Fig 2: The down-modulation of the heme synthesis-export system, obtained by FLVCR1a silencing, sensitizes colorectal cancer cells to metformin. (A)Western blot analysis of FLVCR1a and ALAS1 expression in SKCO1 cells. Vinculin and beta-actin expression are shown as a loading control, respectively. Cells in which the expression of FLVCR1a was down-regulated using a specific shRNA are compared to cells expressing a scramble shRNA. A representative blot is shown. For statistical analyses, a Student t-test was used. Densitometry shows mean ± SEM, n = 3 (FLVCR1a blot) and 2 (ALAS1 blot) biological replicates, ** p < 0.01, **** p < 0.0001. (B,C) Cell viability of SKCO1 cells untreated (NT) or treated with 7 uM 5-FU, 5 uM Oxaliplatin and 50 nM SN38 (B) or with 2.5 mM metformin (C), measured after two days (5-FU, Oxaliplatin and SN38) or two and three days (metformin) of drug exposure without medium refresh. Cells in which FLVCR1a expression was down-modulated using a specific shRNA are compared to cells expressing a scramble shRNA. Cell viability is measured as a fold increase over an untreated calibrator sample and expressed relative to day 0. For statistical analyses a two-way analysis of variance was performed, followed by the Bonferroni correction for multiple groups comparisons. Values represent mean ± SEM, n = 4 biological replicates, *** p < 0.001, **** p < 0.0001. (D) Cell viability of CACO2 cells was assessed by crystal violet staining on the indicated days. Cells in which the expression of FLVCR1a was down-regulated using a specific shRNA are compared to cells expressing a scramble shRNA; 5 mM metformin administration occurred at day 1 of cell proliferation and medium was maintained without refresh till the end of the experiment. Staining quantification is reported in the graph as the percentage of crystal violet stained area versus image total area. For statistical analyses, a two-way analysis of variance was performed, followed by the Bonferroni correction for multiple groups comparisons. Values represent mean ± SEM, n = 3 biological replicates, **** = p < 0.0001.

Fig 3: Characterization of PPO knock-in clones. R38/1, W227/1, U-2 OS, and N2 cell lines were transfected with the PPOX-GFP construct and corresponding knock-in cell lines denoted R38/1-KI, W227/1-KI, U-2 OS-KI and N2-KI, respectively. Control, PPO-KO and PPO-KI samples are highlighted by green, orange and blue background, respectively. (a) Expression levels of PPO-GFP and ALAS-1 were analyzed by Western blotting. Notice markedly higher PPO-GFP expression levels compared to endogenous PPO. Alpha-tubulin was used as a loading control. (b) PPO enzymatic activity in cell lysates was determined by following conversion of protoporphyrinogen IX to protoporphyrin IX. PPO oxidase activity is shown as ratio to the endogenous PPO activity in parent U-2 OS cells (100%). Data represent mean (± S.D.); n = 2. (c) Localization of PPO-GFP (green channel) was assessed by live-cell confocal microscopy. Mitochondria were visualized by vital staining using Mitotracker dye (red channel), cell nuclei were stained by Hoechst 33258 (blue channel). Scale bar 10 µm. (d) Intracellular concentrations of heme and protoporphyrinogen/protoporphyrin IX were determined by RP-HPLC. Data represent mean (± S.D.); n = 2. (e) The oxygen consumption rate was measured by Seahorse. ATP production, maximal respiration, and spare respiratory capacity were determined upon the addition of 1 µM oligomycin, 1 µM CCCP, and 0.5 µM rotenone + 0.5 µM antimycin A, respectively. Statistical significance was calculated by one-way ANOVA and assigned by ***(P < 0.001), **(P < 0.01), *(P < 0.05) and ns (non-significant; P > 0.05).

Fig 4: The down-modulation of the heme synthesis-export system, obtained by ALA treatment, sensitizes colorectal cancer cells to metformin. (A) qRT-PCR analysis (on the left) and Western blot analysis (on the right) of ALAS1 expression in SKCO1 cells upon ALA administration. For qRT-PCR analysis, ALAS1 expression was measured on SKCO1 cells untreated or treated with 5 mM ALA for 24 h. Transcript abundance is normalized on beta-actin mRNA expression and expressed as a fold increase over a calibrator sample. For statistical analyses, a Student-t-test was used. Data represent mean ± SEM, n = 5 biological replicates, ** p < 0.01. For western blot analysis, untreated cells are compared to cells treated with 5 mM ALA for 16, 24 and 48 h. Beta-actin expression is shown as a loading control. Band intensities were measured by densitometry and normalized to beta-actin expression. Densitometry data represent mean ± SEM, n = 2 biological replicates. For statistical analysis, a one-way ANOVA analysis of variance was performed, followed by the Bonferroni correction for multiple groups comparisons; * p < 0.05, ** p < 0.01. (B,C) Cell viability of SKCO1 (B) and CACO2 (C) cells measured at different time points as a readout of cell proliferation. Untreated (NT) cells are compared to cells treated with ALA, metformin or ALA + metformin. Drug administration occurred at day 1 of cell proliferation. Cell viability is measured as a fold increase over an untreated calibrator sample and expressed relative to day 0. Data represent mean ± SEM, n = 5 (SKCO1) or 4 (CACO2) biological replicates. For statistical analyses, a two-way analysis of variance was performed, followed by the Bonferroni correction for multiple groups comparisons; * symbols are used to indicate statistical significance when cells treated with ALA + metformin are compared to cells treated with metformin or ALA alone; # symbols are used to indicate statistical significance when treated cells are compared to untreated cells; # p < 0.05, ** or ## p < 0.01, *** or ### p < 0.001, **** or #### p < 0.0001.