) were calculated with GraphPad Prism and listed on each respective graph. Data are presented as mean ± SD (N=6).

Supplementary Figure S2. Cell confluence images confirm a dose- and time-dependent inhibition of CRC cell proliferation. Colorectal cancer cells (A) DLD-1, (B) LS1034, (C) HT29, and (D) HCT116 were treated in a 6-well plate format with vehicle control (0.1% DMSO) or indicated concentrations of TR-107 for 24, 48, and 72 hours. Phase-contrast live images (10× zoom) of cell confluence were captured at each time point. Images were processed with ImageJ and cell confluence was measured with PHANTAST 2.0 software as described in Materials and Methods. Data are presented as mean ± SD values (N=9). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Supplementary Figure S12. TR-107 treatment reduces integrated stress response and antioxidant protein expression in CRC cells. Immunoblotting of ATF3 and ATF4 and densitometry analysis of colorectal cancer cells (A) LoVo, (B) NCI-H508, (C) LS 174T, and (D) RKO cells treated with TR-107 (50 nmol/L) for 24 and 72 hours indicate a reduction in stress response. Data are presented as mean ± SD values (N=3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Supplementary Table S3. Transcriptomic and proteomic data analysis.

Supplementary Figure S3. Cell confluence images confirm a dose- and time-dependent inhibition of CRC cell proliferation. Colorectal cancer cells (A) LoVo, (B) NCI-H508, (C) LS 174T, and (D) RKO were treated in a 6-well plate format with vehicle control (0.1% DMSO) or indicated concentrations of TR-107 for 24, 48, and 72 hours. Phase-contrast live images (10× zoom) of cell confluence were captured at each time point. Images were processed with ImageJ and cell confluence was measured with PHANTAST 2.0 software as described in Materials and Methods. Data are presented as mean ± SD values (N=9). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Supplementary Figure S6. Washout of TR-107 treatment suggests an irreversible inhibition of CRC cell viability. (A) HCT116 cells were treated in a 96-well plate format with vehicle control (0.1% DMSO) or indicated concentrations of TR-107 for 2, 4, 6, 8, 12, 24, and 72 hours. At each time point, wells were washed out twice with fresh media and replaced with fresh media lacking treatment up until 72 hours. Cell viability was assessed for all treatment durations after 72 hours. Data are presented as mean ± SD values (N=6). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (B) Western blot analysis of HCT116 cells after 24 hours of treatment with washout and 72 hours with no washout. HCT116 cells were treated with vehicle control (0.1% DMSO) or TR-107 10 nmol/L, 50 nmol/L, 1 μmol/L. Immunoblot was performed for ClpX, mtTFA, and β-actin as loading control.