Supplementary Figures 1 - 5 from miR-29a Is Repressed by MYC in Pancreatic Cancer and Its Restoration Drives Tumor-Suppressive Effects via Downregulation of LOXL2

S1.(A) cBioportal analysis of genetic alterations in putative pri-miR-29a/b1 promoter binding transcription factors in CCLE datasets of pancreatic cancer cells. (B) Regression plot indicating negative correlation between miR-29a and MYC expressions in CCLE pancreatic cancer cell lines (CFPAC-1, Capan-1, HUP-T3, KP4, L3_3, PSN1, Panc_02_03, and Panc_08_13) showing MYC alterations. (C) Relative luciferase activity in MIA PaCa-2 cell line. (D) Real-time PCR analysis of DNA fragments precipitated in a CHIP assay using MIA PaCa-2 cell line. S2. (A) Volcano plot for differentially expressed genes in miR-29a overexpressing (OE) and control PaCa-2 cells. (B) Venn diagram of upregulated transcripts in Panc-1 and/or MIA Paca-2 datasets. (C) Genes among the 20 most differentially expressed overlapping genes in Panc-1 and MIA PaCa-2 datasets belonging to one or more of the four tested GO categories. Highlighted genes are predicted miR-29a targets. S3. (A) Western Blot for miR-29a target genes in MIA PaCa-2 cells transfected with different concentrations (5nM, 10nM and 20nM) of control (CTRL) or miR-29a mimics. (B) Migration assays for MIA PaCa-2 cells transfected with siCTRL, siMYBL2, siLOXL2, siCLDN1, siHGK, and siNRAS, or CTRL and miR-29a mimic. S4. (A)Relative luciferase activityin MIA PaCa-2 cell line.(B)Relative miR-29a expressions in Panc-1 and MIA PaCa-2 cells transfected with siLOXL2. (C) Western Blot of LOXL2 and MYC in MIA PaCa-2 cells transfected with siCTRLor siMYC. (D) MIA PaCa-2 cells transfected with CTRL or miR-29a mimic, and siCTRLand siLOXL2 were cultured in serum free media for 48 hrs. Conditioned media (CM) thus obtained were subjected to ELISA for detection of secreted LOXL2. (E) Newly crosslinked pepsin-soluble collagen in ECM of MIA PaCa-2 cells transfected with CTRL or miR-29a mimic, and siCTRLand siLOXL2. (F)Hydroxyproline content representing heavily cross-linked insoluble collagen in ECM of transfected MIA PaCa-2 cells. S5. Correlation analysis between expressions of miR-29a and its target genes (CLDN1, HGK, NRAS and MYBL2) in PDAC patients from the TCGA database.