Figure 2

Functional analysis of the role of FDPS in GBMs. (A,B) Representative Western blot showing pSTAT3, STAT3, p-AKT, AKT, p-ERK, ERK FDPS and PCNA protein levels in 7 human primary glioma cell lines established from the indicated cancer patients (G18, G23, G24, G27, G37, G39, G50) (A), in NHA and in the four indicated glioma cell lines; β-actin or α-Tubulin were used as loading control. Panels show representative blots of three different experiments performed with similar results. The tables below report correlation analysis of western blot results for the protein expression of FDPS and the protein expression of p-STAT3, p-AKT, p-ERK and PCNA. (C) U87MG cells or U87MG cells transfected with siRNA FDPS were cultured for 48 h in presence or absence of EGF in the last 8 minutes before cells lysis; cell lysates were immunoblotted for p-STAT3, STAT3, p-AKT, AKT, p-ERK, ERK, FDPS, Mcl-1, BCL-XL and α-Tubulin as loading control. Data are representative of 3 independent experiments performed with similar results. (D) Distribution of U87MG cells or U87MG cells transfected with siRNA FDPS in the different cell cycle phases. All the results shown are representative of three independent experiments performed in duplicate, expressed as mean ± SD (ANOVA, **p < 0.01vs control). (E) Cytofluorimetric assessment of apoptosis in U87MG cells or U87MG cells transfected with siRNA FDPS or Scramble siRNA. Histograms indicate the total percentage of early (AV + /PI- cells) and late apoptotic events (AV + /PI + cells) as well as necrotic cells (AV-/PI + cells). All the results shown are representative of three independent experiments (ANOVA, *** p < 0.001, ** p < 0.01). (F) Patient-derived primary cell line (GBM39) or GBM39 transfected with siRNA FDPS were cultured for 48 h in presence or absence of EGF in the last 8 minutes before cells lysis; cell lysates were immunoblotted for p-STAT3, STAT3, p-AKT, AKT, p-ERK, ERK, FDPS, Mcl-1, BCL-XL and α-Tubulin as loading control. Data are representative of 3 independent experiments performed with similar results. (G) NHA cells or NHA cells transfected with siRNA FDPS were cultured for 48 h; cell lysates were immunoblotted for p-STAT3, STAT3, p-AKT, AKT, p-ERK, ERK, FDPS, Mcl-1 and α-Tubulin as loading control. Data are representative of 3 independent experiments performed with similar results. (H,I) Detection of apoptosis in patients-derived primary cell line (GBM39), both in basal condition and after FPDS silencing (H) and in NHA cells or NHA cells transfected with siRNA FDPS or Scramble siRNA (I). All the results shown are representative of three independent experiments (ANOVA, ***p < 0.001, **p < 0.01, *p < 0.05).