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Fig. 4 | Breast Cancer Research

Fig. 4

From: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

Fig. 4

NRP1 expression is associated with in vivo tumor progression, cancer stemness and spheroid-initiating potential. a Western blot analysis of NRP1 expression in non-targeting control (shNT) and NRP1 shRNA-silenced (shNRP1 [1]) SUM159 cells inoculated into mice. b Post-inoculation tumor volumes, c tumor volume at week 8 post-inoculation and d Kaplan–Meier analysis of overall survival in SUM159 shNT and NRP1 knockdown groups. e Representative images of NRP1 immunohistochemistry in shNT and shNRP1 tumors and f quantification of NRP1 IHC staining across shNT and NRP1 knockdown groups. g NRP1 expression across CL1, CL2 and CL3 claudin-low subtypes as well PAM50 classifiers in the METABRIC dataset obtained via cBioportal [11, 25]. h qPCR (left and center panel; n = 3) and Western blot (right panel) analysis of ZEB1 expression in HS578T cells after 72 h NRP1 knockdown versus NT control. i qPCR analysis of ITGA6 mRNA expression in HS578T cells (leftmost panel; NRP1 expression shown in h) and SUM159 cells (center and right panel) after 72 h NRP1 knockdown versus NT control (n = 3). j Western blot showing ITGA6 expression in HS578T, SUM159 and MDA-MB-231 cells after 72 h NRP1 knockdown versus NT control. k Western blot showing expression of ZEB1 and NRP1 in FACS sorted CD44+/CD24lo and CD44+/CD24hi populations of SUM159 cells. l Number of spheroids (> 50 µM) formed by day 6 following seeding of single cell SUM159 and Hs578T cell cultures containing 1,200 cells in the presence of 50 µg/ml Vesencumab (red lines) or IgG control (black lines). n = 5, along with m representative images of (mi) SUM159 and (mii) Hs578T spheroid cultures at days 4 and 6 post-seeding. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, ****p ≤ 0.0001, error bars represent SEM

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