Fig 1: CDYL2a and CDYL2b are the predominant isoforms expressed in human breast cancer cell lines and breast tumors. (A-B) qPCR analysis of the relative mRNA expression levels of four CDYL2 transcription variants in the indicated cell lines (A) and 60 primary breast tumor samples (20 samples for luminal, HER2-positive, and TNBC subtype, respectively). The relative expression levels of CDYL2a, CDYL2b, CDYL2c, and CDYL2d mRNA were first normalized to the GAPDH levels. Then, CDYL2a was used as a control in each breast cancer cell line (A) and breast tumor sample (B) as it was detectable in all samples. (C-D) qPCR analysis of the relative mRNA expression levels of CDYL2a (C) and CDYL2b (D) in 60 pairs of primary breast tumor tissues and matched normal breast tissues (20 samples for luminal, HER2-positive, and TNBC subtype, respectively). The expression levels of CDYL2a (C) or CDYL2b (D) in normal tissues were used as a control after normalization to the GAPDH levels. (E) Immunoblotting analysis of CDYL2 protein expression in the indicated cell lines with an anti-CDYL2 antibody. Vinculin was used as a loading control. (F) Immunoblotting analysis of CDYL2 protein expression in 14 pairs of primary breast tumor tissues and matched normal breast tissues with an anti-CDYL2 antibody. Vinculin was used as a loading control.
Fig 2: Re-expression of CDYL2a restores the impaired cell proliferation of CDYL2-depleted cells, while re-expression of CDYL2b reverses the enhanced cell migratory and invasive potential of CDYL2-depleted cells. (A) Immunoblotting analysis of CDYL2 protein expression in BT20 and MCF-7 cells stably expressing shNC, shCDYL2 #1, or shCDYL2 #5 with an anti-CDYL2 antibody. Vinculin was used as a loading control. (B) CDYL2-depleted BT20 and MCF-7 cells were reconstituted with Flag-CDYL2a or Flag-CDYL2b by lentiviral infection. The expression status of CDYL2a and CDYL2b in these cell lines was validated with immunoblotting analysis using an anti-CDYL2 antibody. Vinculin was used as a loading control. (C-E) BT20 and MCF-7 cells stably expressing shNC, shCDYL2 #1, or shCDYL2 #5 were subjected to cell proliferation assays using CCK-8 (C) and colony growth assays (D-E). Representative images of the colonies (D) and quantitative results (E) are shown. (F-H) CDYL2-depleted BT20 and MCF-7 cells were reconstituted with Flag-CDYL2a and Flag-CDYL2b by lentiviral infection and then subjected to cell proliferation assays using CCK-8 (F) and colony growth assays (G-H). Representative images of the colonies (G) and quantitative results (H) are shown. (I-K) BT20 and MCF-7 cells stably expressing shNC, shCDYL2#1, or shCDYL2#5 were subjected to wound-healing assays (I), Boyden's chamber migration assays and Matrigel-coated invasion assays (J-K). Representative images of wound-healing assays are shown in Supplementary Figure S9B and the corresponding quantitative results are shown in I. Representative images of Transwell migration and invasion assays are shown in J and the corresponding quantitative results are shown in K. (L-N) CDYL2-depleted BT20 and MCF-7 cells were reconstituted with Flag-CDYL2a and Flag-CDYL2b by lentiviral infection and then subjected to wound-healing assays (L), Boyden's chamber migration assays, and Matrigel-coated invasion assays (M-N). Representative images of wound-healing assays are shown in Supplementary Figure S9C and the corresponding quantitative results are shown in L. Representative images of Transwell migration and invasion assays are shown in M and corresponding quantitative results are shown in N. ** and *** are significant differences at p<0.01 and p<0.001, respectively.
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