Titles and Legends to Figures

Titles and legends to figures

Figure 1.miRNA expressions in gastrointestinal stromal tumor (GIST) samples.(A)Dendrogram based on unsupervised clustering of miRNA expression profiles by microarray in 17 GISTs. (B) Box plots illustrate the relative expression levels of miR-125a-5p and miR-107 in 14 imatinib-resistant and 16 imatinib-sensitive GIST specimens regardless of KIT/PDGFRA mutational statusby RT-qPCR after normalization to RNU6B. (C) Box plots show the relative expression levels of miR-125a-5p, miR-107,miR-134, miR-301a-3p andmiR-365in 4 imatinib-resistant and 9imatinib-sensitive tumors among GISTs carrying a single KIT mutation. (D)RelativemiR-125a-5p levels in GIST882 (imatinib-sensitive) and GIST48 (imatinib-resistant) cell lines. Differences between groups were calculated using unpaired student’s t-test and p ≤ 0.05 were considered significant. Error bars refer to standard deviation between replicates.

Figure 2.Effects of miR-125a-5p modulation on imatinib response and candidate targets in GIST cells.(A) Proportion of surviving cells in cultures transfected with pre-miR-125a-5p, anti-miR-125a-5p or their respective negative controls after 24 hours of treatment withdifferent concentrations of imatinib, as evaluated by WST-1 assay. (B) Western blot analysis of miR-125a-5p candidate targets in GIST882 cells treated withpre-miR-125a-5p or pre-miR-CTR.Western blots show the protein expressions of PTPN18 and STARD13 in three independent transfection experiments. Bar graphs showing the fold change of protein levels relative to pre-miR-CTR treated cells after normalization to GAPDH. Data represent the mean of three independent experiments, and the error bars refer to standard deviation from the mean.Statistical differences between the two groups were calculated using paired student’s t-test and p ≤ 0.05 was considered significant. FC = Fold Change.

Figure 3.Quantification of miR-125a-5p, PTPN18 and STARD13 expression levels in GIST882 parental cells and its imatinib-resistant subclone (GIST882R).(A) Quantification of miR-125a-5p, PTPN18 and STARD13 expressions by RT-qPCR after normalization to RNU6B or 18S rRNA. (B) Quantification of PTPN18 and STARD13 protein expression levels by Western blot analysis. GAPDH was used as normalization control. Data indicate mean values of three independent replicates and error bars refer to standard deviation between replicates. Statistical differences between the two groups were calculated using paired student’s t-test and p ≤ 0.05 was considered significant.

Figure 4.Quantification of PTPN18 and STARD13 protein expression levels in GIST clinical samples by Western blot analysis.(A) Western blot images for PTPN18 and STARD13 (160 kDa and 125 kDa) in clinical samples. Bar graphs showing the protein levels after normalization to GAPDH. (B)Correlationof protein expressions and miR-125a-5p expression levels in imatinib resistant and sensitive GISTs. r = Correlation coefficient. Statistical differences between the two groups were calculated using paired student’s t-test, and the expression correlation was evaluated by Pearson`s correlation analysis.p ≤ 0.05 were considered significant.

Figure 5.Effect of PTPN18 silencing on imatinib response in GIST882 cells.Proportion of surviving cells in cultures transfected with short hairpin RNAs against PTPN18 (shPTPN18) or shRNA control plasmid (shControl) after 72 hours of treatment with different concentrations of imatinib, as evaluated by WST-1 assay. Statistical differences between the two groups were calculated using paired student’s t-test and p ≤ 0.05 was considered significant.

Figure 6.RT-qPCR analysis of miRNA expressions in relation to metastasis, KIT mutational status and survival in GISTs. Box plots show relative expression levels of (A)miR-150-3p and miR-301a-3p in 20 cases of metastatic and 10 non-metastatic GISTs, and (B) miR-150-3pin 13single and 11 double KIT mutated tumors. Comparisons between groups were calculated using unpaired student`s t-test and p ≤ 0.05 were considered significant. (C) Kaplan-Meier curves show significant association of miR-1915 expression with disease-free survival and overall survival in GIST patients. Differences in survival were calculated using log-rank test. High and low expressions refer to above and below the median level of all tumors.

Supplementary figure legends

Supplementary Figure S1. Measurement of transfection efficacies by RT-qPCR. (A) miR-125a-5p expression levels in GIST882 and GIST48 cell lines upon overexpression and/or inhibition of miR-125a-5p. (B)PTPN18 expression level in GIST882 cells upon transfection with short hairpin against PTPN18 (shPTPN18) or control plasmid (shControl). Left:PTPN18 mRNAlevel was measured by RT-qPCR after normalization to 18S rRNA.Right:Western blot showing PTPN18 protein level in cellswith and without silencing of PTPN18. GAPDH was used as loading control.Data represent mean of three independent experiments. Error bars refer to standard deviation of triplicates.FC=fold change.

Supplementary Figure S2. Unsupervised clustering analysis of miRNA expression in 17 GIST specimens. Tumors were clustered based on Pearson correlation and complete linkage. Red and green colors indicate relatively high and low expression, respectively. Missing values are indicated in grey.

Supplementary Figure S3. RelativemiR-125a-5p and miR-107 expression levels in resistant and sensitive tumors of the patients with multiple tumors (GIST9 and GIST10). Tumors 9-3 and 10-3 were sensitive to imatinib, and the remaining tumors were imatinib-resistant.

Supplementary Figure S4. RelativemiRNA expression levels in GIST882 (imatinib-sensitive) and GIST48 (imatinib-resistant) cell lines. Differences between cell lines were calculated using unpaired student’s t-test and p ≤ 0.05 were considered significant. Error bars refer to standard deviation between independent replicates.

Supplementary Figure S5. Analyses of cell survival by WST-1 assay following overexpressionof miR-211 (top) and miR-944 (bottom) in GIST882 cells. The proportion of surviving cells is shown for cultures transfected with pre-miR-211 or -944 compared to pre-miR-negative control after 24 hours of treatment with different concentrations of imatinib. Statistical differences between groups were calculated using paired student’s t-test and p≤ 0.05 was considered significant.