Additional file 1 of Upregulated expression of miR-4443 and miR-4488 in drug resistant melanomas promotes migratory and invasive phenotypes through downregulation of intermediate filament nestin
Creators: name: Castaldo, Vittorio
name: Minopoli, Michele
name: Di Modugno, Francesca
name: Sacconi, Andrea
name: Liguoro, Domenico
name: Frigerio, Rachele
name: Ortolano, Arianna
name: Di Martile, Marta
name: Gesualdi, Luisa
name: Madonna, Gabriele
name: Capone, Mariaelena
name: Cirombella, Roberto
name: Catizone, Angiolina
name: Del Bufalo, Donatella
name: Vecchione, Andrea
name: Carriero, Maria Vincenza
name: Ascierto, Paolo Antonio
name: Mancini, Rita
name: Fattore, Luigi
name: Ciliberto, Gennaro
Publisher: name: figshare
Year: 2024
Subjects: subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology
subject: Computational Biology

description: Additional file 1: Suppl. Fig. 1. a. A375 sensitive cells (SEN) and their resistant counterparts (RES) were plated in a 96-wells plate. The number of viable cells was measured by quantification of the cellular ATP present at different time points (from 24 to 96 hours). Results were calculated as fold change (± SD) relative to the time point of 24 hours. b, c. To perform migration assays, LOX IMVI SEN and RES cells or A375 SEN and DRES cells (double resistant to BRAFi and MEKi) were seeded in serum-free media into the upper chamber of a transwell, whereas the lower chamber was filled with 10% FBS RPMI. After 8 hours, cells remaining on the top side of the membrane were counted and the average number ± SD of cells are reported as fold change respect to control considered as 100. d, e. To perform invasion assays, LOX IMVI SEN and RES cells or A375 SEN and DRES cells were seeded into the upper chamber of a transwell coated with Matrigel, whereas the lower chamber was filled with 10% FBS RPMI. After 24 hours, invading cells were counted and the average number ± SD of cells are reported as fold change respect to control considered as 100. Three independent experiments were performed; each experiment was performed at least in quadruplicate. All the experiments have been performed at least in triplicate ± standard deviation (SD) and p-value &lt; 0.05 was considered as significant (Student’s t-test). Suppl. Fig. 2. a-d. xCELLigence RTCA technology was used to measure cell migration and invasion of LOX IMVI SEN and RES cells or A375 SEN and DRES cells (double resistant to BRAFi and MEKi). To evaluate migration (a, c), cells were seeded in serum-free medium on filters in the upper chamber, whereas the lower chamber was filled with 10% FBS RPMI. Cell migration was monitored for 8 hours. To evaluate invasion (b, d), cells were seeded in serum-free medium on filters coated with Matrigel in the upper chamber, whereas the lower chamber was filled with 10% FBS RPMI. Cell invasion was monitored for 18 hours. Each experiment was performed at least twice in quadruplicate. The slope represents the change rate of cell index values generated in a 0–1 hour time frame. All the experiments have been performed at least in triplicate. Suppl. Fig. 3. Representative images by confocal microscopy of F-Actin and DAPI immunostaining of A375 SEN and DRES cells (double resistant to BRAFi and MEKi) that were cultured into 8-well μ-slides for 48 hours (left panels). Magnification 63x. Quantification analyses of the long cell axis measured from the aforementioned confocal images were performed by Zeiss Zen control software (right graph). All the experiments have been performed at least in triplicate ± standard deviation (SD) and p-value &lt; 0.05 was considered as significant (Student’s t-test). Suppl. Fig. 4. a. Total RNAs were extracted from LOX IMVI sensitive cells (SEN) and their resistant counterparts (RES) to perform qRT-PCR analyses for miR-4443 and miR-4488 expression levels. Each miRNA transcript was quantified using the comparative Ct method for relative quantification (ΔΔCt) using U6 as normalizer. The values were calculated as “fold change” (± SD) compared to SEN considered as 1. b, c. A375 SEN and LOX IMVI SEN cells were transiently transfected with miR-4443, miR-4488 or the relative scrambled (SCR) for 48 hours. Cells have been then harvested and the expression levels of miR-4443 and miR-4488 was determined as described above. d. To perform migration assays, LOX IMVI SEN cells, transfected as described above, were seeded in serum-free media into the upper chamber of a transwell, whereas the lower chamber was filled with 10% FBS RPMI. After 8 hours, cells remaining on the top side of the membrane were counted and the average number ± SD of cells are reported as fold change respect to control (SCR) considered as 100. e. To perform invasion assays, LOX IMVI SEN cells, transfected as described above, were seeded into the upper chamber of a transwell coated with Matrigel, whereas the lower chamber was filled with 10% FBS RPMI. After 24 hours, invading cells were counted and the average number ± SD of cells are reported as fold change respect to control (SCR) considered as 100. All the experiments have been performed at least in triplicate ± standard deviation (SD) and p-value &lt; 0.05 was considered as significant (Student’s t-test). Suppl. Fig. 5. a. LOX IMVI SEN cells were transiently transfected with miR-4443, miR-4488 or with the SCR for 72 hours and then cells have been stained with an antibody PE mouse anti-nestin for 45 minutes to perform flow cytometry analyses. Non-stained cells were used as negative control. Black arrows indicate the peaks relative to cells transfected with miR-4443 or miR-4488 mimics (left graphs). Quantitative analyses were calculated as “fold change” (± SD.) compared to SCR considered as 1 (right graph). Data were analyzed using CytExpert version 2.2 software. b. LOX IMVI SEN cells, transfected as described above, have been harvested and lysed to extract total proteins that have been subjected to Western blot analyses with the indicated antibodies. Tubulin levels have been estimated for the protein equal loading. c. LOX IMVI SEN and RES cells were subjected to Western blot analyses as described above. Tubulin levels have been estimated for the protein equal loading. d. LOX IMVI SEN and RES cells were subjected to flow cytometry analyses as described above. Non-stained cells were used as negative control. Black arrow indicates the peaks relative to RES cells (left graph). Quantitative analyses were calculated as “fold change” (± S.D.) compared to SEN cells considered as 1 (right panel). Data were analyzed using CytExpert version 2.2 software. All the experiments have been performed at least in triplicate ± standard deviation (SD) and p-value &lt; 0.05 was considered as significant (Student’s t-test). Suppl. Fig. 6. a. A375 SEN, RES and DRES cells (double resistant to BRAFi and MEKi) were harvested and lysed to extract total proteins to perform Western blot analyses with the indicated antibodies. Tubulin levels have been estimated for the protein equal loading. b. Representative images by confocal microscopy of phalloidin, nestin and DAPI immunostaining of A375 SEN and DRES cells that were cultured into 8-well μ-slides for 48 hours. Magnification 63x. Suppl. Fig. 7. a. The online software GEPIA 2.0 (http://gepia2.cancer-pku.cn/#index) was interrogated to unveil nestin prognostic value based on Skin Cutaneous Melanoma (SKCM) data from The Cancer Genome Atlas (TCGA). Patients have been stratified according to the mutational status and the Kaplan Meier (KM) curves represent the data relative to the BRAF-wild type subtypes (n=164). b. Nestin levels (NES) were evaluated by immunohistochemistry (IHC) in 14 melanoma patients’ biopsies collected before starting MAPKi therapy together with the expression levels of miR-4443 and miR-4488 (miRNAs) quantified by qRT-PCR. Data have been used to plot KM curves to assess the predictive value of NES+miRNAs for therapy response (internal validation cohort). High and low levels were assessed by considering positive and negative z-scores, respectively. The hazard ratio, Cox models and the log-rank p values were evaluated to plot KM curves.