Fig. 4

Depletion of Noggin after brain colonization causes reduced motility and invasiveness of lung cancer cells. A Heatmap showing RNA sequencing data obtained from M0 and M2 cells (left). Red: up-regulated; Blue: down-regulated in M2 cells. Gene set enrichment analysis of genes differentially expressed between A549-M0 and M2 cells (right). Normalized enrichment score (NES) and false discovery rate (FDR) are shown in the graph. B qRT-PCR of NOG mRNA in M0 and M2 cells. Expression levels were normalized to RPL32 mRNA levels. P, unpaired two-tailed Student’s t-test. C Overexpression of Noggin in M2 cells. qRT-PCR analysis of NOG mRNA in M2 cells transfected with NOG cDNA or an empty vector (right, pLVX-Neo). Expression levels were normalized to RPL32 mRNA levels. P, two-tailed Student’s t-test. Western blot results of Noggin in M2 cells transfected with NOG cDNA (right). β-actin was used as a loading control. D Transwell migration assay of M0, M2, and Noggin-transfected M2 cells. Cells (1 × 10⁵/insert) were cultured in upper wells for 24 h. Migrated cells were counted after staining with crystal violet. Mean ± SD (n = 3). P, unpaired two-tailed Student’s t-test. E Wound healing assay of Noggin-transfected M2 cells. Confluent cell cultures were subjected to scratch wounds and then incubated for 48 h in the presence of mitomycin C (1 μg/mL) to attenuate the proliferation-caused effect. Mean ± SD (n = 12). P, unpaired two-tailed Student’s t-test. F Co-cultures of Noggin-transfected M2 cells on astrocyte feeder layers. Spheroids generated with Noggin-transfected M2 cells or control (green) were overlaid on top of confluent astrocyte feeder layers (red). Invasion of spheroids was observed after 12 h. Spheroid invasion area was measured by ImageJ. G qRT-PCR of NOG mRNA in A549 cells transfected with nontargeting control (NTC) or Noggin siRNAs (#1, #2, and #3). Expression levels were normalized to RPL32 mRNA levels. **P < 0.01, ***P < 0.001; unpaired two-tailed Student’s t-test. H Cell attachment assays of A549 cells transfected with NTC or Noggin siRNAs. Mean ± SD (n = 3). P, two-way ANOVA with Bonferroni post hoc tests. The adjusted P-values for NTC versus #1, NTC versus #2, and NTC versus #3 are 0.082, 0.0064, and < 0.0001, respectively. I Transwell migration assay of A549 cells transfected with NTC or Noggin siRNAs. Mean ± SD (n = 3). P, unpaired two-tailed Student’s t-test