Fig. 2. Knockdown of the α6A subunit in human CRC cells

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Fig. 2. Knockdown of the α6A subunit in human CRC cells Fig. 2. Knockdown of the α6A subunit in human CRC cells. (A) Representative gel showing the results of a competitive reverse transcription–polymerase chain reaction for the detection of α6A and α6B transcripts in stably expressing shctl and shα6A Caco-2/15, DLD-1, T84 and HT29 cells. (B) qPCR using probes specific for total α6, α6A and α6B confirming the specificity of abolition of α6A variant expression by shα6A relative to shctrl; *P ≤ 0.05, **P ≤ 0.01, ANOVA, n = 3. (C) Representative WB for detection of α6A and α6B subunits in shctl- and shα6A-infected Caco-2/15, DLD-1, T84 and HT29 cells. Keratin 18 (K18) was used as loading control. Densiometric analysis of α6A and α6B protein levels in shctl- versus shα6A-infected cell populations; *P ≤ 0.05, **P ≤ 0.01, t-test, n = 3. From: Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells Carcinogenesis. 2014;35(6):1217-1227. doi:10.1093/carcin/bgu006 Carcinogenesis | © The Author 2014. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 1. Expression of the α6 integrin subunit in human CRCs Fig. 1. Expression of the α6 integrin subunit in human CRCs. (A) qPCR analysis of α6, α6A and α6B transcript levels in 97 primary colorectal tumours (Tu) relative to their matched RMs. Data were normalized using B2M levels as the reference gene. ***P ≤ 0.001. (B) Graph showing the correlation between α6 and α6A transcript expression in the 97 primary colorectal tumours. Pearson r = 0.588, P ≤ 0.0001. From: Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells Carcinogenesis. 2014;35(6):1217-1227. doi:10.1093/carcin/bgu006 Carcinogenesis | © The Author 2014. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 3. Knocked down α6A splice variant decreases cell proliferation Fig. 3. Knocked down α6A splice variant decreases cell proliferation. (A) Cell counts over a 3–6 day period after the seeding of Caco-2/15, DLD-1, T84 and HT29 stably expressing shα6A or shctl. Cells were counted at the indicated times. (B) BrdU labelling assay in Caco-2/15, DLD-1, T84 and HT29 shα6A and shctl cells at 2 days post-seeding. (C) ISEL assay in Caco-2/15, DLD-1, T84 and HT29 shα6A and shctl cells at 2 days post-seeding. Cytochalasin D (CD)-treated cells were used as positive control for apoptosis. Statistical analysis between shctrl and shα6A: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, t-test, n = 3. From: Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells Carcinogenesis. 2014;35(6):1217-1227. doi:10.1093/carcin/bgu006 Carcinogenesis | © The Author 2014. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 4. Knockdown of α6A variant in human CRC cells inhibits their growth in xenografts. (A and B) Tumour growth (mm<sup>3</sup>) following subcutaneous injection of 2×10<sup>6</sup> T84 and HT29 stably expressing shα6A and shctl cells into nude mice. Tumour volumes were determined by external measurement [V = (d<sup>2</sup> × D)/2]. (C and D) Weight (g) of tumours from T84 and HT29 shctl and shα6A cells at the time of killing. Statistical analysis between shctrl and shα6A: *P ≤ 0.05, **P ≤ 0.01, t-test, n = 4. (E) Representative haematoxylin and eosin staining images for T84, HT29 and DLD-1 shctl and shα6A xenograft tumours. Scale bars = 200 μm for main panels and 50 μm for inserts. Hash symbols denote necrosis/oedema regions. (F) qPCR analyses for the expression of α6A and α6B transcript levels in xenograft tumours from T84, HT29 and DLD-1 shctl and shα6A cells. Data are expressed by α6A normalized to α6B levels. **P ≤ 0.01, <sup>§</sup>P = 0.0783, t-test, n = 4. From: Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells Carcinogenesis. 2014;35(6):1217-1227. doi:10.1093/carcin/bgu006 Carcinogenesis | © The Author 2014. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 5. Regulation of the Wnt/β-catenin pathway by the α6A variant subunit. Representative WB and graph of the densiometric analysis for the detection of active β-catenin and total β-catenin in the whole cell extract (A) and β-catenin in nuclear extracts (B). β-Actin served as loading control in cell extracts and histone H1 for nuclear extracts. Statistical analysis between shctrl and shα6A: *P ≤ 0.05, **P ≤ 0.01, t-test, n = 3. (C) TOPflash assay of the response of β-catenin/TCF4 promotor activity in the α6A variant knocked down cell lines and their corresponding shctrl. Results showed the net luciferase/renilla ratio (Topflash − FOPflash). Statistical analysis between shctrl and shα6A: *P ≤ 0.05, **P ≤ 0.01, t-test, n = 3. From: Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells Carcinogenesis. 2014;35(6):1217-1227. doi:10.1093/carcin/bgu006 Carcinogenesis | © The Author 2014. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Fig. 6. Regulation of the Wnt/β-catenin pathway Fig. 6. Regulation of the Wnt/β-catenin pathway. (A and B) Inhibition of GSK3β rescues Wnt/β-catenin activity. (A) Response of β-catenin/TCF4 promotor activity in α6A knocked down T84 cells and controls ± SB216763. Statistical analysis between shctrl and shα6A: **P ≤ 0.001; statistical analysis between − versus + SB216763: <sup>$$</sup>P ≤ 0.001; ANOVA, n = 3. (B) qPCR analysis for the expression of LGR5 and CCD2 transcript levels in shctl and shα6A cells. T84 cells were treated for 24h ± SB216763 prior to analysis. Data were normalized to RPLPO as reference gene. Statistical analysis between untreated shctrl and shα6A: **P ≤ 0.001; statistical analysis between SB216763-treated shctrl and shα6A: <sup>##</sup>P ≤ 0.001; statistical analysis between − versus + SB216763: <sup>$</sup>P ≤ 0.05, <sup>$$</sup>P ≤ 0.001; ANOVA, n = 3. (C and D) Knockdown of α6A reduces DVL2 protein levels in the four cell lines tested. (C) Representative WB and graph of the densiometric analysis of the detection of DVL2 protein levels in shctl and shα6A cells. Statistical analysis between untreated shctrl and shα6A: *P ≤ 0.05, ***P ≤ 0.0001, t-test, n = 3. (D) qPCR analysis for the expression of DVL2 transcript levels in shctl and shα6A cells. Data were normalized to RPLPO levels. (E) Working model for the involvement of the α6Aβ4 integrin in the regulation of the Wnt/β-catenin pathway. (Left) The α6Aβ4 integrin is over-expressed in CRC. When present, α6A regulates positively DVL2 at the protein level. DVL2, which inhibits GSK3β-mediated β-catenin phosphorylation, enhances β-catenin stability and translocation into the nucleus for the activation of the transcription of specific target genes involved in cell proliferation. (Right) Knockdown of the α6A subunit in CRC cells results in a decrease in DVL2 levels, thus allowing β-catenin phosphorylation by GSK3β. β-Catenin being targeted to degradation is no longer translocated to the nucleus resulting in repression of the transcription of Wnt/β-catenin-specific target genes. In this context, pharmacological inhibition of GSK3β with SB216763 restores Wnt/β-catenin pathway activity by bypassing the regulation of DVL2 by the α6A integrin subunit. The ‘?’ box denotes a still unknown mechanism by which α6A could be involved in the repression of key proteins regulating DVL2 degradation. Pathways/molecules activated are in black, whereas those inhibited are in grey. From: Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells Carcinogenesis. 2014;35(6):1217-1227. doi:10.1093/carcin/bgu006 Carcinogenesis | © The Author 2014. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com