KSHV infection leads to Par3 nuclear localization.

<p>(A) KSHV negative BJAB, Ramos, (B) KSHV positive BCBL1, BC-3, JSC-1 cell lines and (D) PBMCs uninfected and infected with KSHV at day 6 were used to determine the localization of Par3. LANA staining was used as positive control. In KSHV positive cells graphs were plotted with the percent of LANA foci colocalized with Par3. (C) Co-localization of LANA with Par3 was performed in KSHV negative Ramos cell line. The graphs represent the percent of LANA foci co-localized with Par3.</p

KSHV can induce epithelial to mesenchymal transition markers in infected B-cells.

<p>(A, B) HEK-293 and BAC-KHSV cells were probed with the antibodies against the EMT markers E-cadherin, and SNAIL. DAPI was used to stain the nucleus. (C) Ramos cells were transfected with an increasing dose of LANA to evaluate the transcript levels of E-cadherin and SNAIL. (D) KSHV infection was carried out in primary PBMCs and monitored up to 7 days post-infection. Control cells without KSHV infection was measured for days 1, 2, 4 and 7 for the measurement of SNAIL and E-cadherin transcripts. (E) Stable LANA knockdown cells were compared to controls for the measurement of Par3, E-cadherin, MMP9 and SNAIL. LANA blots were used as a positive control to monitor its expression in cells, and GAPDH was used as a protein loading control. (F) HEK-293-BAC-KSHV compared to HEK293 cells were evaluated at the protein levels for the expression of Par3, E-cadherin, MMP9 and SNAIL. LANA blots were used as a positive control for the expression of cells and GAPDH was used as a protein loading control. (G) SNAIL knockdown in BC-3 cells were evaluated with control vector for the measurement of the expression of LANA, Par3, and E-cadherin. GAPDH was used as a protein loading control.</p

LANA interacts and colocalizes with Par3.

<p>(A) Co-Immunoprecipitation of Par3 with LANA was examined in HEK-293 cells. Myc tagged LANA and HA tagged Par3 constructs were used in this experiment. (B) GST pull-down assays shows binding of Par3 with N- (1–340 aa) and C-terminus (930–1162 aa) of LANA. GST-N-LANA and GST-C-LANA bound to Glutathione Sepharose beads were used to pull down Par3 from HA-Par3 transfected HEK-293 cell lysates. (C) Endogenous immunoprecipitation assays with Par3 antibody in KSHV negative BJAB and KSHV positive (BC-3 and BCBL1) cell line was performed. (D) Par3 domains and truncations used for mapping the interaction domain. (E) Co-Immunoprecipitation of Par3 truncations (1–1266, 1–373, 1–653 and 511–1266 aa) with LANA was examined in HEK-293 cells.</p

KSHV infection leads to Par3 up-regulation.

<p>(A) Expression of Par3 was examined at the transcript and protein levels by real-time PCR and Western blot in KSHV infected PBMCs at day 2 and 6. Here RQ and RD terms are using for relative quantification and relative density, respectively. (B) Par3 were measured at the transcript and protein levels in BJAB (KSHV negative) and BCBL1 and BC-3 (KSHV positive) cell lines. (C) Par3 levels were measured in HEK-293 and HEK-293-BACKSHV at the transcript and protein levels. (D) KSHV positive cells (BC-3 and JSC-1) were knockdown with LANA and compared with controls to assessed transcript of Par3.</p

Expression of SNAIL and Par3 in BJAB and BC-3 tumors in mouse xenograft model.

<p>(A, B) NOD/SCID mice were injected with ten millions of BJAB and BC-3 cells intraperitoneal. (C, D). After 5 weeks, the mice were scarified and tumor dissected for analysis of transcripts and proteins of SNAIL and Par3 in these tumors. (E) Immunohistochemistry were performed on BJAB and BC-3 generated tumors. Here we used primary antibodies against LANA, E-cadherin and Par3. DAPI was used for nuclear staining. (F) H and E staining was shown for a similar group of tumor tissues capitalize to Fig 8F.</p

Status of EMTs in KSHV infected PBMCs and Par3 knockdown cells.

<p>(A) PBMCs were subjected to infection with KSHV and analyzed using days post-infection for the screening of epithelial (E-cadherin, Zo-1, Dsp) and mesenchymal (Snail, Lef1, B-catenin, Cdh2). qRT-PCR was performed for the transcript analysis of EMTs (epithelial to mesenchymal markers). (B) HEK293-KSHV-shControl and HEK-293KSHV-shPar3 cells were used to study these EMTs. qRT-PCR was performed to determine the fold changes for EMTs and to confirm the Par3 knockdown in HEK-293KSHV stable cell lines. (C) BJAB-shContol and BJAB-shPar3 cells were generated to study these EMTs. qRT-PCR was performed to analyze the fold changes for EMTs and to confirm Par3 expression in transiently transfected BJAB cells.</p

LANA stabilizes Par3 in KSHV positive cells.

<p>(A) HEK-293-BAC-KSHV cells were transfected with sh-LANA and sh-Control. Endogenous Par3 expression was measured and presented in graph. GAPDH was used as a protein loading control. (B, C) Stabilization of Par3 was examined with cyclohexamide in HEK-293 and BJAB cells. Left panels and right panels were used as vector control and LANA expression, in a time dependent manner. (D) The proteosome inhibitor MG132 was used to determine if Par3 stability was linked to the proteosome degradation pathway. GFP and GAPDH were used as transfection and endogenous protein loading controls. (E) BC-3-shControl and BC-3-shLANA cells were treated with cyclohexamide and observed Par3 endogenous on hours dependent manner. (F) BC-3-shControl and BC-3shLANA cells were treated with MG132 and followed immunoprecipitation of Par3. Endogenous ubiquitin and Par3 were detected using specific antibodies in both the cell lines. GAPDH was monitored as an internal control for loading in the input section.</p

NF-kB is regulated by SNAIL in KSHV positive cells.

<p>(A) BC-3 and (B) BJAB cells were treated with DMSO and SNAIL inhibitor for 48 hours and assessed through Western blots for LANA, NF-kB, SNAIL and endogenous GAPDH. (C) BC-3 shControl and BC-3-ShLANA were treated with DMSO and SNAIL inhibitor for 48 hour and blotted for Par3, NF-kB (p65), SNAIL and endogenous control GAPDH. (D) Ubiquitination assays for SNAIL were performed in BC-3sh-control and sh-NF-kB cells treated with DMSO and MG132. (E) sh-Control and sh-Par3 in presence/absence of LANA were measured in the BJAB cell background. Left panel was observed in the presence of DMSO and the right panel was treated with SNAIL inhibitor at the same time. Caspase3 and GAPDH was observed in both panels.</p

Primer sequence for ChIP analysis.

<p>Primer sequence for ChIP analysis.</p

Schematic represents a putative model illustrating the contribution of Par3 and SNAIL to KSHV-associated cancers.

<p>KSHV infected endothelial or B-cells expresses LANA important for establishment of latent infection. LANA up-regulates Par3 and SNAIL which leads to epithelial to mesenchymal transition through down-regulation of E-cadherin and enhanced expression of MMP9 in B-cells. This model suggests that KSHV-infection can regulate the EMTs important for progression of infected cells to an oncogenic and invasive phenotype.</p