Fig 1: (A) Q-PCR analysis of COL3A1 transcript expression in the W1, W1PR1, W1PR1-C7, W1TR, W1TR-1p17, W1TR-2p17 cell lines. The figure presents the relative gene expression in the resistant cell lines with respect to that in the sensitive cell line, which has been assigned a value of 1. (B) Q-PCR analysis of COL3A1 transcript in the W1TR, W1TR-1p17 and W1TR-2p17 cell lines. The figure presents the relative gene expression in the CRISPR-Cas9-edited W1TR-1p17 resistant to TOP (topotecan) cell line (gray bar) and ALDH1A1+ W1TR-2p17 cell line (black bar) with respect to that in the resistant to TOP W1TR cell line, which has been assigned a value of 1 (white bar). The values were considered significant at * p < 0.05, ** p < 0.01, and *** p < 0.001. (C) Western blot analysis of COL3A1 protein expression in the W1, W1PR1, W1PR1-C7, W1TR, W1TR-1p17 and W1TR-2p17 cell lines. A primary anti-GAPDH Ab served as a loading control for the cell lysates; M-protein molecular weight marker. (D) Immunofluorescence visualization of COL3A1 expression in in W1TR, W1TR-1p17 and W1TR-2p17 cell lines. COL3A1 was detected using the anti-COL3A1 Ab and an Alexa Fluor®488-conjugated secondary Ab (green). DAPI was used to counterstain nuclei (blue). Objective 40×. Scale bar = 20 µm.
Fig 2: (A) ALDH1A1 protein expression analysis in the W1 drug-sensitive cell line and drug resistant W1PR1, W1PR1-C7, W1TR, W1TR-1p17, W1TR-2p17 cell lines. The cellular proteins were separated using 7% PAGE and transferred to a nitrocellulose membrane, immunoblotted with a primary antibody (Ab) and HRP-conjugated secondary Ab. A primary anti-GAPDH Ab served as a loading control for the cell lysates; M-protein molecular weight marker (B) Immunofluorescence visualization of ALDH1A1 expression in the W1 drug-sensitive cell line, drug resistant sublines and CRISPR-Cas9-edited variants. ALDH1A1 was detected using the anti-ALDH1A1 Ab and an Alexa Fluor®488-conjugated secondary Ab (green). DAPI was used to counterstain nuclei (blue). Objective 40×. Scale bar = 20 µm.
Fig 3: (A) Expression analysis of ALDH1A1 transcript (Q-PCR) in the W1, W1PR1, W1PR1-C7, W1TR, W1TR-1p17 and W1TR-2p17 cell lines. The figure presents the relative gene expression in the PAC (paclitaxel)-resistant cell lines (gray bars), TOP (topotecan)-resistant cell lines (black bars) with respect to that in the sensitive cell line (W1) (white bar), which has been assigned a value of 1. (B) Expression analysis of ALDH1A1 transcript (Q-PCR) in the W1PR1 and W1PR1-C7 cell lines. The figure presents the relative gene expression in W1PR1-C7 cell line (gray bar) with respect to that in the W1PR1 cell line (white bar), which has been assigned a value of 1. (C) Expression analysis of ALDH1A1 transcript (Q-PCR) in the W1TR, W1TR-1p17, W1TR-2p17. The figure presents the relative gene expression in the W1TR-1p17 cell line (gray bar) and W1TR-2p17 cell line (black bar) with respect to that in W1TR cell line (white bar), which has been assigned a value of 1. The values were considered significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.
Fig 4: (A) Q-PCR analysis of BCRP transcript expression in the W1 cell line (white bar), W1PR1 and W1PR1-C7 cell line (gray bars), W1TR cell line, W1TR-1p17 and W1TR-2p17 cell lines (black bars). The figure presents the relative gene expression in the resistant cell lines with respect to that in the sensitive cell line, which has been assigned a value of 1; (B) Q-PCR analysis of BCRP transcript expression in the W1TR, W1TR-1p17 and W1TR-2p17 cell lines. The figure presents the relative gene expression in the CRISPR-Cas9-edited W1TR-1p17 resistant to TOP (topotecan) cell line (gray bar) and ALDH1A1+ W1TR-2p17 cell line (black bar) with respect to that in the resistant to TOP W1TR cell line, which has been assigned a value of 1 (white bar). The values were considered significant at ** p < 0.01 and *** p < 0.001. (C) Western blot analysis of BCRP protein expression in the W1, W1TR, W1TR-1p17 and W1TR-2p17 cell lines. A primary anti-GAPDH Ab served as a loading control for the cell lysates; M-protein molecular weight marker. (D) Immunofluorescence visualization of BCRP expression in W1TR, W1TR-1p17 and W1TR-2p17 cell lines. BCRP was detected using the anti-BCRP Ab and an Alexa Fluor®488-conjugated secondary Ab (green). DAPI was used to counterstain nuclei (blue). Objective 40×. Scale bar = 20 µm.
Supplier Page from OriGene Technologies for ALDH1A1 Human Gene Knockout Kit (CRISPR)