Depletion of fibroblast growth factor receptor type 1 inhibits pulmonary tumor outgrowth. (A) Real-time PCR analysis of fibroblast growth factor receptor type 1 (FGFR1) in D2.A1 cells expressing a nontargeting control short hairpin RNA (shRNA; scram) or three unique shRNA sequences targeting FGFR1 (sh#1 to sh#3). Data are the mean (±SD) of three independent experiments. (B) RT-PCR analysis of D2.A1 cells expressing the FGFR1 shRNA targeting sequences as described in (A). Primers used flank the α exon of FGFR1 and thus depict inclusion (α) or exclusion (β) of this exon. GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; sc, scrambled short hairpin RNA. (C) Three-dimensional outgrowth of the FGFR1-depleted D2.A1 cells described in (A) and (B) was quantified by bioluminescence over the course of 11 days (D0 to D11). Data are the means (±SE) of two independent experiments completed in triplicate, resulting in the indicated P value. RLU, Relative light units. (D) D2.A1 cells expressing FGFR1 shRNAs #2 or #3 were injected into the lateral tail vein of female BALB/c mice, and pulmonary tumor formation was quantified by bioluminescence. Data are the mean (±SE) area flux values at the indicated time points expressed as a percentage of the injected value (% of T0; P < 0.01; n = 5 mice/group). Insets: images of representative lungs 21 days after tail vein injection with the indicated cells. (E) Pulmonary tissues from mice injected with control (scram) and FGFR1-depleted (shFr1#2) D2.A1 cells were stained with hematoxylin and eosin (H&E) or analyzed by immunohistochemistry for phosphorylation of extracellular signal-regulated kinases 1 and 2 (pErk1/2), the proliferation marker Ki67 or FGFR1. H&E images were taken at a 1x magnification, while all immunohistochemistry images were taken at a 400x magnification. (F) RT-PCR analysis of D2.A1 cells expressing a scrambled shRNA (sc) or the #2 shRNA targeting murine FGFR1 (sh). Depletion of endogenous FGFR1 in these cells was rescued by expression of a nontargeted human full-length form of FGFR1-α-IIIc (Fr1-α) or green fluorescent protein (GFP) as a control. The relative ratio of α to β FGFR1 transcripts is shown. (G) Three-dimensional outgrowth of the FGFR1-depleted and rescued D2.A1 cells described in (C) was quantified by bioluminescence. Data are the mean (±SE) of three independent experiments completed in triplicate, resulting in the indicated P value.