Leukemia inhibitory factor increases the invasiveness of trophoblastic cells through integrated increase in the expression of adhesion molecules and pappalysin 1 with a concomitant decrease in the expression of tissue inhibitor of matrix metalloproteinases  Pankaj Suman, Ph.D., Nachiket Shembekar, M.Sc., Satish Kumar Gupta, Ph.D.  Fertility and Sterility  Volume 99, Issue 2, Pages 533-542.e2 (February 2013) DOI: 10.1016/j.fertnstert.2012.10.004 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 LIF-mediated invasiveness of HTR-8/SVneo cells and activation of downstream signaling pathways. (A) Invasion assay was performed as mentioned in Materials and Methods, and the data are expressed as fold changes in invasion as compared with untreated controls. Values are expressed as mean ± SEM of six experiments performed in duplicates. (B and C) HTR-8/SVneo cells were treated with LIF (50 ng/mL) for varying periods of time, and Western blots were done as mentioned in Materials and Methods. Representative blots for different signaling molecules are shown in the panel B. (C) The densitometric plots corresponding to the Western blots performed for the activated signaling molecule. Band intensities were normalized with respect to the respective unphosphorylated proteins, and the data are expressed as fold changes with respect to the controls. The data are shown as mean ± SEM of three experiments (*P<.05; **P<.001 with respect to the unstimulated control). (D) For immunolocalization of activated STATs, HTR-8/SVneo cells (0.05 × 106) were cultured for 24 hours on a coverslip placed in a 24-well plate. Cells were starved for 2 hours and then treated with LIF (50 ng/mL) for 10 minutes, and immunostaining was carried out as mentioned in Materials and Methods. Phase: phase contrast image; Alexa Fluor 488: immunolocalization of activated signaling molecules mentioned on the left of each panel; DAPI: nuclear staining by DAPI; merge: overlap of the respective Alexa Fluor 488 and DAPI images. Scale bar represents 20 μm size. Fertility and Sterility 2013 99, 533-542.e2DOI: (10.1016/j.fertnstert.2012.10.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Microarray analysis of the gene expression in LIF-stimulated HTR-8/SVneo cells and validation of a few by qRT-PCR. (A) Scatter plot showing the average gene expression data obtained from microarray analysis in the form of a point with respect to X and Y coordinates. (B) Heat map of the selected family of genes associated with the invasive and angiogenic properties of trophoblast cells. Furthermore, to validate the microarray findings and to analyze the expression of TIMPs, HTR-8/SVneo cells (∼105) were cultured in a 24-well plate and treated with LIF (50 ng/mL) for 24 hours. After that, total RNA was isolated, and cDNA was prepared. To analyze the change in the expression of PDPN (C), integrin β3 (D), PAPPA (E), SERPINB3 (F), ID1 (G), ICAM1 (H), TIMP1 (I), TIMP2 (J), and TIMP3 (K) upon LIF treatment, qRT-PCR was performed on the cDNAs prepared as mentioned in Materials and Methods. In the bar diagram, values are expressed as mean δCt values ± SEM of three experiments performed in triplicate after normalization with the 18S rRNA. *P<.05; **P<.001. Fertility and Sterility 2013 99, 533-542.e2DOI: (10.1016/j.fertnstert.2012.10.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Immunolocalization of invasion-associated proteins in HTR-8/SVneo cells treated with LIF and in human placenta. (A) HTR-8/SVneo cells were treated with LIF for 24 hours and probed for immunofluorescence using specific antibodies for PAPPA, podoplanin, ICAM1, ITGB3, ID1, SPB3, and LIF. For a given protein, images were captured at the same exposure time for both the samples. (B) Human placenta (22 weeks) sections were immunostained for the expression of PAPPA, podoplanin, ICAM1, ITGB3, ID1, SPB3, and LIF, and the figures in the panel are the representative light and fluorescent micrographs of human placenta after performing the immunohistochemistry using specific antibodies against the above-mentioned proteins. In both panels, blue fluorescence signifies the nuclear staining by DAPI and the green fluorescence is specific for the particular protein mentioned to their left side. Scale bar represents 50 μm. Fertility and Sterility 2013 99, 533-542.e2DOI: (10.1016/j.fertnstert.2012.10.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Role of PAPPA in LIF-mediated invasion of HTR-8/SVneo cells. HTR-8/SVneo cells were transfected with either PAPPA siRNA or nongenomic siRNA for 72 hours, and qRT-PCR was done to check the level of silencing in them keeping 18S rRNA as the internal control (A). Each bar represents the ΔCt values after normalization with the 18S rRNA. The data are expressed as mean ± SEM of three experiments performed in triplicate. The transfected cells were used to study their invasive behavior in the presence or absence of LIF as described in Materials and Methods (B). The results are expressed as mean ± SEM of fold change in invasion as compared with nongenomic siRNA-transfected cells in the absence or presence of LIF, observed in three independent experiments. *P<.05; **P<.001. Fertility and Sterility 2013 99, 533-542.e2DOI: (10.1016/j.fertnstert.2012.10.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplementary Figure 1 Activation of STAT3(tyr705) and ERK1/2 by LIF. HTR-8/SVneo cells were treated with LIF (50 ng/mL) for varying periods of time, and Western blot was performed using specific antibodies on the cell lysates. Band intensities were normalized with respect to the respective unphosphorylated proteins, and the data are expressed as fold changes with respect to the controls. The data are shown as mean ± SEM of three experiments (*P<.05; **P<.001 with respect to the unstimulated control). Fertility and Sterility 2013 99, 533-542.e2DOI: (10.1016/j.fertnstert.2012.10.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions