Fig 1: Silencing of Wnt signaling pathway eliminates the effect of OTX1 on cervical cancer cells. (A) Western blot analysis of protein expression of ß-catenin in CaSki cells. *P<0.05 vs. si-NC. (B) Western blot for protein expression of ß-catenin, APC, GSK-3ß and AXIN2 in CaSki cells. (C) EdU assay for proliferation, (D) wound healing assay for migration (scale bar, 100 µm) and (E) Transwell assay for invasion of CaSki cells (scale bar, 200 µm). All experiments were performed in triplicate. *P<0.05 vs. pc-NC + si-NC; #P<0.05 vs. pc-OTX1 + si-NC. OTX1, orthodenticle homolog 1; si, small interfering; NC, negative control; APC, adenomatous polyposis coli; GSK, glycogen synthase kinase; AXIN, axis inhibition protein.
Fig 2: Expression profiles and correlation analysis of multiplexed signaling proteins in A549 cells(A) The correlation analysis pipeline of protein intensity at the single-cell level. Created with BioRender.com.(B) Raw staining images in A549 cells from 23 protein markers and DAPI from 11 cycles were shown.(C) A workflow of data analysis was presented. The raw images were segmented and masked using WGA, concanavalin A, WNT1, APC, and p-EGFR. The signaling intensity was quantified based on the cytoplasmic and nuclear masks.(D) Normalized mean intensity per cell of 25 markers for one ROI. Sixty-three regions of interest and a total of 3,457 numbers of A549 cells were analyzed. The normalized total intensity was shown in Figure S10.(E) Pairwise marker correlation based on single-cell mean intensity in A549 cells was evaluated using Pearson Correlation and average linkage method on Euclidean distance. The correlation of total intensity per marker per cell was shown in Figure S11, and the correlation of total intensity was shown in Figure S12. The pairwise Pearson correlations were corrected by the Holm-Šídák method, yielding significance with a p value of p<=0.0001 (****).(F) Pairwise marker correlation based on single-cell mean intensity in the cytoplasm and nucleus-dependent manner was evaluated using Pearson Correlation using average linkage method on Euclidean distance from the same cell as (D). The pairwise Pearson correlations were corrected by the Holm-Šídák method, yielding significance with a p value of p<=0.0001 (****).
Fig 3: Inhibition of Wnt signaling pathway eliminates the effect of OTX1 on cervical cancer cells. (A) Western blot for protein expression of ß-catenin, APC, GSK-3ß and AXIN2 in CaSki cells. (B) EdU assay for proliferation, (C) wound healing assay for migration (scale bar, 100 µm) and (D) Transwell assay for invasion of CaSki cells (scale bar, 200 µm). All experiments were performed in triplicate. *P<0.05 vs. pc-NC; #P<0.05 vs. pc-OTX1. OTX1, orthodenticle homolog 1; si, small interfering; NC, negative control; APC, adenomatous polyposis coli; GSK, glycogen synthase kinase; AXIN, axis inhibition protein.
Fig 4: OTX1 activates the Wnt signaling pathway. (A) OTX1 co-expressed genes in cervical cancer were analyzed by Pearons's test (LinkedOmics). (B) Top 50 genes associated with OTX1 expression in cervical cancer (LinkedOmics). (C) Pathway analysis of OTX1 co-expressed genes by LinkedOmics using GSEA. (D) GSEA showed that OTX1 activated the Wnt signaling pathway. (E) Interaction network between OTX1 and Wnt proteins using GeneMANIA. (F) Co-IP assay was used to analyze the interaction between OTX1 and Wnt9b. Western blot for protein expression of Wnt9b, ß-catenin, APC, GSK-3ß and AXIN2 in (G) C-33A and (H) CaSki cells. All experiments were performed in triplicate. *P<0.05 vs. si-NC; #P<0.05 vs. pc-NC. OTX1, orthodenticle homolog 1; si, small interfering; NC, negative control; GSEA, gene set enrichment analysis; APC, adenomatous polyposis coli; GSK, glycogen synthase kinase; AXIN, axis inhibition protein; co-IP, co-immunoprecipitation; FDR, false discovery rate.
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