*4.1. Chemicals and Antibodies*

1-(3'-(4"-ethylthio-1",8"-naphthalimid-*N*"-yl))-propyl-3-benzylimdazolium bromide (MC5), Dichloro[1-(3'-(4"-ethylthio-1","-naphthalimid-*N*"-yl))-propyl-3-benzyl-imidazol-2-ylidene] (η6-*p*-cymene)ruthenium(II) (MC6), and Chlorido[(η2, η2-cycloocta-1,5-diene)-1-(3'-(4"-ethylthio-1", 8"-naphthalimid-*N*"-yl))-propyl-3-benzyl imidazol-2-ylidene] rhodium(I) (MC7) were synthesized and purified, as previously described [6,15]. Raptinal, antimycin, GSH, NAC, Mito TEMPO, rotenone, CCCP, and JC-1 were purchased from Sigma-Aldrich (Steinheim, Germany). Pharmacological inhibitors of p38α, VX-702 and Ralimetinib (LY2228820) were from selleckchem (Munich, Germany) and DHE was from Biomol GmbH (Hamburg, Germany). MitoTracker Green, MitoSox Red, and the

transfection reagent, Lipofectamine 3000 were obtained from Thermo Fischer (Darmstadt, Germany). Primary antibodies against Bcl-xL (#2764), pATF2 (T71; #9221), pp38 MAPK (T180, Y182; #9211), PARP (#9542), Bax (#5023), phospho-Bad sampler kit (#9105), caspase 3 (#9662), cleaved caspase 3 (#9661), pERK (T202, Y204; #5683), pStat1 (Y701; #7649), pStat1 (S727; #8826), pStat3 (Y705; #9145), pStat3 (S727; #9134), Stat1 (#9176), Stat3 (#9139), as well as anti-mouse and rabbit IgG horseradish peroxidase (HRP)-linked antibodies were purchased form Cell Signaling Technologies. Anti-vinculin (SC-73614), anti-p38 (SC-7972), and anti-ERK (SC-135900) were from Santa Cruz Biotechnology.

#### *4.2. Cell Culture*

Human breast- (MCF-7, MDA-MB-231) and colon (HCT116 WT, HCT116 p53-/-, HT-29) cancer cell lines, as well as HFF were maintained in Dulbecco's modified Eagle medium (DMEM)-GlutaMax (Gibco, Darmstadt, Germany) supplemented with 10% (*v*/*v*) FCS (Gibco) and 1% penicillin/streptomycin (*v*/*v*) (Gibco) and they were incubated under 5% CO2 and at 37 ◦C. All of the treatments were performed in the same media at the indicated conditions.

### *4.3. Cell Viability Assay*

The SRB assay was employed to measure the inhibitory effects of MC5, MC6, and MC7 on the proliferation of HCT116 WT, HCT116 p53-/-, HT-29, MCF-7, and MDA-MB-231, as well as the influence of anti-oxidants (GSH and Mito TEMPO) and p38 knock-down on the toxicity of MC6-treated HCT116 CRC cells. Cells were seeded in either 96-well plates or 24-well plates at a density of 10,000 and 60,000 cells/well, respectively. At the end of treatments, plates were fixed with 10% ice-cold trichloroacetic acid (TCA), followed by incubation at 4 ◦C for at least one hour. Afterwards, well contents were washed three times with water and were dried at 60 ◦C. Next, 0.054% SRB sodium salt was added to the wells and incubated at room temperature for 30 mins. Plates were then washed three times with 1% acetic acid and were dried at 60 ◦C. Finally, the SRB dye was dissolved in 10 mM Tris (pH 10.5) and measurement was performed using the Tecan Ultra plate reader (Tecan, Crailsheim, Germany) at 535 nm absorbance wavelength.

#### *4.4. Cell Cycle Analysis*

For analysis of cell cycle phase distribution, 10<sup>6</sup> cells/well were harvested, washed with PBS, then fixed with 70% ice-cold ethanol, and incubated at −20 ◦C overnight. After twice washing with ice-cold PBS (Gibco), cells were treated with 200 μg/mL of RNase (Sigma-Aldrich) for 30 mins at 37 ◦C, followed by 15 min incubation with PI (50 μg/mL) (Sigma-Aldrich) in the dark for nucleic acid staining. Samples were analyzed by FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA) and data analysis was performed using the software, CellQuest™ Pro (Becton Dickinson).

#### *4.5. Intracellular ROS Measurement Using Flow Cytometry (FACS) and Live Cell Imaging*

DHE and MitoSox Red were used for the detection of whole cell- and mitochondrial superoxide generation, respectively. For DHE staining, the HCT116 cells were seeded in 12-well plates at a density of 200,000 cells/well. After the indicated treatments, cells were incubated with phenol red-free DMEM containing 15 μM of DHE for 20 mins, harvested, and resuspended in fresh media. For MitoSox Red staining, 60,000 cells/well were seeded in 24-well plates. At the time points indicated, media were replaced with phenol red-free DMEM containing 5 μM of MitoSox Red for 15 mins. Cells were then harvested and resuspended in fresh media. FACS analysis was immediately performed using Guava easyCyte HT sampling flow cytometer (Guava Technologies, Hayward, CA, USA) and data were analyzed using GuavaSoft 3.1.1 software.

Live cell imaging of mitochondrial ROS production was performed using the Incucyte ZOOM live cell analysis system (Essen BioScience, Broadwater Road Welwyn Garden City, United Kingdom). HCT116 cells were seeded in a 24-well plate at a density of 60,000 cells/well. On the following day, the cells were first incubated with 2 nM of MitoTracker Green dissolved in FCS- and pheno red-free

DMEM for 20 mins, after which with fresh media containing 5 μM of MitoSox Red as well as the treatments. Time-lapse images were then taken every 30 mins for a period of 24 h. Data were analyzed using the Incucyte software 2016b. Briefly, the signal intensity was calculated based on a fluorescent area mask; with a top hat filter being used for excluding dead cells due to the higher auto-fluorescence. Nine pictures/well were used to determine the overall signal/well and each condition was performed in quadruplicates.

#### *4.6. Fluorescence Microscopy*

For assessing mitochondrial localization of naphthalimide-NHC analogues, HCT116 cells were cultured in 12-well plates at a density of 150,000 cells/well. After the indicated treatments, the cells were washed with PBS and were incubated with 2 nM of MitoTracker Green dissolved in FCSand phenol red-free media for 20 mins in order to stain mitochondria. Cells were then washed with PBS and were imaged using the BIOREVO fluorescence microscope (BZ9000, KEYENCE; Neu-Isenburg, Germany).

### *4.7. Mitochondrial Membrane Potential Assessment*

200,000 cells/well were seeded in 12-well plates. Treated cells were harvested and then incubated with phenol red-free DMEM containing 2 μM of the JC-1 dye for 15 mins in the dark. After resuspension in fresh media, FACS analysis was performed by Guava easyCyte HT sampling flow cytometer using GuavaSoft 3.1.1 software for data analysis.

#### *4.8. RNA Isolation, Reverse Transcription, and Quantitative Real Time (qRT) PCR*

At the end of treatments, total RNA was isolated using QIAzol lysis reagent (Qiagen, Hilden, Germany) and the quality of RNA samples was determined by NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Darmstadt, Germany). cDNA synthesis was performed from 500–1000 ng of total RNA using ProtoScript II first strand cDNA synthesis kit (New England Biolabs, Frankfurt am Main, Germany), according to the manufacturer's instructions. The thermal cycler q-Tower (Analytik Jena AG, Jena, Germany) was used to analyze gene expression levels. The amplification reaction solutions (5 μL) were prepared with 2.5× of ready to use master mix LightCycler® 480 SYBR Green I (Roche, Mannheim, Germany), 1× of nuclease-free H2O and cDNA templates, as well as 1× of the following primer mixtures (Eurofins Genomics, Ebersberg, Germany): *CDKN1A* (5s: GACACCACTGGAGGGTGACT; 3as: CAGGTCCACATGGTCTTCCT), *Bax* (5s: GGG GACGAACTGGACAGTAA; 3as: CAGTTGAAGTTGCCGTCAGA), *Bad* (5s: GGTTCTGAGGGGAG ACTGAGG; 3as: GCTTCCTCTCCCACCGTAGC, *ATF2* (5 s: CAGCGTTTTACCAACGAGGA; 3as: GA ATCTTGTTGGTGTTGGGGTC), *Stat1* (5 s: GGAAAAGCAAGCGTAA TCTTCAGG; 3as: GAATATTCCCCGACTGAGCC), and *vinculin* (as reference gene) (5s-CAGTCAGACCCTTACTCA GTG-3 ; 3as-CAGCCTCATCGAAGGTAAGGA).

### *4.9. Immunoblotting*

After the respective treatments, cells were lysed using a urea-based lysis buffer supplemented with multiple phosphatase/protease inhibitors, namely sodium orthovanadate, aprotinin, PMSF, pepstatin, and sodium pyrophosphate. Protein concentration was determined with Bradford reagent (Sigma-Aldrich). 20–50 μg of total protein was separated on 10% SDS-PAGE, then transferred onto a PVDF membrane (GE Healthcare, Munich, Germany), after which it was blocked with 5% (*w*/*v*) milk in TBST (Tris-Buffered Saline Tween-20) for at least 1 h. Membranes were then incubated with primary antibody solutions (diluted following the manufacturer's instructions) overnight at 4 ◦C and were visualized by further incubation with the corresponding HRP-linked secondary antibodies (diluted at 1:5000 in 5% (*w*/*v*) milk TBST) for 1 h at room temperature, followed by three washing steps with TBST. Target proteins were finally detected by Western Lightning™ Plus ECl (Perkin Elmer, Waltham, USA) using the Fujifilm LAS-3000 imaging system and were quantified with ImageJ software.

### *4.10. Protein ELISA-Microarray Analysis*

We have previously reported a detailed protocol on the assay [29]. Briefly, treated cells were lysed in a similar manner to that of immunoblotting. Proteins were then diluted 1:6 in a PBS-based buffer (containing 1 mM EDTA, 0.5% (*v*/*v*) Triton X-100, and 5 mM NaF). Protein concentration was assessed using the Pierce BCA Protein Assay kit (Thermo Fischer). The levels of phosphorylated ERK1/2 and JNK were quantified using sandwich ELISA microarrays that were based on the ArrayStrip platform (Alere Technologies GmbH, Jena, Germany). Signals were detected by the Arraymate reader (Alere Technology GmbH). Data were analyzed using the KOMA software [39] and were normalized to the amount of total proteins used.

#### *4.11. siRNA-Mediated Knock Down*

siRNA oligos against *p38α* (*MAPK14*) as well as the non-targeting siRNA were obtained from Thermo Fischer. HCT116 cells were transfected with 40 pmol of the respective siRNA diluted in 100 μL/well Opti-MEM Reduced Serum Medium (Gibco) and complexed with 1.5 μL/well of Lipofectamine 3000 in a 24-well plate and they were incubated for 20 mins at room temperature. Cell suspension was then added at a density of 60,000 cells/well and treatments were performed on the following day, as indicated.

#### *4.12. Cell Death Analysis (AV/PI Staining)*

AV- and PI staining were performed in parallel for the detection of early apoptotic and late apoptotic/necrotic cells, respectively. 250,000 cells/well of the non-transfected HCT116, HCT116 cells transfected with either a negative control- or anti-*p38α* siRNA, as well as HCT116 cells in the absence/presence of p38α pharmacological inhibitors were harvested, washed with AV binding buffer (BD Biosciences, Heidelberg, Germany), and then resuspended in 50 μL binding buffer containing 5 μL of both FITC-conjugated AV and PI (BD Biosciences). Samples were incubated for 15 mins in the dark, after which they were resuspended in binding buffer up to 500 μL for FACS analysis, which was done using Guava easyCyte HT sampling flow cytometer. Data were analyzed by GuavaSoft 3.1.1 software.

#### *4.13. Caspase 3 Activation Assay*

At the end of the treatments, 60,000 cells/wells were harvested, fixed, and permeabilized with 4% paraformaldehyde and 90% ice-cold methanol, respectively, as previously described [40]. Cells were then incubated overnight with cleaved caspase 3 antibody at a dilution of 1:800 and they were further incubated with secondary antibody (goat anti-rabbit Alexafluor 488-conjugated; Dianova, Hamburg, Germany) for 1 h at room temperature, thereafter analyzed using Guava easyCyte HT sampling flow cytometer and the GuavaSoft 3.1.1 software.

#### *4.14. TUNEL Assay*

TUNEL assay was performed for the detection of DNA fragmentation as a characteristic of late stage apoptosis using Cell Meter TUNEL Apoptosis Assay Kit (Biomol GmbH, Hamburg, Germany). Briefly, 60,000 cells/well were harvested, fixed, and permeabilized by 4% paraformaldehyde and 0.2% Triton X-100, respectively. After washing with PBS, cells were incubated with the tunnelyte reaction mixture for 1 h at 37 ◦C, thereafter the fluorescence intensity was measured by the Tecan Ultra plate reader (Tecan) at an excitation/emission wavelength of 550/650 nm, respectively. Additionally, the cells were analyzed using the BIOREVO Fluorescence microscope (BZ9000, KEYENCE).

#### *4.15. Statistical Analyses*

Data were analyzed using GraphPad Prism and Microsoft Excel. Densitometric analyses were performed by the ImageJ software. Statistical significance between the respective treatment and DMSO was determined by student's *t*-test. Multiple comparisons were performed using either one-way or two-way ANOVA, followed by a post-hoc Tukey test as indicated. *p*-values less than or equal to 0.05, 0.01, 0.001, and 0.0001 are presented as \*, \*\*, \*\*\*, and \*\*\* on the figures, respectively.
