Figure 1.
NSCLC and SCLC cells differ in sensitivity to radiation treatment and display differential expression of proteins involved in the regulation of miRNA biogenesis.
(A) Western blot analysis of the level of protein expression of Drosha, Dicer, exportin 5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), fragile X mental retardation syndrome-related protein 1 (FXR1) and Argonaute 2 (AGO2) in a panel of NSCLC (U1810, U1299, A549, H661, H157, H23) and SCLC (U1285, H82, H69, U1690, U1906, U2020) cell lines. (B) Densitometric analysis of relative levels of protein expression in H23, H1299, U1810 and H661 cell lines. Cell lines distributed according to radiosensitivity, measured as the fraction surviving at 2 Gy (SF2). Equal loading was verified using anti-β-actin antibodies. Results are representative of three independent experiments.
Figure 2.
Expression of components of miRNA machinery in NSCLC following treatment with ionizing radiation.
The cleavage of PARP and the level of expression of Drosha, Dicer, exportin 5 (XPO5), Tudor-SN (TSN), protein activator of the interferon-induced protein kinase (PACT), and fragile X mental retardation syndrome-related protein 1 (FXR1) in U1810, H661 and H23 cells were detected by Western blot at 6, 24 and 48 h post-irradiation with 8 Gy. Equal loading was verified using anti-β-actin antibodies. Data are representative of three independent experiments.
Figure 3.
Knock-down of Dicer and Drosha is not sufficient to sensitize NSCLC cells to irradiation.
(A) The level of Dicer expression, cleavage of PARP and processing of caspase-3 and -9 in U1810 cells transfected (48 h) with control (si scr) or Dicer (siDicer) siRNA assessed by Western blot 48 h after irradiation. Equal loading was verified using anti-GAPDH antibodies. Data are representative of three independent experiments. (B) Detection of apoptotic cell death in U1810 assessed by measuring the sub-G1 population after transfection (48 h) with control or Dicer siRNA and irradiation treatment (48 h). (C) Caspase-3-like activity (fold increase with respect to control) in U1810 cells after treatment with either irradiation alone or in combination with transfection of control or Dicer siRNA (for details see Materials and methods). (D) The level of Drosha expression and cleavage of PARP in U1810 cells transfected (48 h) with control (si scr) or Drosha (siDrosha) siRNA analyzed by Western blot 48 h after treatment with irradiation. Equal loading was verified using anti-GAPDH antibodies. All data are representative of three independent experiments. (B) Apoptotic cell death in U1810 measured by analysis of the sub-G1 population after transfection (48 h) with control or Drosha siRNA and irradiation treatment (48 h). Results shown are the mean±S.E.M. of three independent experiments.
Figure 4.
Depletion of a component of the RNA-induced silencing complex (RISC), Argonaute2, does not affect the radioresistance of NSCLC.
(A) The level of Argonaute2 (AGO2) mRNA in U1810 cells transfected with control (scram) or AGO2 siRNA normalized against 18S ribosomal RNA. Results are the mean±S.E.M. of three independent experiments. (B) Cleavage of PARP and processing of caspase-3 and -9 in U1810 cells transfected (48 h) with control (si scr) or AGO2 siRNA, and then subjected to irradiation for 48 h. Equal loading was verified using anti-GAPDH antibodies. All data are representative of three independent experiments. (C) Detection of apoptotic cell death in U1810 cells after transfection (48 h) with control or AGO2 siRNA and subsequent treatment with irradiation (48 h). (D) Caspase-3-like activity (fold increase with respect to control) in U1810 cells after treatment with either irradiation alone or in combination with transfection with control or AGO2 siRNA. All results shown are the mean±S.E.M. of three independent experiments.
Figure 5.
The knock-down of a component of the RNA-induced silencing complex (RISC), Tudor-SN, is insufficient to sensitize NSCLC to irradiation.
The level of Tudor-SN expression and cleavage of PARP in U1810 (A), A549 (C) and H661 (D) cells transfected (48 h) with control (si scr) or Tudor-SN (si TSN) siRNA analyzed by Western blot 48 h after irradiation. Equal loading was verified using anti-GAPDH antibodies. (B) The apoptotic cell death in U1810 cells after transfection with TSN siRNA and irradiation. All data are representative of three independent experiments.