Fig 1: Effect of PLEKHA7 re-expression on SUM149 cell adherens junctions. (A) SUM149 cells expressing LZRS ms neo (control) or LZRS PLEKHA7 were grown to confluence and immunofluorescence was performed for E-cadherin, p120-catenin, PLEKHA7, a-catenin, or ß-catenin. Images were obtained by confocal microscopy. Images are maximum projection intensity. Scale bar is 10µM. Representative images are shown. (B) Intensity of p120-catenin or ß-catenin staining is expressed as a ratio of the signal at apical AJs to cytoplasmic staining. N > 75 cells per condition per group. ** indicates p < 0.001, Student’s t-test (p < 0.0001 for both p120-catenin and ß-catenin). (C) Protein levels of PLEKHA7, E-cadherin, p120-catenin, ß-catenin, and a-catenin in SUM149 LZRS ms neo or SUM149 LZRS PLEKHA7 cells were obtained by Western blot. A representative image is shown. No consistent change was observed for any junctional protein in repeated experiments. (D) A representative graph displaying Wnt/ß-catenin signaling in SUM149 LZRS ms neo and SUM149 LZRS PLEKHA7 cell lines using dual luciferase reporter assay. * indicates p < 0.05, Student’s t-test (p = 0.022).
Fig 2: PLEKHA7 expression in inflammatory breast cancer (IBC) patient samples and cell lines. (A) Pie chart displaying the number of IBC patient samples demonstrating solid tumor patterns in 0–24% (N = 1 tumor), 25–74% (N = 4 tumors), or 75–100% (N = 41 tumors) of the total tumor. (B) Pie chart displaying the number of IBC patient samples demonstrating glandular tumor patterns in 0% (N = 5 tumors), 1–5% (N = 28 tumors), 6–25% (N = 8 tumors), 26–50% (N = 3 tumors), or 51–100% (N = 2 tumors) of the total tumor. (C) Pie chart depicting the average percentage of PLEKHA7 expression as lost (56.4%), cytoplasmic (26.4%), or basal (24.1%) in the regions of solid tumor from all IBC patient samples. No apical staining of PLEKHA7 was observed in areas of solid tumor. Note that total percentage is 106.9% since some tumor cells demonstrated both a cytoplasmic and basal staining pattern. (D) Pie chart depicting the average percentage of PLEKHA7 expression as lost (68.6%) or apical (31.4%) in the regions of glandular tumor from all IBC patient samples. No cytoplasmic or basal staining of PLEKHA7 was observed in areas of glandular tumor. (E) Expression of PLEKHA7 by immunohistochemistry (IHC) in normal breast tissue. Scale bar = 100 µm in all images. (F–I) Examples of expression patterns for PLEKHA7 in IBC patient samples. (F) Apical staining, (G) loss of staining, (H) cytoplasmic staining, and (I) basal staining. (J) A representative gel image demonstrating expression of PLEKHA7, E-cadherin, and p120-catenin by Western blot is shown.
Fig 3: PLEKHA7 effects on SUM149 tumor growth in orthotopic xenografts. (A) Tumor volume of xenografts from SUM149 LZRS ms neo (control) or SUM149 LZRS PLEKHA7 cells implanted into the fourth mammary gland of NOD/SCID immunocompromised mice measured at eight weeks post-implantation. * indicates p = 0.034, Student’s t-test. n = 6 for control group, n= 8 for PLEKHA7 group. (B) Fraction of Ki-67 positive cells in tumors from SUM149 LZRS ms neo or SUM149 LZRS PLEKHA7 xenografts at the eight week endpoint. p = 0.082, Student’s t-test. n = 6 for control group, n= 8 for PLEKHA7 group. (C) Representative IHC images from SUM149 LZRS ms neo or SUM149 LZRS PLEKHA7 xenografts for H & E, PLEKHA7, and Ki67. Scale bar represents 100 µm.
Fig 4: Effects of PLEKHA7 re-expression on SUM149 cell growth and survival in 3D culture. (A) SUM149 LZRS ms neo (control) and SUM149 LZRS PLEKHA7 cells were grown in Matrigel for approximately two weeks. Representative images at 4×, 10×, and 20× magnification are shown. Scale bar in each image = 100µm. (B) Quantification of SUM149 LZRS ms neo or SUM149 LZRS PLEKHA7 colonies from 3A are shown as the fold change from SUM149 LZRS ms neo. * indicates p < 0.05, Student’s t-test. (C) Representative images from SUM149 LZRS ms neo and SUM149 LZRS PLEKHA7 suspension cultures undergoing sphere compaction over 18 h. Images taken at 4×. Scale bar in each image = 100µm. (D) A representative graph of SUM149 LZRS ms neo and SUM149 LZRS PLEKHA7 suspension cultures compacting into spheres over 18 h under ultralow attachment conditions. *** indicates p < 0.001, Student’s t-test (p < 0.0001). (E) A representative graph of normalized ATP content produced by SUM149 LZRS ms neo and SUM149 PLEKHA7 spheres after treatment with various concentrations of DOXIL/doxorubicin for 72 h. ** indicates p < 0.01, Student’s t-test (p = 0.002 for 1 µM doxorubicin and 10 µM doxorubicin). Each group was normalized to no treatment.
Supplier Page from MilliporeSigma for Anti-PLEKHA7 antibody produced in rabbit