Figure 2

(A) Cell surface expression of CD44 was analyzed by flow cytometry. The violet area in the plot is relative to human IgG1; CD44 signals are showed in green for WT MIA PaCa-2, in pink for PGS MIA PaCa-2 and in black for ANXA1 KO MIA PaCa-2. (B) RT-PCR for K18 mRNA expression measured on levels of HPRT. Values are expressed using the delta-delta Ct method to derive relative fold change. ***p < 0.001. (C,D) Western blots showing K8, ANXA1, lamin A/C, vimentin and GAPDH expression in WT, PGS and ANXA1 KO MIA PaCa-2 cells. (E,F) Vimentin and lamin A/C relative expression was analyzed by densitometry. The optical density of the protein bands was normalized on GAPDH levels giving to the control band an arbitrary value of 100. The blots were exposed to Las4000 (GE Healthcare Life Sciences) and the relative intensities of bands were determined using ImageQuant software (GE Healthcare Life Sciences). (G) Immunofluorescence analysis to detect ANXA1 (red; panels a, b, c), vimentin (yellow; panels d, e, f), lamin A/C (purple; panel g, h, i) and F-actin (green; panels l, m, n) in WT, PGS and ANXA1 KO MIA PaCa-2. Nuclei were stained with DAPI. Magnification 63 × 1.4 NA. Bar = 10 μm. (H) Fluorescence intensity for ANXA1, vimentin and lamin A/C signals (arbitrary units, A.U.) using ImageJ software; determined on 150 cells (for three independent experiments). The results relative to ANXA1 KO MIA PaCa-2 are representative ± SEM of almost three analyzed clones with a similar behaviour.