Figure 1.

Inhibition of tubulogenesis by L2HGDH shRNA AND L2HGDH siRNA. Primary RPT cell cultures were either (A) transduced with lentivirus containing either L2HGDH shRNA or Control TRC shRNA vectors, or (D) transfected with either Silencer Select L2HGDH siRNA, or Negative Control siRNA. Prior to culturing in matrigel, the shRNA transduced cells were selected 1 week with 1.6 µg/ml puromycin, while the siRNA transfected cells were cultured 1 day to allow for gene expression. Subsequently, the primary cultures were trypsinized and passaged into matrigel in DMEM/F12-IT further supplemented with 5 ng/ml EGF, as described in Materials and Methods. Representative microscope fields of matrigel cultures are illustrated including (A1) matrigel cultures transduced with either Control TRC shRNA or L2HGDH shRNA, and (B1) matrigel cultures transfected with either Silencer Select L2HGDH siRNA or Negative Control siRNA. (B) the effect of the L2HGDH shRNA on number of tubules was quantitated, relative to Control shRNA, and (C) the relative levels of L2HGDH mRNA determined in the two conditions. Similarly, in (E) the effect of Silencer Select L2HGDH siRNA on the number of tubules was quantitated, relative to Negative Control siRNA, and (F) the relative levels of L2HGDH mRNA determined in the same 2 conditions. Values are averages +/- SEM of triplicate determinations. (*) p < 0.05 relative to either the TRC control shRNA (B, C) or the Negative Control siRNA (E, F), respectively. Scale Bars, 50 µm.