Post-transcriptional regulation of aortic calcification in KLOTHO deficient mice: impact of miR-145 and miR-378 Ying Tang1, *, Tapan A. Shah1, *, †, Edward J. Yurkow2, and Melissa B. Rogers1, ‡ Fig. S1. KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of differentially expressed miRNAs in male Klotho homozygous mutant mice. Fig. S2. KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of differentially expressed miRNAs in female Klotho homozygous mutant mice. Fig. S3. Gene Ontology (GO) analyses of differentially expressed miRNAs in male Klotho homozygous mutant mice. Fig. S4. Gene Ontology (GO) analyses of differentially expressed miRNAs in female Klotho homozygous mutant mice. Fig. S5. Interaction network of miRNAs down-regulated in male Klotho homozygotes relative to healthy controls. Fig. S6. Interaction network of miRNAs down-regulated in female Klotho homozygotes relative to healthy controls. Fig. S7. Experimental Controls. Table S1. Average miRNA abundance in aorta from Klotho mutant homozygotes relative to healthy control (p<0.05). Table S2. Genes targeted by selected miRNAs that may influence aortic calcification. Table S3. Known modulators of BMP signaling. Table S4. Klotho wild type and Klotho heterozygous mice are equivalent controls Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta S2 Legend on next page Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta Fig. S1. KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of differentially expressed miRNAs in male Klotho homozygous mutant mice. The genes potentially targeted by up (A) or down-regulated (B) miRNAs were predicted by DIANA TOOLS. Target genes were grouped into gene pathways using the KEGG database. The y-axis represents the name of the KEGG pathway, and the x-axis represents the number of the genes in a certain pathway. Low p adjust values are in red and high p adjust values are in blue. KEGG pathways that are directly related to the osteogenic differentiation that occurs in vascular calcification are marked with an asterisk (*). S3 Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta S4 Legend on next page Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta Fig. S2. KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of differentially expressed miRNAs in female Klotho homozygous mutant mice. The genes potentially targeted by up (A) or down-regulated (B) miRNAs were predicted by DIANA TOOLS. Target genes were grouped into gene pathways using the KEGG database. The y-axis represents the name of the KEGG pathway, and the x-axis represents the number of the genes in a certain pathway. Low p adjust values are in red and high p adjust values are in blue. KEGG pathways that are directly related to the osteogenic differentiation that occurs in vascular calcification are marked with an asterisk (*). S5 Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta S6 Legend on next page Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta Fig. S3. Gene Ontology (GO) analyses of differentially expressed miRNAs in male Klotho homozygous mutant mice. The genes potentially targeted by up-regulated (A) or down-regulated (B) miRNAs were predicted by DIANA TOOLS. Target genes were grouped into GO molecular function terms using the GO database. The y-axis represents the name of the GO molecular function term, and the x-axis represents the number of genes associated with each GO molecular function term. Low p adjust values are in red and high p adjust values are in blue. S7 Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta S8 Legend on next page Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta Fig. S4. Gene Ontology (GO) analyses of differentially expressed miRNAs in female Klotho homozygous mutant mice. The genes potentially targeted by up- regulated (A) or down-regulated (B) miRNAs were predicted by DIANA TOOLS. Target genes were grouped into GO molecular function terms using the GO database. The y- axis represents the name of the GO molecular function term, and the x-axis represents the number of genes associated with each GO molecular function term. Low p adjust values are in red and high p adjust values are in blue. S9 Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta Fig. S5. Interaction network of miRNAs down-regulated in male Klotho homozygotes relative to healthy controls. These miRNAs were down-regulated in diseased Klotho male homozygotes according to microarray measurements and are predicted to target Bmp2 and/or the downstream BMP signaling intermediaries, Smads1, 5, 9. The subset of miRNAs whose abundance decreased in both male and female Klotho homozygotes are in bold. S10 Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta Fig. S6. Interaction network of miRNAs down-regulated in female Klotho homozygotes relative to healthy controls. These miRNAs were down-regulated in diseased Klotho female homozygotes according to microarray measurements and are predicted to target Bmp2 and/or the downstream BMP signaling intermediaries, Smads1, 5, 9. The subset of miRNAs whose abundance decreased in both male and female Klotho homozygotes are in bold. S11 Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta Fig. S7. Experimental Controls. A, B. Grinding tissue with glass beads does not significantly affect calcium or protein yield. Whole aorta was ground in liquid nitrogen either with or without glass beads. Frozen ground powder tissue was split two ways for protein and calcium assays. A. Effect of glass beads on calcium, protein and pSMAD1/5/9 levels. Duplicate measurements are presented with range. Experiments were repeated twice with similar results. B. Representative blots showing pSMAD1/5/9, actin levels and the Ponceau S stained membrane after transfer. The positive control lane (+ Ctrl) was loaded with lysate from MC3T3-E1 cells transfected with a Bmp2 expression plasmid. C, D. Validation of the phospho-SMAD1/5/9 antibody. MC3T3-E1 (C) and C3H10T1/2 (D) cells were transfected with an expression plasmid encoding BMP2 (B2) or luciferase (Luc) (1). Cells were then lysed in RIPA buffer and subjected to western blotting as described in the experimental procedures section. These representative blots show that pSMAD1/5/9 levels were induced in cells transfected with the Bmp2 expressing plasmid relative to the luciferase plasmid. Actin levels and a Ponceau S stained membrane are shown as loading controls. S12 Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta Table S1. Average miRNA abundance in aorta from Klotho mutant homozygotes relative to healthy control (p<0.05). Raw data is available in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/, GSE135759). % Male control Female % control mmu-miR-16-1-3p 1344 mmu-miR-205-5p 15398 mmu-miR-711 1338 mmu-miR-429-3p 2226 mmu-miR-5620-3p 687 mmu-miR-16-1-3p 1331 mmu-miR-466f 630 mmu-miR-141-3p 1061 mmu-miR-466j 585 mmu-miR-205-3p 581 mmu-miR-669m-5p 532 mmu-miR-6359 581 mmu-miR-466m-5p 532 mmu-miR-200a-5p 563 mmu-miR-669b-5p 491 mmu-miR-675-3p 543 mmu-miR-466f-5p 489 mmu-miR-466f-5p 540 mmu-miR-669o-5p 473 mmu-miR-296-3p 507 mmu-miR-195a-3p 470 mmu-miR-1946b 450 mmu-miR-7030-5p 466 mmu-let-7f-1-3p 438 mmu-miR-466h-5p 465 mmu-miR-1947-3p 427 mmu-miR-665-5p 433 mmu-miR-291b-5p 386 mmu-miR-3082-5p 432 mmu-miR-6241 383 mmu-miR-669k-5p 415 mmu-miR-3092-3p 366 mmu-miR-696 415 mmu-miR-6546-3p 350 mmu-miR-467h 414 mmu-miR-183-3p 339 mmu-miR-7654-3p 405 mmu-miR-1946a 333 mmu-miR-669l-5p 398 mmu-miR-669k-5p 331 mmu-miR-8119 378 mmu-miR-18a-3p 316 mmu-miR-5620-5p 369 mmu-miR-1930-3p 312 mmu-miR-669f-5p 366 mmu-miR-489-3p 302 mmu-miR-714 360 mmu-miR-376a-3p 296 mmu-miR-6968-5p 328 mmu-miR-744-5p 294 mmu-miR-2137 327 mmu-miR-539-5p 274 mmu-miR-5130 321 mmu-miR-7047-3p 274 mmu-miR-7023-5p 295 mmu-miR-7049-5p 273 mmu-miR-7016-5p 286 mmu-miR-7079-5p 265 mmu-miR-92b-5p 286 mmu-miR-181c-3p 263 mmu-miR-3547-5p 278 mmu-miR-381-5p 263 mmu-miR-146b-5p 278 mmu-miR-7004-5p 249 mmu-miR-7033-5p 271 mmu-miR-191-3p 246 mmu-miR-6980-5p 271 mmu-let-7b-3p 242 mmu-miR-297a-5p 265 mmu-miR-141-5p 234 mmu-miR-669e-5p 245 mmu-miR-7684-3p 231 S13 Tang et al. Supporting Information, MicroRNA profiles in calcified vs. healthy aorta mmu-miR-669a-5p 243 mmu-miR-7241-5p 227 mmu-miR-669p-5p 243 mmu-miR-5129-3p 227 mmu-miR-7648-3p 237 mmu-miR-6958-5p 225 mmu-miR-1982-5p 236 mmu-miR-7684-5p 223 mmu-miR-15a-3p 224 mmu-miR-203-5p 223 mmu-miR-6971-5p 223 mmu-miR-7669-3p 220 mmu-miR-7005-5p 221 mmu-miR-383-5p 215 mmu-miR-762 220 mmu-miR-7082-3p 209 mmu-miR-7083-5p 220 mmu-miR-17-5p 205 mmu-miR-6912-5p 217 mmu-miR-7682-3p 200 mmu-miR-466c-5p 215 mmu-miR-704 198 mmu-miR-6909-5p 215 mmu-miR-702-3p 197 mmu-miR-574-5p 213 mmu-miR-468-3p 195 mmu-miR-669d-5p 212 mmu-miR-92a-1-5p 194 mmu-miR-1892 209 mmu-miR-8103 191 mmu-miR-1224-5p 209 mmu-miR-7077-5p 188 mmu-miR-7085-5p 208 mmu-miR-7008-5p 186 mmu-miR-346-3p 208 mmu-let-7i-3p 183 mmu-miR-5119 204 mmu-miR-450b-5p 180 mmu-miR-7686-5p 201 mmu-miR-200c-5p 179 mmu-miR-7036-5p 201 mmu-miR-673-3p 176 mmu-miR-6921-5p 197 mmu-miR-6929-3p 167 mmu-miR-6349 197 mmu-miR-3098-5p 167 mmu-miR-8095 195 mmu-miR-7057-3p 167 mmu-miR-7048-5p 194 mmu-miR-18b-5p 163 mmu-miR-21a-5p 193 mmu-miR-1983 159 mmu-miR-1894-3p 192 mmu-miR-7074-3p 159 mmu-miR-6391 189 mmu-miR-465a-5p 157 mmu-miR-7684-3p 185 mmu-miR-3109-5p 157 mmu-miR-1187 184 mmu-miR-146b-3p 156 mmu-miR-7042-5p 184 mmu-miR-147-3p 156 mmu-miR-6240 183 mmu-miR-297c-5p 154 mmu-miR-3470a 181 mmu-miR-7054-5p 153 mmu-miR-7011-5p 176 mmu-miR-7115-3p 150 mmu-miR-7235-5p 174 mmu-miR-7007-3p 149 mmu-miR-5100 173 mmu-miR-6959-5p 148 mmu-miR-8101 171 mmu-miR-7225-3p 147 mmu-miR-744-5p 171 mmu-miR-7080-3p 145 mmu-miR-6991-5p 169 mmu-miR-1930-5p 143 mmu-miR-7069-5p 169 mmu-miR-743a-5p 142 mmu-miR-6935-5p 168 mmu-miR-292-3p 142 S14 Tang et al.