pone.0240746.s006.pdf (953.54 kB)

Post transcriptional regulation of E-cadherin by MMP14 and MMP19.

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journal contribution
posted on 15.10.2020 by Lauren E. King, Hui-Hua Zhang, Cathryn M. Gould, Daniel W. Thomas, Lachlan W. Whitehead, Kaylene J. Simpson, Antony W. Burgess, Maree C. Faux

(A) Differential RNAseq analysis of MMP gene expression for SW480, SW480+APC and SW480 +control (SW480+ctrl) cells. Shown is the MEAN ± Std Dev of triplicate samples. (B) Whole cell lysate immunoblot analysis of MMP14 and E-cadherin in Difi, SW480 and SW480+APC cells. β-tubulin serves as a loading control. (C) siMMP14 knockdown (duplex#2) in SW480 cells promotes E-cadherin. E-cadherin immunoblot analysis from cells transfected with siMMP14 duplexes for 72 h. Quantification is shown in the plot below, Mean± SEM (n = 4, *p = 0.05, one-tailed unpaired Student’s t-test vs mock control). (D) MMP14 mRNA expression from SW480 cells transfected with siRNAs #1–4 or siZEB1 (SMARTpool) for 72 hours. Shown is MEAN ± SEM (n = 4), **p<0.01 (p = 0.006), ***p<0.001), one-tailed unpaired Student’s t-test vs mock control. Note only duplex #2 results in depletion of MMP14. (E) CDH1 mRNA expression from SW480 cells transfected with siMMP14 #2 or siZEB1 (SMARTpool) for 72 hours. Shown is mean ± SEM (n = 4), *p<0.05 (p = 0.028), one-tailed paired Student’s t-test vs mock control. (F) Whole cell lysis analysis of ZEB1 expression after knockdown of MMP14 #1–4. Quantification of ZEB1 protein levels (Mean ± SD (n = 2)) is shown below the representative blot. (G) siMMP19 knockdown in SW480 cells promotes E-cadherin. E-cadherin immunoblot analysis from cells transfected with siMMP19 duplexes for 72 h. Cells were harvested 72 hours post-transfection and whole cells lysates probed with antibodies against E-cadherin and β-tubulin. Quantification is shown in the plot below. Mean± SEM (n = 5) *p<0.05, **P<0.005 (exact p values are indicated) one-tailed unpaired Student’s t-test vs mock control. (H) MMP19 mRNA expression from SW480 cells transfected with siMMP duplexes #1–4 or siZEB1 (SMARTpool) for 72 hours. Shown is MEAN ± SEM (n = 4) *p<0.05 (p = 0.048), ***p<0.001 one-tailed paired Student’s t-test vs mock control. (I) CDH1 mRNA expression from SW480 cells transfected with siMMP19 duplexes #1–4 or siZEB1 (SMARTpool) for 72 hours. Shown is MEAN ± SEM (n = 4), *p<0.05 (p = 0.011), one-tailed unpaired Student’s t-test vs mock control. (J) Whole cell lysis analysis of ZEB1 expression after knockdown of MMP19 #1–4. For immunoblot analysis, cells were harvested 72 hours post-transfection and whole cells lysates probed with antibodies against ZEB1 and β-tubulin. Quantification of ZEB1 protein levels is shown below the representative blot. Mean ± SD (n = 4). Protein levels were determined using densitometry normalised to the loading control β-tubulin. For RNA expression, the data is normalised to GAPDH and shows the average of four independent experiments displayed as the Mean± SEM. Shown are cropped blots. Uncropped blots are included in S1 Raw images.

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