Figure 1.
Figure 1. Effects of miR-221 and miR-222 transfection in HUVECs. c-Kit protein expression in cells transfected with ssEGFP or with the miR-221/miR-222 mix was detected 48 hours after transfection by (A) FACS (**P < .01) or (B) by Western blot analysis (band intensity normalized to that of tubulin; *P < .05). (C) RT-PCR of c-Kit. (top) c-kit mRNA level was detected by RT-PCR in control and miR-221/miR-222 mix–transfected cells by amplification of a 331-bp fragment. (bottom) A 232-bp fragment of ATP1A1 mRNA was amplified from the same RT samples to ensure equal loading per lane. The absence of the 372-bp fragment in lanes 1 to 3 indicates that the cDNA is not contaminated by genomic DNA. A representative experiment of 3 performed is reported. L1 indicates 100-bp ladder. (D) Quantification of c-Kit mRNA. RT-PCR band intensity of c-Kit and ATP1A1 was quantified using image analysis software, and their ratio was used to compare relative expression. (E) SCF-induced tube formation (left panel): 48 hours after transfection, HUVECs were seeded onto Matrigel and were exposed to 100 ng/mL SCF + 2% FBS for 6 hours. Images of wound healing were taken after staining with crystal violet. An EPSON Expression 1680 Pro scanner (Seiko Epson, Nagano, Japan) equipped with a transparent unit was used to acquire the images. EPSON Twain Network version 2.00E and Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA) were used for image acquisition and elaboration. Results of 1 of 3 representative experiments are reported. SCF-induced wound healing (right panel): HUVECs were collected 24 hours after transfection, counted, and seeded at a density of 2 × 105 on a coated 12-well multiplate. Six hours later, the monolayers were wounded and healing was observed after overnight incubation. The tube formation was photographed after staining with crystal violet. Images were acquired with a Wilovert inverted microscope (Hund, Wetzlar, Germany) equipped with 10×/0.25 objective lenses and a CoolPix 4500 digital camera (Nikon, Tokyo, Japan). (F) SCF-induced migration: HUVECs transfected with ssEGFP or miR-221/miR-222 mix were allowed to migrate through type I collagen–coated filters in response to 100 ng/mL SCF for 5 hours. The number of migrated cells per field (magnification, × 200) is reported. **P < .01. (G) Cell viability: cells were collected at the end of transfection (light gray bars) and after 48 hours of exposure to SCF (gray bars), stained with trypan blue, and counted. Total number of viable cells is reported. Dead cells (black bars) in control and miR-221/miR-222–transfected plates are also reported. *P < .05.

Effects of miR-221 and miR-222 transfection in HUVECs. c-Kit protein expression in cells transfected with ssEGFP or with the miR-221/miR-222 mix was detected 48 hours after transfection by (A) FACS (**P < .01) or (B) by Western blot analysis (band intensity normalized to that of tubulin; *P < .05). (C) RT-PCR of c-Kit. (top) c-kit mRNA level was detected by RT-PCR in control and miR-221/miR-222 mix–transfected cells by amplification of a 331-bp fragment. (bottom) A 232-bp fragment of ATP1A1 mRNA was amplified from the same RT samples to ensure equal loading per lane. The absence of the 372-bp fragment in lanes 1 to 3 indicates that the cDNA is not contaminated by genomic DNA. A representative experiment of 3 performed is reported. L1 indicates 100-bp ladder. (D) Quantification of c-Kit mRNA. RT-PCR band intensity of c-Kit and ATP1A1 was quantified using image analysis software, and their ratio was used to compare relative expression. (E) SCF-induced tube formation (left panel): 48 hours after transfection, HUVECs were seeded onto Matrigel and were exposed to 100 ng/mL SCF + 2% FBS for 6 hours. Images of wound healing were taken after staining with crystal violet. An EPSON Expression 1680 Pro scanner (Seiko Epson, Nagano, Japan) equipped with a transparent unit was used to acquire the images. EPSON Twain Network version 2.00E and Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA) were used for image acquisition and elaboration. Results of 1 of 3 representative experiments are reported. SCF-induced wound healing (right panel): HUVECs were collected 24 hours after transfection, counted, and seeded at a density of 2 × 105 on a coated 12-well multiplate. Six hours later, the monolayers were wounded and healing was observed after overnight incubation. The tube formation was photographed after staining with crystal violet. Images were acquired with a Wilovert inverted microscope (Hund, Wetzlar, Germany) equipped with 10×/0.25 objective lenses and a CoolPix 4500 digital camera (Nikon, Tokyo, Japan). (F) SCF-induced migration: HUVECs transfected with ssEGFP or miR-221/miR-222 mix were allowed to migrate through type I collagen–coated filters in response to 100 ng/mL SCF for 5 hours. The number of migrated cells per field (magnification, × 200) is reported. **P < .01. (G) Cell viability: cells were collected at the end of transfection (light gray bars) and after 48 hours of exposure to SCF (gray bars), stained with trypan blue, and counted. Total number of viable cells is reported. Dead cells (black bars) in control and miR-221/miR-222–transfected plates are also reported. *P < .05.

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