qPCR analysis of <i>Hb-α</i> and <i>Hb-β</i> expression in hPVECs and VK2/E6E7 cells before and after induction with LPS.

<p>Cells seeded at a density of 10⁶/well in 24-well plates were treated with LPS (10 μg/ml for 6 hrs). Expression of <i>Hb-α</i> and <i>Hb-β</i> was up-regulated in LPS-induced cells. Each bar represents the mean ± SD of three independent replicates (***p< 0.001 Vs Untreated).</p

Immunofluorescence localization of cytokeratin-13.

<p>Confocal images showing cytoplasmic localization of cytokeratin-13(green) in hPVECs (a-c) and VK2/E6E7 cells (d-f). Nucleus was stained with DAPI (blue). FITC (a, d), FITC and DAPI merge (b,e) and no primary antibody controls (c,f) are shown. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).</p

Cultures of human primary vaginal epithelial cells (hPVECS) and VK2/E6E7 cells.

<p>Different phases of growing cells of hPVECs are shown (a-d). a: Cells attached to the surface of the culture flask on day 10 (10x), b: on day 15 (40x), c: confluent cells on day 25 (10x) and d: confluent cells on day 25 (40x); e, f, g: confluent hPVECs from three different patient samples; h: confluent cultures of VK2/E6E7 cell line on day 10 (40x).</p

<i>Hb-α</i> and <i>Hb-β</i> expression in hPVECs and VK2/E6E7 cells.

<p>(A) RT-PCR analysis; 1: Untreated hPVECs, 2: hPVECs treated for 6 hrs with LPS (10 μg/ml), 3: Untreated VK2/E6E7 cells and 4: VK2 cells treated with LPS (10 μg/ml) for 6 hrs. Loading control, <i>Gapdh</i> (238 bp) expression in hPVECs. The gels shown are one of the representative pictures from three independent experiments performed on three different days. (B) Densitometric analysis of bands from RT-PCR amplification products of Hb-α and Hb-β mRNAs shown in figure-5A.</p

Expression of NF-<i>κ</i>B in hPVECs and VK2/E6E7 cells.

<p>(A). NF-κB levels in un-stimulated, LPS stimulated and Bay 11–7082 treated hPVECs and VK2/E6E7 cells analyzed by ELISA. Cells were seeded at a density of 10⁶/well in 24-well plates and induced with LPS (10 μg/ml for 6 hrs) or Bay-11-0782 (5 μM for 24 hrs). Levels of p65-NF-κB were up-regulated in LPS-induced cells, where as Bay-11-7082 reversed this effect (a). Values represent mean ± SD of three experiments performed on different days. Values are statistically significant (***p<0.001 over Bay 11–7082 treated cells). (B). Western blot analysis of p65-NF-κB expression in hPVECs and VK2/E6E7 cells. hPVECs (i, iii) and VK2/E6E7 cells (ii, iv)1: Untreated cells, 2: LPS induced cells, 3: Bay 11–7082 treated cells, 4: Bay 11–7082 treated cells induced with LPS. Results were normalized to the β-actin (iii, iv), which is constitutively expressed in cells and serves as an internal standard. The blots shown are the representative pictures from three independent experiments. (C). Densitometric analysis of bands from western blots of p65-NF-κB reported in Fig 9B. The levels of p65-NF-κB were up-regulated in LPS-induced cells, whereas Bay 11–7082 repressed p65-NF-κB levels.</p

Expression of hemoglobin-α and β subunits in human vaginal epithelial cells and their functional significance

<div><p>Hemoglobin (Hb) is a major protein involved in transport of oxygen (O₂). It consists of Hb-α and Hb-β subunits, which are normally expressed by cells of erythroid lineage. However, till recently, it was not known whether non-erythroid cells like vaginal cells synthesize Hb and whether it has any functional significance. Therefore, we designed the following objectives: (1) to establish <i>in-vitro</i> culture system of human primary vaginal epithelial cells (hPVECs), (2) to determine whether Hb-α and Hb-β proteins are truly synthesized by hPVECs, (3) to evaluate the effect of LPS (lipopolysaccharide) on the expression of Hb-α and Hb-β proteins (4) to decipher the significance of the Hb-α and Hb-β expression in hPVECs and (5) to determine the molecular mechanism regulating the expression of Hb-α in hPVECs. To accomplish these studies, we applied a battery of assays such as RT-PCR, qRT-PCR, Flow cytometry, western blot, and immunofluorescence, Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). The results revealed the expression of Hb-α and Hb-β at both mRNA and protein level in hPVECs. The expression was significantly upregulated following LPS treatment (10μg/ml for 6 hrs) and these results are comparable with the expression induced by LPS in human vaginal epithelial cell line (VK2/E6E7). These cells constitutively produced low levels of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines. Also, the response of phosphorylated (p65)-NF-<i>κ</i>B to LPS was upregulated with increased expression of IL-6, Toll-like receptor-4 (TLR4) and human beta defensin-1 (hBD-1) in hPVECs and VK2/E6E7 cells. However, Bay 11–7082 treatment (5μM for 24 hrs) could neutralize the effect of LPS-induced p65-NF-κB activity and represses the production`of Hb-α and Hb-β. The results of EMSA revealed the presence of putative binding sites of NF-κB in the human Hb-α promoter region (nt-115 to -106). ChIP analysis confirmed the binding of NF-<i>κ</i>B to Hb-α promoter. In conclusion, the present findings revealed for the first time that hPVECs synthesized Hb-α and Hb-β and the expression is comparable with the expression of VK2/E6E7 cells. The identification of NF-<i>κ</i>B regulatory sequences in Hb-α promoter, whose activation is associated with immune response of hPVECs, indicating Hb-α and Hb-β may act as an endogenous antimicrobial defense protein against vaginal inflammation/infections.</p></div

Electrophoretic mobility shift assay (EMSA) of Hb-α with LPS.

<p>EMSA was performed using oligonucleotide probes corresponding to NF-κB regulatory elements. Nuclear extract from LPS stimulated hPVECs was used. Nucleoprotein containing NF-κB was incubated with a fragment encompassing nt-115 to -106 bp upstream of Hb-α promoter start site-2. We observed a strong DNA binding to NF-κB protein with LPS treatment. Presence of super-shift bands in the presence of the p65- NF-κB antibody demonstrating the specific binding. (***: denotes unbound DIG oxygenase labelled DNA; 1- Only wild type labelled probe; 2- Wild type labelled Probe + Protein (1μg); 3- Wild type labelled Probe + Protein (+2μg); 4- Wild type labelled Probe + Protein (++ 4μg); 5- Wild type labelled Probe + Protein (+++ 5μg); 6- Mutant labelled Probe + Protein (1μg); 7- Wild type labelled Probe + unlabelled Probe 1x + Protein (1μg); 8- Wild type labelled Probe + unlabelled Probe 2x + Protein (1μg); 9- Wild type labelled Probe + unlabelled Probe 4x + Protein (1μg); 10- Standard labelled Probe 1x + Protein (1ug); 11- Standard labelled Probe 2x + Protein (1μg); 12- Standard labelled Probe 4x + Protein (1μg); 13- Wild type labelled Probe + Protein (1μg) + p65- NF-κB antibody(1x); 14- Wild type labelled Probe + Protein (1μg) + p65-NF-κB antibody(2x) and 15- Wild type labelled Probe + Protein (1μg) + anti rabbit IgG(1x). (*:NF-κB -antibody- NF-κB -DNA complexes and **: NF-κB -DNA complexes.</p

Immunofluorescence localization of vimentin.

<p>hPVECs (a-c) and VK2/E6E7 cells (d-f) did not express vimentin. Cytoplasmic localization of vimentin (green) is seen in HeLa cells (positive control) (g-i). Nucleus was stained with DAPI (blue), FITC (a, d, g), FITC and DAPI merge (b,e,h) and no primary antibody controls (c,f,i) are indicated. The figure shown is one of the representative pictures from three independent experiments (Mag. 63X).</p

Immunofluroscence of phosphorylated p65-NF-κB expression in hPVECs and VK2/E6E7 cells.

<p>hPVECs (A) and VK2/E6E7 (B) cells were treated with LPS (10 μg/ml 6 hrs) or Bay 11–7082 (5 μM for 24 hrs). Stimulation with LPS activates p65-NF-κB expression in hPVECs (b,e) and VK2/E6E7 cells (h,k) as compared to unstimulated hPVECs (a,d) and VK2/E6E7 cells (g,j). Treatment with Bay 11–7082 attenuated NF-κB expression in hPVECs (c,f) and VK2/E6E7 cells (i,l). Nucleus was stained with DAPI (blue), p65-NF-κB was stained with FITC (green), FITC and DAPI merged (d,e,f,j,k,l). The images shown are the representative pictures of one of three identical experiments performed on three different days (Mag. X 63).</p

TLR4 expression in hPVECs and VK2/E6E7 cells.

<p>(A). RT-PCR analysis of TLR4 expression in VK2/E6E7 cells and hPVECs. expression of TLR4 (182 bp) in VK2/E6E7 cells (a), (c), and hPVECs (b),(d) was observed. 1: Untreated cells (control); 2: DMSO treated cells (control); 3: TLR4 antibody treated cells; 4: LPS treated cells (10 μg/ml for 6 hrs) and 5: Bay 11–7082 treated cells (5μM for 24 hrs) stimulated with LPS (10μg/ml for 6 hrs) and (c), (d) Housekeeping gene <i>Gapdh</i> (238 bp) considered as an internal standard. The gel picture shown is one of the representative pictures from three independent experiments. (B). Densitometric analysis of bands from RT–PCR amplification products of <i>TLR4</i> mRNA reported in Fig 10A. The expression of <i>TLR4</i> mRNA was up-regulated in LPS-induced cells. Bay 11–7082 treatment has no effect on the expression of TLR4 mRNA in cells induced with LPS.</p