HCT116 SW48 Nutlin: p53

Transcription

1 Figure S HCT6 SW8 Nutlin: p GAPDH Figure S. Nutlin- treatment induces p protein. HCT6 and SW8 cells were left untreated or treated for 8 hr with Nutlin- ( µm) to up-regulate p. Whole cell lysates were analyzed by immunoblotting. p protein was strongly induced following Nutlin treatment. GAPDH was used as loading control.

2 Figure S Human GCCTCAGTTGCCCCATCTGTGC------CCTGGGTAGGAATATCCTGGATCCCCTTG------GGTCT GATGGGGTAGCCGATGC Chimp GCCTCAGTTGCCCCATCTGTGC------CCTGGGTAGGAACATCCTGGACCCCCTTG------GGTCT GATGGGGTAGCCGATGC Orangutan GCCTCAGTTGCCCCATCTGTGC-nt-CCTGGGTAGGAACATCCTGGATCCCCTTG------GGTCT GATGGGGTAGCTGATGC Gibbon GCCTCAGTTGCCCCATCTGTGC------CCTGGGTAGGAACATCCTGGACCCCCTTG------GGTCT GATGGGGTAGCTGATGC Gorilla GCCTCAGTTGCTCCATCTGTGC------CCTGGGTAGGAACATCCTGGACCCCCTTG------GGTCT GATGGGGTAGCCGATGC Green GCCTCAGTTGCCCTGTCTGTGC------CCTGGGTAGGAACGTCCTGGACCCCCTTG------GGTCT AATGGGGTAGCTGATGC Rhesus GCCTCAGTTGCCCCGTCTGTGC------CCTGGGTAGGAACGTCCTGGACCCCCTTG------GGTCT GATGGGGTAGCTGATGC Crabeat_monk GCCTCAGTTGCCCCGTCTGTGC------CCTGGGTAGGAACGTCCTGGACCCCCTTG------GGTCT GATGGGGTAGCTGATGC aboon GCCTCAGTTGCCCCGTCTGTGC------CCTGGGTAGGAACGTCCTGGACCCCCTTG------GGTCT GATAGGGTAGCTGATGC Marmoset ACCTCAGTTGCTCCGCCTGTGC------CCTGGGTAGGAACATTCTGGACCCCTTTA------GGTCT GAAACCCGATGC Squirrel_monk ACCTCAGTTGCCCC-CCTGTGC------CCTGGGTAGAAACATCCTGGACCCCCTTA------GGTCT GATGGGGTGCCCGATGC Mouse GACTCAGTTTCCCCTGCTGTGA------CCT---CTGGAGCTGCCAGCAGC GGGCTCAGCCAG---AGTAGGACGGATGA--- Rat GCCTCAGTTTCCCCTGCTGTGA------CCT---CTGGAGTTGCCAGCAGTCCCCCGTGCAGTGGGCTCAGCCAG---AGTAGGAAGGACGA--- Dog GCCTCAGTCTCCCCACCTGTGA------ATCGGACAAGAACATCT-----CCCCTTG------GGGCTGACGGTGGGGGGGAGGGCGGCGGGCGG Cat GCCTCAGTTTCCTTATCTGTGA------ACCGGACAAGAACATCC-----CCCCTAG------GGGCTTCGGGGGGTGGGGGGGGGAGGTGGTG- ****** * ***** * ** ** * * Figure S. mir-89 is conserved among apes and old-world monkeys. Multiple alignment of mir-89 candidate sequence between mammalian species. Mammalian orthologous regions for the human mir-89 precursor showed a mosaic pattern of similarity wherein conserved sites are located at the end of hairpin. The rest of the precursor sequence is poorly conserved in mammals, although synteny of genomic alignments in the vicinity of this region is unambiguous. Mature human mir-89-p and -p sequences are shown in red. Positions of % sequence conservation are marked by stars.

3 Figure S Human Rhesus Squirrel Monkey Marmoset Apes OWM NWM NWM Figure S. Pre-miR-89 structure is conserved among apes and old-world monkeys. (A) Phylogenetic tree of mir-89 hairpin regions based on the multiple alignment of primate candidate sequences (apes, old-world monkey, and new-world monkey. () Secondary structure prediction for mir-89 stemloop sequence. Only apes and old-world monkey (OWM), but not new-world monkey (NWM) sequences adopt stable mirna-like structures.

4 Figure S Dox Anti-miR Figure S. mir-89-p knockdown reduces colony formation after Doxorubicin treatment. As shown in Figure, HCT6 cells were reverse transfected with Anti-miR-89-p for 8 hr, then treated with nm Dox for 8 hr and seeded for colony formation on plastic for days.

5 Fold change normalized to GAPDH si sigdf Fold change normalized to U si sigdf Figure S GDF mir-6 mir-89-p C Relative cell growth 6 sigdf Anti-miR-89-p sigdf/anti-mir-89-p mir-89-p mimic/ mir-89-p/sigdf day day day day Figure S. Comparison of mir-89 and GDF knockdowns HCT6 cells were reverse transfected with sirna targeting GDF. (A) 8 hours after transfection, GDF mrna levels were measured by SYR-Green qrt-pcr. GDF was down-regulated by over %7. () mir-89-p levels were quantitated following GDF knockdown by TaqMan qrt-pcr. The unrelated mir-6 is shown as a control. (C) HCT6 cells were reverse transfected with the indicated molecules and seeded in 96-well plates. Relative proliferation was measured over a day period using WST-8 colorimetric assays.

6 si Anti-89 Figure S6 mock dox mock dox CDK CCNA HDAC CDCA GAPDH Fold change normalized to GAPDH..... _untreated Anti-miR-89_untreated _Dox Anti-miR-89_Dox UC p MDM GADDa Figure S6 Effects of mir-89 knockdown on gene expression following DNA damage. A) HCT6-pWT or pko cells were transfected with nm Anti-miR-89 for 8 hours, then treated with nm doxorubicin for 8 hours. Whole cell lysates were probed with the indicated antibodies, GAPDH was used as a loading control. ) HCT6 cells were transfected and treated with doxorubicin as in (A), RNA was isolated and the expression of p pathway genes was measured using qrt-pcr.

7 Figure S7 Fold enrichment normalized to U6 mir-89-p IgG AGO Figure S7. mir-89-p is enriched in RISC after mimic transfection To ensure that the levels of mir-89-p incorporated into RISC after mir-89-p over-expression achieved by transient mimic transfection were physiologically reasonable, we immunoprecipitated Ago 8 hr after transfecting HCT6-pWT cells with mir-89-p mimics. RNA was isolated from the IP material and TaqMan RT-qPCR quantitation of mir-89-p in the RISC IP compared to input showed approximately -fold enrichment.

8 Figure S8 Relative absorbance 6 mir-89-p Day Day Day Day MDA-M- Relative colony formation MDA-M- mir-89-p mir-89-p Figure S8. MiR-89-p over-expression reduces the viability of MDA-M- cells. (A) The mutant p cell line MDA-M-, was transiently transfected with mir-89-p mimic or control and seeded in a 96-well plate. MTT assays were performed over days to measure proliferation. () MDA-M- cells were transfected as above, and re-seeded at cells/well in a -well plate (n=). After days, colonies were fixed with ice-cold methanol, stained with crystal violet, and counted (lower panel).

9 Figure S9.9.8 mir-89 Expression relative to U WI8 MCFa Relative cell growth mir-89 sideath day day day day Relative cell growth 6 mir-89 sideath day day day day Figure S9. mir-89-p is less potent in normal fibroblasts A) RNA isolated from the indicated normal tissues was purchased from Clontech. MiR-89 expression was measured in the tissue panel by TaqMan qrt-pcr and expression values are given as a fraction of U6 expression. () The normal fibroblast cell lines WI8 and MCFA were transfected with mir-89-p or control mimic, and their proliferation over days was measured using MTT colorimetry. The effect of mir- 89-p was much less than that of sideath.

10 KO WT Figure S mimic GAPDH AX PUMA Figure S. mir-89 mimic transfection induces p-associated mediators of apoptosis HCT6-pWT or pko cells were transfected with nm mir-89 mimic for 8 hours. Whole cell lysates were probed with the indicated antibodies, GAPDH was used as a loading control.

11 C Figure S Sub G G-M S G. nm nm nm nm Cell (%) 8 6 Relative cell growth mir-89 mimic sicelldeath Fold chance normalized to GAPDH nm nm nm nm Figure S. Dose-dependence of mir-89 mimic transfection In order to test the effects of different concentrations of mir-89 mimic, we transfected HCT6-pWT cells with,,, or nm mir-89-p mimic or nm mimic. (A) After 8 hours, cells were fixed and stained with PI, and cell cycle was analyzed by flow cytometry. () RNA was harvested 8 hours after transfection, and the expression of mir-89 targets was measured by SYR-Green qrt-pcr. SDHA is shown as a negative control. (C) Cells were reverse transfected and seeded in 96 well plates, and relative growth after a further 7 hours was measured by WST-8 assay.