Recombinant purified buffalo leukemia inhibitory factor plays an inhibitory role in cell growth

<div><p>Leukemia Inhibitory Factor (LIF) is a polyfunctional cytokine, involved in numerous regulatory effects <i>in vivo</i> and <i>in vitro</i>, varying from cell proliferation to differentiation, and has therapeutic potential for treating various diseases. In the current study, a COS-1 cell line overexpressing recombinant Buffalo LIF (rBuLIF) was established. The rBuLIF was purified to homogeneity from the total cell lysate of COS-1 cells using a two-step affinity chromatography. The purified LIF was confirmed by western blot and mass spectrometer (MS/MS). Particularly, high-resolution MS has identified the rBuLIF with 73% of sequence coverage with highest confidence parameters and with the search engine score of 4580. We determined the molecular weight of rBuLIF protein to be 58.99 kDa and 48.9 kDa with and without glycosylation, respectively. Moreover, the purified rBuLIF was verified to be functionally active by measuring the growth inhibition of M1 myeloid leukemia cells, revealing a maximum inhibition at 72 hours and half-maximal effective concentration (EC50) of 0.0555 ng/ml, corresponding to a specific activity of >1.6×10⁷ units/mg. Next, we evaluated the effect of rBuLIF on buffalo mammary epithelial cell lines for its role in involution and also identified the IC50 value for BuMEC migrating cells to be 77.8 ng/ml. Additionally, the treatment of MECs (BuMEC and EpH4) displayed significant (<i>P</i> < 0.05) reduction in growth progression, as confirmed by qRT-PCR analysis, suggesting its strong involvement in the involution of the mammary gland <i>in vivo</i>. Thus, we conclude that the glycosylated rBuLIF, purified from COS-1 cells was found to be functionally active as its natural counterpart.</p></div

Temporal expression and secretion of albumin in condition media (CM) of hepatocytes.

<p>Panel A—Western blot analysis of albumin using condition media obtained from different time points of hepatocytes culture using FBS-free William’s E media. Lane 1: pre-stained protein marker; Lane 2: fresh hepatocytes lysate; Lane 3: 1^st day CM; Lane 4: 3^rd day CM; Lane 5: 4^th day CM; Lane 6: 5^th day CM; Lane 7: 7^th day CM. Panel B—Control experiment. Lane 1: pre-stained protein marker; Lane 2: pure hepatocytes (positive control); Lane 3: condition media (test); Lane 4: FBS-free William’s E media (negative control).</p

Immunostaining of 5 days old cultured buffalo hepatocytes with α1-antitrypsin antibody.

<p>Panel A shows light microscopic image of hepatocytes, and panel B shows immunostained hepatocytes with α1-antitrypsin antibody labelled with FITC (green) and nuclear stain DAPI (blue) dyes.</p

Hepatocyte growth on fibroblast feeder layer.

<p>Hepatocyte proliferation curve represented by the absorbance at 540 nm of BrdU-labelled hepatocytes at different days of culture.</p

Immunostaining of 5 days old cultured buffalo hepatocytes with anti-cytokeratin-18 and anti-albumin.

<p>Immunostaining with (A) CY3 labelled anti-cytokeratin-18 antibodies (fluorescence signal in red); (B) FITC labelled anti-albumin antibodies (green); (C) staining of hepatocytes nuclei with DAPI (blue). Panel D shows the merged images from panels A, B and C. Panel E shows light microscopic image of hepatocytes; panel F shows negative control (Isotype control), and panels G shows staining with DAPI, while panel H shows images merged from panels F and G.</p

Agarose gel electrophoresis of RT-PCR products of hepatocyte-specific marker genes expressed in 5 days old cultured hepatocytes.

<p>Panel A shows 293 bp amplicon of albumin; B—130 bp amplicon of hepatocyte nuclear factor 4α; C—240 bp amplicon of glucose-6-phosphatase; D—136 bp amplicon of CYP1A1; E—164 bp amplicon of CYP3A4; F—142 bp amplicon of tyrosine aminotransferase. Lane 1: 100 bp ladder; Lane 2: RT-PCR of liver tissue (positive control) by using the gene-specific primers; Lane 3: RT-PCR of respective genes from cultured buffalo hepatocytes; Lane 4: RT-PCR from skin fibroblasts (negative control). Amplification of Glyceraldehyde 3–phosphate dehydrogenase (GAPDH) was used as housekeeping gene.</p

Chromatograms of rBuLIF-GFP protein during affinity purification using Anti-GFP antibodies from whole cell lysate.

<p>(A) First run of chromatography for purification (SDS profile shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198523#pone.0198523.g001" target="_blank">Fig 1B and 1C</a>). (B) The second run of the purification, sample obtained from the first run peak was injected into the column for the second round of purification (respective SDS profile is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198523#pone.0198523.g001" target="_blank">Fig 1D</a>). The blue line in the chromatogram indicates the elution profile of the proteins while the green line shows the gradient applied.</p

Western blot analysis of 5 days cultured buffalo hepatocytes.

<p>Blots of buffalo hepatocytes lysate by using antibodies against albumin (Panel A); lane 1: HepG2 (Positive control), Lane 2: buffalo hepatocytes (test); Lane 3: skin fibroblast (negative control); Lane 4: pre-stained protein marker; cytokeratin-18 (Panel B); Lane 1: pre-stained protein marker; Lane 2: HepG2 cells; Lane 3: buffalo hepatocytes; Lane 4: skin fibroblast; and α1-antitrypsin (Panel C), order of lanes is similar to that shown in panel B.</p

Expression of LIF in COS-1 cells.

<p><b>(A)</b> GFP-tagged full-length LIF was expressed in COS-1 cells and detected by western blot as a secretory protein in the media as well as in total cell lysate. (B) SDS-PAGE Profile: Gel showing the initial washing fractions of the rBuLIF protein during affinity purification of whole cell lysate from the first run of purification. (C) Initially, collected fractions during the chromatographic run from Lane 1, 2 and 3 all the proteins obtained were concentrated in a lyophilizer 1:20 folds to visualize clearly in the SDS gel. M lane for the marker, first run elution profile of the peak shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198523#pone.0198523.g002" target="_blank">Fig 2A</a>-lanes 4–8. (D) The second run of the chromatographic elution: Eluted fractions from the first run were subjected to the same packed column for the separation of the proteins, based on the affinity. (E) Western blot image of purified rBuLIF protein for confirmation. M lane for the marker, PT lane for PNGase F treated BuLIF without glycan moieties showing 10 kDa reduction in size, PN lane for non-PNGase treated showing intact purified rBuLIF protein of 58.9 kDa molecular weight.</p

Oil-red staining of lipid droplets in 5 days old cultured buffalo hepatocytes.

<p>Panel A shows lipid droplets (indicated by arrow) in hepatocytes in phase contrast at 200X magnification; and panel B shows oil red stained hepatocytes containing lipid droplets (arrow).</p