Skip to main content
. 2018 Jan 19;9(13):11126–11144. doi: 10.18632/oncotarget.24277

Figure 3. Sema4D-Plexin B1 pathway plays a role in TGF-β1 production by tumor cells.

Figure 3

(A) IHC of stage III laryngeal SCC showing Sema4D+ve/high tumor cells (2x). (B) The peri-tumoral stroma is dense fibrotic and non-inflamed (20x). (C) PSR stain examined in bright field microscopy (2x). (D) The PSR stains the dense peri-tumoral fibrotic stroma red (20x). (E) PSR stain examined under polarized light (2x). (F) Orange red to yellow green birefringence specific to collagen I and III respectively, is observed in the peri-tumoral stromal (20x). (G) In vitro fibrosis assay. Graph illustrates decreased collagen production by fibroblasts when incubated in HN6 tumor conditioned media of Sema4D-shRNA for 72hrs compared to ctl-lentivirus. PSR stained the extra cellular collagen and was estimated using OD. Immunoblot illustrates the extent of inhibition of Sema4D using shRNA. (H) Downregulation of TGF-β1 production by HN6 cells upon Sema4D-shRNA, compared to transfection control, NT and NOK. TGF-β1 detected using ELISA. Immunoblot shows the extent of Sema4D inhibition. (I) Upper panel shows immunoblot of siRNA silencing of Plexin-B1, Sema4D and combined Plexin-B1/Sema4D, which reflected with a decrease in the activated TGF-β1 level in HN4 CM as detected by ELISA (lower panel). The immunoblot shows 2 titrations of Plexin-B1 siRNA. The CM tested for TGF-β1 was obtained from the siRNA titrations labeled with asterisks. (J) TGF-β1 upregulation in CM upon treatment of HN6 tumor cells with rhSema4D for 48hrs. CM; culture medium, NT; non-transfected, NOK; normal oral keratinocytes and NM; Normal media, PSR; Picro sirus red stain, rhSema4D; recombinant human Sema4D.