Figure 4.
Figure 4. Increased soluble autotaxin production by EBV-infected HL cells is associated with increased use of LPC and generation of LPA. (A) (upper panel) Immunoblotting demonstrates increase in autotaxin protein levels in the supernatant of EBV-infected KM-H2 cells compared with controls. Other EBV-negative HL cells produced lower but detectable levels of soluble autotaxin compared with EBV-positive variants. (lower panel) EBV-negative KM-H2 cells cultured in supernatant from EBV-infected KM-H2 cells (neo cells, EBV media) showed a transient increase in growth compared with control (EBV-negative KM-H2 cells grown in their own supernatant; neo cells, neo media). EBV-positive KM-H2 cells grown in medium from EBV-negative KM-H2 cells (EBV cells, neo media) showed reduced growth compared with control (EBV-positive KM-H2 cells grown in their own supernatant EBV cells, EBV media). ▪ indicates Neo cells, Neo media; ▦, Neo cells, EBV media; ▨, EBV cells, Neo media; and □, EBV cells, EBV media. (B) KM-H2 cells treated with exogenously supplied LPAfollowed by cell growth assay (WST-1). LPA significantly increased the growth of EBV-negative KM-H2 cells (KM-H2-neo) but not of EBV-positive cells (KM-H2-EBV). ▪ indicates KM-H2-neo; ▦, KM-H2-neo + LPA; ▨, KM-H2-EBV; and □, KM-H2-EBV + LPA. (C) (top panel) Microarray analysis of LPA/SIP receptor (EDG receptor) expression in EBV-negative KMH-2 cells and EBV-positive KM-H2 cells (KM-H2 Akata) represented as Affymetrix “calls.” P indicates mRNA present; A, mRNA absent; M, mRNA called marginal. Although EBV-negative and EBV-positive KM-H2 cells expressed the S1PR1 receptor (EDG1), they lacked expression of the major LPA receptor, LPAR1 (EDG2). The other known LPA or S1P receptors (EDG3-8) were lacking in these cells. (bottom panel) These findings were confirmed by RT-PCR analysis of a range of EBV-positive and EBV-negative HL cells (data for EDG1 and EDG2 shown). (D) Hydrolysis of 20 μM LPC by serum-free-conditioned medium, collected after 16 hours of culture over a 5-hour incubation. (E) Generation of LPA by incubation of 20 μM LPC for 3 hours with KM-H2 or KM-H2-EBV cell-conditioned medium. (F) Acyl species analysis of LPA generated by incubation of egg LPC with conditioned media. The 18:1 and 18:0 LPAs were generated by the cells without added LPC. □ indicates KM-H2 neo; and ▪, KMH2-EBV. See the “Statistical analysis” section under “Materials and methods” for explanation of error bars and asterisks.

Increased soluble autotaxin production by EBV-infected HL cells is associated with increased use of LPC and generation of LPA. (A) (upper panel) Immunoblotting demonstrates increase in autotaxin protein levels in the supernatant of EBV-infected KM-H2 cells compared with controls. Other EBV-negative HL cells produced lower but detectable levels of soluble autotaxin compared with EBV-positive variants. (lower panel) EBV-negative KM-H2 cells cultured in supernatant from EBV-infected KM-H2 cells (neo cells, EBV media) showed a transient increase in growth compared with control (EBV-negative KM-H2 cells grown in their own supernatant; neo cells, neo media). EBV-positive KM-H2 cells grown in medium from EBV-negative KM-H2 cells (EBV cells, neo media) showed reduced growth compared with control (EBV-positive KM-H2 cells grown in their own supernatant EBV cells, EBV media). ▪ indicates Neo cells, Neo media; ▦, Neo cells, EBV media; ▨, EBV cells, Neo media; and □, EBV cells, EBV media. (B) KM-H2 cells treated with exogenously supplied LPAfollowed by cell growth assay (WST-1). LPA significantly increased the growth of EBV-negative KM-H2 cells (KM-H2-neo) but not of EBV-positive cells (KM-H2-EBV). ▪ indicates KM-H2-neo; ▦, KM-H2-neo + LPA; ▨, KM-H2-EBV; and □, KM-H2-EBV + LPA. (C) (top panel) Microarray analysis of LPA/SIP receptor (EDG receptor) expression in EBV-negative KMH-2 cells and EBV-positive KM-H2 cells (KM-H2 Akata) represented as Affymetrix “calls.” P indicates mRNA present; A, mRNA absent; M, mRNA called marginal. Although EBV-negative and EBV-positive KM-H2 cells expressed the S1PR1 receptor (EDG1), they lacked expression of the major LPA receptor, LPAR1 (EDG2). The other known LPA or S1P receptors (EDG3-8) were lacking in these cells. (bottom panel) These findings were confirmed by RT-PCR analysis of a range of EBV-positive and EBV-negative HL cells (data for EDG1 and EDG2 shown). (D) Hydrolysis of 20 μM LPC by serum-free-conditioned medium, collected after 16 hours of culture over a 5-hour incubation. (E) Generation of LPA by incubation of 20 μM LPC for 3 hours with KM-H2 or KM-H2-EBV cell-conditioned medium. (F) Acyl species analysis of LPA generated by incubation of egg LPC with conditioned media. The 18:1 and 18:0 LPAs were generated by the cells without added LPC. □ indicates KM-H2 neo; and ▪, KMH2-EBV. See the “Statistical analysis” section under “Materials and methods” for explanation of error bars and asterisks.

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