Then, a second pair of primers were used to amplify a fragment of 975 bp from your 1264 bp template of the KLF4 3 UTR. protein levels in stable clones of A549 cells that overexpress pcDNA, miR-7 or miR-7+KLF4 (B). U6 and ERK2 were used as loading controls for RT-PCR and Western blot assays, respectively.(TIFF) pone.0103987.s004.tiff (4.8M) GUID:?3EDBDD20-95AD-4E55-A0BE-9763246128AA Physique S5: miR-7 overexpression promotes cell cycle progression of HaCaT cells. 1.6105 HaCaT cells were seeded in 35 mm cell culture. Once attached, cells were deprived of growth factors for 24 hours to induce cell cycle arrest and then, growth factors were added. Cells were harvested at 0, 12 and 24 hours after arrest and stained with propidium iodide (PI) to determine their cell cycle profile by circulation cytometry. h, hours.(TIFF) pone.0103987.s005.tiff (4.8M) GUID:?55B598B0-9112-453F-8607-0A128C7B9217 Figure S6: miR-7 regulates the protein levels of KLF4 target genes. Whole cell lysates from A549 cells stably overexpressing miR-7 or the vacant vector (pcDNA) were analyzed to determine Cyclin D (CycD) (A) and p27 (B) protein levels by Western blot assays using specific antibodies. ERK2 protein was used as loading control. The relative expression of each protein was calculated by dividing its densitometric Pamiparib transmission by the ERK2 transmission. All values were normalized considering the value of pcDNA transfected cells at the 0 hours time point as 100%. Data symbolize the imply of three impartial experiments, *pcDNA.(TIFF) pone.0103987.s006.tiff (482K) GUID:?F5C566B9-59DE-4773-8B94-D8D4DB4C0432 Physique S7: miR-7 overexpression in tumors inhibits KLF4 and regulate Cyclin D and p21 protein levels. Representative Western blots showing protein levels of KLF4, Cyclin D and p21 in a tumor derived from pcDNA transfected cells and three impartial tumors derived from miR-7 overexpressing cells (A) and in pools of three impartial tumors derived from either miR-7 or miR-7+KLF4 expressing clones (B). ERK2 protein was used as loading control.(TIFF) pone.0103987.s007.tiff (431K) GUID:?D7D09CEE-BB92-40E9-BBAC-624285880F80 Table S1: miRNAs with predicted binding sites within the KLF4 3 UTR are listed with their G values as calculated by PITA. (DOC) pone.0103987.s008.doc (34K) GUID:?7261269C-E5FE-42F9-8560-111D96FD9136 Table S2: RT-PCR and qPCR primers. (DOC) pone.0103987.s009.doc (40K) GUID:?73A35848-DBBC-45B5-A762-0DF9533FDF5D Movie S1: Wound healing process of a pcDNA HaCaT clone recorded by using a Nikon TE300 inverted bioluminescence microscope. (MPG) pone.0103987.s010.mpg (1.3M) GUID:?74EE969E-0179-4482-8A29-6C95B1F68D99 Movie S2: Wound healing process of a miR-7 HaCaT clone recorded by using a Nikon TE300 inverted bioluminescence microscope. (MPG) pone.0103987.s011.mpg (1.3M) GUID:?1BED7032-1AF6-4B97-AF34-B9DB7D8C840A Movie S3: Wound healing process of a miR-7+KLF4 HaCaT clone recorded by using a Nikon TE300 inverted bioluminescence microscope. (MPG) pone.0103987.s012.mpg (1.2M) GUID:?3C92B9FD-9B66-4438-8372-F30170A95452 Movie S4: Wound healing process of a pcDNA A549 clone recorded by using a Nikon TE300 inverted bioluminescence microscope. (MPG) pone.0103987.s013.mpg (2.6M) GUID:?02D4F8D0-9947-4A44-A169-F2CE0CA4B0AA Movie S5: Wound healing process of a miR-7 A549 clone recorded by using a Nikon TE300 inverted bioluminescence microscope. (MPG) pone.0103987.s014.mpg (2.7M) GUID:?47F6ADCC-2EDD-4039-9D6B-E18B3D8EE9EA Abstract MicroRNAs (miRNAs) are endogenous small non-coding RNAs that have a pivotal role in the post-transcriptional regulation of gene expression and their misregulation is common Pamiparib in different types of malignancy. Although it has been shown that miR-7 plays an oncogenic role in different cellular contexts, the molecular mechanisms by which miR-7 promotes cell transformation are not well understood. Here we show that this transcription factor KLF4 is a direct target of miR-7 and present experimental evidence indicating TNFSF10 that the regulation of KLF4 by miR-7 has functional implications in epithelial cell transformation. Stable overexpression of miR-7 into lung and skin epithelial cells enhanced cell proliferation, cell Pamiparib migration and tumor formation. Alteration of these cellular functions.