LIF-induced expression of iNOS expression was LIFR-dependent.

<p><b>A.</b> Cells treated with LIF in presence and absence of a blocking antibody to LIFR which reduced immunofluorescence for LIFR (green) and iNOS (red). Blue: nuclear staining with DAPI. Scale bar: 50 µm. <b>B</b>.-Quantification of the average iNOS fluorescence intensity shown in A normalized respect to the control group in three independent experiments. * p<0.05.</p

LIF-induced proliferation depends on iNOS.

<p><b>A</b>. Immunofluorescence for BrdU (green) in cells grown in control medium (CM, a), EGF-containing medium (b) and LIF-containing medium (c). Blue: nuclear staining with DAPI. Scale bar:100 µm. <b>B</b>. BrdU immunofluorescence (green) in the presence of the LIFR blocking antibody (d), the LIFR blocking antibody plus the NO donor SNAP (e), the iNOS inhibitor L-NIL (f) and L-NIL plus SNAP (g). Blue, nuclear staining with DAPI. Scale bar: 100 µm. <b>C.</b> Quantification of data shown in A and B. ≠ * p<0.05 between the groups.</p

Olfactory neurosphere-derived cells expressed LIF.

<p><b>A.</b> iNOS immunofluorescence (red) in a neurosphere. Scale bar: 20 µm. <b>B</b>. Co-localization of LIFR (green) and iNOS (red) in cells treated with LIF. DAPI-stained nuclei are blue. Bar: 25 µm. <b>C</b>. Immunofluorescence for iNOS in cells grown in control medium (a), EGF-containing medium (b) and LIF-containing medium (c). Scale bar: 50 µm. <b>D</b>. Flow cytometric analysis showing that LIF increased the iNOS expression measured by the mean intensity of fluorescence of the cells in each group. * p<0.05 respect to the control groups. <b>E</b>. qPCR revealed iNOS mRNA in rat olfactory neurosphere cells grown in control medium (CM), EGF-containing medium and LIF containing medium expressed as the mean ± SEM of the iNOS expression increment fold respect to the control group (CM). *p<0.05 respect to the control groups.</p

Temporal course of LIF-induced iNOS expression.

<p><b>A.</b> Immunofluorescence of iNOS in cells treated with LIF for 0 (a), 6 (b), 12 (c) and 24 h (d). Blue: DAPI nuclear staining. Scale bar: 50 µm. <b>B.</b> qPCR iNOS mRNA treated as in A of three independent experiments expressed as the mean ± SEM of the iNOS expression increment fold respect to the control group (CM). * p<0.05.</p

Primary cultures of olfactory epithelial cells expressed iNOS and LIFR.

<p><b>A</b>. iNOS Immunofluorescence in cells grown for 24 h in control medium and in the same medium containing LIF. Blue: nuclei stained with DAPI. Red: Immunoreactivity to iNOS. Scale bar: 50 µm. <b>B</b>. Co-localization of iNOS (red) and LIFR (green) in cells grown in LIF. Blue: DAPI stained nuclei. Scale bar: 50 µm. <b>C</b>. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed mRNA for iNOS (upper panel) and YWHAZ, as a loading control (lower panel) in rat olfactory mucosal cells grown in control medium (CM), and in medium containing EGF, LIF and L-NIL. The negative control contained no mRNA. MW: DNA size markers; the predicted band for iNOS is 370 bp. <b>D</b>: Quantification of Brdu immunofluorescence of cell grown in control medium (CM), LIF containing medium and L-NIL containing medium. Data were expressed as mean of % Brdu positive cells ± SEM. *p<0.05 with respect to the control group.</p