Figure 4.

OGFr was necessary for OGF's inhibitory action on growth. (A) Northern blot analysis, (B) semiquantitative densitometry, and (C) Western blot analysis demonstrating the specificity and level of OGFr knockdown in RTCFs. Log-phase cells were transfected for 24 hours with either scrambled siRNA or OGFr siRNA. Forty-eight hours after transfection, the cells were harvested and the RNA isolated. Data (percent of OGFr/GAPDH ratio) represent the mean ± SE of two blots from independent experiments. ***Significantly different from nontransfected cultures at P < 0.001. (B) Photomicrographs of log-phase RTCFs stained with a polyclonal antibody to OGFr, demonstrating the extent of OGFr protein knockdown. The cells were transfected for 24 hours with OGFr siRNA or scrambled siRNA and incubated in medium for an additional 48 hours. Photomicrographs of cells stained with OGFr were taken with the same exposure time. Semiquantitative measurement of OGFr immunocytochemistry demonstrated the level of protein knockdown in the RTCFs. Decreased OGFr staining intensity (mean gray value) is indicative of decreased OGFr protein expression. Data represent the mean ± SE. ***Significantly different from nontransfected cells at P < 0.001. (C) Western blot analysis and quantitative densitometry demonstrating the level of OGFr knockdown. The cells were transfected with OGFr siRNA or scrambled siRNA. Seventy-two hours after the start of transfection, the cells were harvested and the protein isolated. Data (% of OGFr/actin) represent the mean ± SE of three independent blots. *Significant difference between nontransfected and scrambled-siRNA–transfected cultures at P < 0.05. (D) Growth of RTCFs cultures transfected with OGFr siRNA or scrambled siRNA for 24 hours and treated with OGF (10⁻⁶ M), NTX (10⁻⁶ M), or an equivalent volume of sterile water for 72 hours; compounds and media were changed daily. Values represent the mean ± SE cell counts of at least two aliquots/well and least two wells/treatment. ***Significantly different at P < 0.001 from cultures that were not transfected.