*2.1. MC5, MC6, and MC7 Exhibit Cytotoxic Effects in Tumor Cells of Different Tissue Origins*

To determine the cytotoxicity of the naphthalimide-NHC complexes (Figure 1A) in different cancer models, the cell lines of breast-(MCF-7 and MDA-MB-231) and colorectal (HCT116) tissue origins were treated with increasing concentrations of the compounds for three incubation periods (24, 48, and 72 h), after which Sulforhodamine B (SRB) assay was performed. The metal-free naphthalimide ligand—MC5—as well as the rapid apoptosis inducer, raptinal [16] were included as references. In all of the investigated cell lines, cellular survival was found to decrease time- and concentration-dependently in response to the three compounds, with HCT116 cells showing the highest sensitivity to metal-containing analogues in the first 24 h (Figure 1B). After 48 and 72 h of incubation with MC6 and MC7, cell viabilities became comparable among MCF-7 and HCT116 cells, while they remained substantially higher in the case of MDA-MB-231 at most of the tested concentrations (Figure 1B). Moreover, MC5 was found to be particularly active against MCF-7 breast cancer cells, but not in HCT116 and MDA-MB-231. This is in good agreement with the previous report, showing significantly lower IC50 values of metal-free naphthalimide species in MCF-7 as compared to that of HT-29 CRC cells [15]. Notably, the compounds exhibited extremely low cytotoxic effects in HFF cells, even at the highest tested concentrations, which might suggest the preferential toxicity of the naphthalimide-NHC conjugates towards cancer cells (Figure S1).

We also tested the cytotoxicity of the three analogues in the context of p53-deficiency or mutant p53 using the p53-null HCT116 and HT-29 cell lines, respectively. As shown in Figure S2, mutant p53 harboring HT-29 cells are less sensitive to the cytotoxic effects of the compounds at most tested concentrations and time points.

**Figure 1.** Naphthalimide-*N*-heterocyclic carbene (NHC) analogues exhibit cytotoxic effects against human breast- and colon cancer cells. (**A**) Chemical structures of the compounds; (**B**) Increasing concentrations of each of the complexes, as well as the rapid apoptosis inducer, raptinal [16] (as positive control) were applied to the different cell lines and Sulforhodamine B (SRB) assay was performed after 24, 48, and 72 h of treatment. The Ru(II)- and Rh(I)-containing complexes show the highest and the least efficacy against HCT116 and MDA-MB-231, respectively, in most tested concentrations. 0.1% DMSO-treated cells served as mock. Data represent mean ± SD of three independent experiments, each was done in quadruplicates.

#### *2.2. MC5, MC6, and MC7 Inhibit Cell Cycle Progression in HCT116 CRC Cells*

To shed light on the mechanism that is responsible for the inhibitory activity of the compounds on cellular viability, we sought to assess changes in the cell cycle regulation. HCT116 cell line was selected for further investigation based on its higher susceptibility to the MC6- and MC7-mediated cytotoxic effects (Figure 1B). A 24 h post-treatment analysis of the DNA content revealed a G1 arrest in response to treatments (Figure 2A). Although all three compounds caused a significant increase in the G1 phase cell population as compared to mock (0.1% DMSO), this effect was found to be more pronounced in the case of the Rh(I) analogue (MC7), followed by the metal-free ligand (MC5), and finally the Ru(II) complex (MC6) (Figure 2B).

Additionally, we examined the mRNA levels and protein expression of p21, an inhibitor of the complexes of cyclin D and cyclin-dependent kinases (CDKs), which have a key role in the G1 to S phase transition [17]. As expected, p21 mRNA and protein levels were induced after 24 h of treatment, with no statistically significant difference being found across the three compounds (Figure 2C–E).

**Figure 2.** Naphthalimide-NHC analogues induce cell cycle arrest and p21 expression in HCT116 CRC cells. (**A**) Representative histogram plots show the distribution of cell cycle phases in HCT116 cells treated with either 0.1% DMSO (as mock) or the three complexes, MC5, MC6, and MC7 at a concentration of 50, 12, and 25 μM, respectively for 24 h; (**B**) All of the analogues were found to induce a G1 phase arrest as compared to mock treatment. Comparison of the percentage cell population of G1, S, and G2/M phases between mock and each of the three complexes was performed by two-tailed student's *t*-test. Error bars represent the SD of two biological replicates, one of which is depicted in (**A**); (**C**) p21 mRNA levels are up-regulated in response to 24 h of treatments, analyzed by qRT-PCR. Relative expression was calculated using the ΔΔ Ct method where the Ct values of p21 were normalized to those of the housekeeping gene (vinculin). Lower and upper ends of the bars indicate the minimum and maximum values, respectively, and the "+" in the middle represents the mean. Error bars ± SD; *n* = 4; (**D**) p21 protein levels upon 24 h of treatment with the three complexes at the indicated concentrations, determined by immunoblotting; (**E**) Densitometric quantification of p21 bands normalized to those of the loading control (vinculin). Error bars indicate the SEM of two biological replicates, one of which is presented in (**D**). Multiple comparisons were made using one-way ANOVA test and a post-hoc Tukey test. \*, \*\*, \*\*\*, and \*\*\*\* denote *p*-values less than or equal to 0.05, 0.01, 0.001, and 0.0001, respectively.
