Figure 1.
miR-21 (A) was upregulated and PTEN (B) was downregulated in xenografts, derived from miR-21 over-expressing HCT116 cells (miR-21) and corresponding vector controls (pcmv) in SCID mice as described previously [12].
Quantitative real-time RT PCR was performed with RNA isolated from formalin fixed paraffin embedded xenografts.
Figure 2.
Photomicrograph of attached parental (P) and floating colonospheres (Sp) of HCT116 (A) and HT29 (B) colon cancer cells (upper panel); the middle panel represents the results of quantitative real time RT-PCR showing upregulation of miR-21 in colonospheres (Sp) when compared with the corresponding parental control (P), and western blot (lower panel) showing the relative expression of PTEN, pAKT, total Akt in parental cell and their corresponding colonospheres.
Figure 3.
Quantitative real-time RT-PCR (upper panel) showing downregulation of miR-21 in 5-FU+Oxaliplatin (chemo-resistant) colon cancer (A) HCT116 and (B) HT29 cells in response to CDF for 72 hours.
Controls were incubated with DMSO. Western-blot (lower panel) showed changes in the relative concentrations of PTEN, pAkt and total Akt in chemo-resistant HCT116 and HT29 cells in response to CDF or DMSO. (C) CDF treatment or anti-miR-21 transfection increases luciferase activity in chemo-resistant HCT116 cells transfected with pGL3-luc construct having PTEN-miR21 seed sequence. NC = Negative Control.
Figure 4.
Quantitative real-time RT-PCR (left panel) showing downregulation of miR-21 in response to CDF for 72 h in SW620, a metastatic colon cancer cell line; while control was incubated with an equivalent volume of DMSO.
Western-blots (right panel) showing changes in relative expression of PTEN, pAkT and total Akt in SW620 cells following 72 h incubation with CDF or DMSO (controls). Human β-actin served as the loading control.