Additional file 1 of Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy
Creators: name: Alcalá, Sonia
name: Villarino, Lara
name: Ruiz-Cañas, Laura
name: Couceiro, José R.
name: Martínez-Calvo, Miguel
name: Palencia-Campos, Adrián
name: Navarro, Diego
name: Cabezas-Sainz, Pablo
name: Rodriguez-Arabaolaza, Iker
name: Cordero-Barreal, Alfonso
name: Trilla-Fuertes, Lucia
name: Rubiolo, Juan A.
name: Batres-Ramos, Sandra
name: Vallespinos, Mireia
name: González-Páramos, Cristina
name: Rodríguez, Jéssica
name: Gámez-Pozo, Angelo
name: Vara, Juan Ángel Fresno
name: Fernández, Sara Fra
name: Berlinches, Amparo Benito
name: Moreno-Mata, Nicolás
name: Redondo, Ana María Torres
name: Carrato, Alfredo
name: Hermann, Patrick C.
name: Sánchez, Laura
name: Torrente, Susana
name: Fernández-Moreno, Miguel Ángel
name: Mascareñas, José L.
name: Sainz, Bruno
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Figure S1. NMR studies of the aquation of [Ru(terpy)(bpy)Cl]Cl complex (Ru0) using deuterium oxide. (A) 1H-NMR of [Ru(terpy)(bpy)Cl]Cl complex (Ru0) in water at t= 0. (B) 1H-NMR of [Ru(terpy)(bpy) H2O]+2Cl-2 complex (Ru1) in water. (C) 1H-NMR of [Ru(terpy)(bpy)Cl]Cl complex (Ru0) in water at different times (starting concentration of Ru0 = 2mM). (D) 1H-NMR of [Ru(terpy)(bpy)Cl]Cl complex (Ru0) after dissolving in water (t = 0 min) and after irradiation with visible light for 60-120 min (starting concentration of Ru0= 2mM). Figure S2. Analysis of Ru1 toxicity in vivo. (A-C) Average values ± SEM of indicated hematocrit parameters determined from blood of mice extracted 2h (A), 4h (B), or 8h (C) post treatment with diluent control (Ctl) or Ru1(1.4mg/kg, r.o). No significant differences were found, as determined by unpaired two-sided Student’s t-test. (D) Picomoles of Ru1 per mg of tumor, determined by ICP-MS, from tumors extracted at indicated time points post treatment initiation. Dashed line indicates the background of the assay. Figure S3. Ru1 negatively regulates MT-encoded genes. (A) Table summarizing the 14 mtDNA-encoded genes modulated in PancA6L spheres treated with Ru1(100μM, 24 h) compared to untreated Controls. Shown are the gene name, description, Log2 fold change, p value and p adjusted (adj). (B) Mean ± SD of normalized Fragments Per Kilobase Million (FPKM) values for the indicated target genes in Ctl-, Ru1and Ru1-met-treated Panc185 or PancA6L spheres. (ns=not significant, as determined by one-way ANOVA with Dunnett post-test, compared to Control). Figure S4. Ru1 affects PaCSC oxygen consumption and mitochondrial properties and morphology. (A) Measured and calculated mean ± SD oxygen consumption rate (OCR) parameters (Resp = Respiration; Max = Maximum; SRC = Spare Respiratory Capacity; OC =Oxygen consumption) in Ctl- and Ru1-treated PancA6L or Panc215 spheres (n = 3 biological replicates with 3 readings). * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001, **** p &lt; 0.0001, ns = not significant, as determined by unpaired two-sided Student’s t-test. (B) Mean fold-change ± SD in lactate (mM/total protein) in untreated (-), Ru1(100μM) or Ru1-met (100μM)-treated Panc185 and PancA6L cells compared to control, set as 1.0. *** p &lt; 0.001, ns = not significant, as determined by one-way ANOVA with Dunnett post-test, compared to Control. (C) Representative IF confocal images of TMRE (mitochondria membrane potential) or CellROX DeepRed (ROS) staining in untreated (Control), Ru1(100μM) or Ru1-met(100 μM)-treated PancA6L cells (24 h). (D) Representative fluorescence confocal images of MitoGreen (mitochondrial mass) and DAPI (Blue) staining in Ru1(100μM)- treated PancA6L cells (24 h). Scale bar = 10 μM. (E) Representative transmission electron micrographs of Control (untreated) or Ru1(100μM)-treated Panc185 cells (24 h). Mitochondria are better defined in the Control compared to Ru1-treated samples. (F) Amount of Ru1 molecules per 1000bp of DNA, determined by ICP, in mtDNA isolated from gradient-purified mitochondria from untreated (CTL) or Ru1-treated (100μM, 2 h) Panc185 cells. Figure S5. GQ in the mtDNA determined with G4 hunter. Figure S6. Ru1 inhibits mtDNA transcription. (A) Sequence of 9 predicted GQs, their positions and G4Hunter score indicating G4-prone structures. Isolated guanines (G) are shown in red, and cysteines (C) in blue. (B) Mean fold-change ± SD in the relative mRNA expression of the indicated mtDNA- and nuclear-encoded genes as a function of increasing concentrations of Ru1 in Panc185 or PancA6L cells (48h treatment). Values were normalized to ß-actin levels. * p &lt; 0.05, ** p &lt; 0.01, *** p &lt; 0.001, as determined by one-way ANOVA with Dunnett post-test, compared to untreated (Ctl). Table S1. Antibodies. Table S2. RTqPCR primer sequences. Table S3. PCR primer sequences.