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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: Mol Cancer Res. 2019 May 10;17(8):1735–1747. doi: 10.1158/1541-7786.MCR-19-0070

Figure 2. Loss of SPCA2 impairs E-cadherin biogenesis.

Figure 2.

(A) Knockdown of SPCA2 in MCF7 cells was confirmed by qPCR. There was no change in E-cadherin transcript. **p<0.01, Student’s t-test. (B) Western blot of cell lysates probed with antibody against E-cadherin and β-Actin. (C) E-cadherin protein expression from densitometric analysis of Western blots, normalized against β-Actin. *p<0.05, Student’s t-test, n=3. (D) Immunofluorescence of surface E-cadherin in non-permeabilized MCF7 cells treated with both control and shSPCA2. Note that the antibody recognizes an extracellular epitope. (E) Total mean fluorescence was quantified. *p<0.05, Student’s t-test, control n=276, shSPCA2 n=47. (F) Immunofluorescence of E-cadherin in permeabilized MCF7 cells treated with both control and shSPCA2. Note that the antibody recognizes a cytoplasmic epitope (G) Total mean fluorescence from (F) was quantified. ***p<0.001, Student’s t-test, control n=91, shSPCA2 n=44. (H) Immunofluorescence of total E-cadherin in permeabilized MCF7 shSPCA2 cells expressing vector control, or FLAG-tagged SPCA2R (shRNA-resistant SPCA2), with quantification (I). *p<0.05, **p<0.01, Student’s t-test, vector n=34, SPCA2R n=44. Scale bars are 20 μm. (J) Flow cytometry analysis of MCF7 shSPCA2 cells transfected with empty vector or SPCA2R showing percentages of cells in each quadrant (Q1-Q4). (K) Number of cells with high E-cadherin fluorescence found in the pink shaded area, representing 0.78% and 5.07% of total cells in Vector or SPCA2R transfected cells, respectively. (L) Histogram showing shift in total E-cadherin fluorescence in shSPCA2 cells expressing SPCA2R. *p<0.05, ***p<0.001, Student’s t-test, n=3 for each condition. See SI Appendix, Fig. S2.