Mutual regulation of B-cell associated NKL homeobox genes.

(A) RQ-PCR analysis of 13 DLBCL cell lines for MSX1 demonstrating elevated expression levels in DOHH-2 while OCI-LY1 and WILL-1 showed low MSX1 levels. (B) RQ-PCR analysis of DOHH-2 cells treated for siRNA-mediated knockdown of MSX1 demonstrated concomitantly reduced expression levels of MSX1 and NKX6-3 (left). RQ-PCR analysis of DOHH-2 cells treated for siRNA-mediated knockdown of NKX6-3 demonstrated reduced expression levels of NKX6-3 while the MSX1 transcript levels increased (right). (C) Reporter gene assay in NIH-3T3 cells using a construct which contained a potential binding site for MSX1 at position -6.011 bp of the NKX6-3 gene. In comparison to the vector control forced expression of MSX1 resulted in increased expression levels of the reporter gene, indicating that MSX1 activates NKX6-3 transcription. (D) RQ-PCR analysis for NKX6-3 of DOHH-2 and WILL-1 after treatment with BMP-receptor inhibitor dorsomorphin (DM) (left). RQ-PCR analysis for MSX1 of DOHH-2 and WILL-1 after treatment with DM (right). (E) RQ-PCR analysis for MSX1 of DOHH-2 (left) and WILL-1 (right) after treatment with recombinant BMP7 protein. (F) RQ-PCR analysis of 13 DLBCL cell lines for HHEX demonstrating reduced expression levels in DOHH-2, OCI-LY1 and WILL-1 (indicated in red). (G) RQ-PCR analysis for NKX6-3 of OCI-LY1 cells treated by forced expression with HHEX (left). RQ-PCR analysis for MSX1 of DOHH-2 cells treated by forced expression with HHEX (middle). RQ-PCR analysis of DOHH-2 cells treated by siRNA-mediated knockdown of NKX6-3 (right). While this treatment resulted in reduced expression levels of NKX6-3, no change in HHEX expression levels was detectable. (H) RQ-PCR analysis of 13 DLBCL cell lines and one HL control cell line (L-540) for HLX demonstrating reduced expression levels in all DLBCL cell lines. Of note, WILL-1 showed low transcript levels of HLX. (I) RQ-PCR analysis for NKX6-3 of OCI-LY1 treated by forced expression of HLX (left). RQ-PCR analysis for MSX1 of DOHH-2 treated by forced expression of HLX (middle). These data indicated that HLX repressed the expression of both NKX6-3 and MSX1. RQ-PCR analysis for HLX of WILL-1 treated by forced expression of MSX1 (right), indicating that MSX1 repressed the expression of HLX. (K) Reporter gene assay in NIH-3T3 cells using a construct which contained a potential binding site for HLX/MSX1 at position -6.011 bp of the NKX6-3 gene. In comparison to the control, siRNA-mediated knockdown of HLX resulted in increased expression levels of the reporter gene, indicating that HLX represses NKX6-3 transcription.