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. 2022 May 17;12:877194. doi: 10.3389/fonc.2022.877194

Figure 5.

Figure 5

Dual oxaliplatin plus palbociclib synergistically inhibited emergence of drug-resistant cells. (A) Combination index matrices of indicated doses of palbociclib and oxaliplatin combination treatment in KKU-055wt and resistant clone R29. Colors in the matrix indicate different levels of drug effect (synergistic: blue, antagonistic: red). (B) Isobologram of 0.13 µM palbociclib and 1.25 µM oxaliplatin combination treatment in KKU-055 resistant clone R29. (C) Annexin V assay of the KKU-055wt and resistant clones treated with 0.13 µM palbociclib, 4 µM oxaliplatin, the combination of both, or vehicle for 48 hours. Percent annexin V-positive cells quantification is shown in bar graphs. The bars represent the average of 4 replicates ± SD. Analysis for statistical significance was performed using Student’s t-test (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001). (D) Clonogenic survival assay of KKU-055wt and resistant clones that were treated with 1 µM palbociclib, 5 µM oxaliplatin, the combination of both, or vehicle (left). The resistant clones were cultured in medium without drug for six weeks (drug holiday), and then treated with 1 µM palbociclib, 5 µM oxaliplatin, the combination of both, or vehicle (middle). The results are representative wells of triplicates. Percent intensity quantification is shown in bar graphs (right), and the bars represent the average of 3 replicates ± SD. (E) The emergence of drug-resistant cells was demonstrated by crystal violet staining. Two-month cultures of KKU-055wt cells under 0.5 µM palbociclib, 5 µM oxaliplatin, and the combination of both in 2 of 96-well plates (192 wells). The number of emergences well was counted and plotted in a bar graph (right). (F) KKU-055 pooled R and KKU-213B pooled R were treated with the combination of 0.5 µM palbociclib and 5 µM oxaliplatin (upper), or sequentially treated with 5 µM Oxaliplatin for 7 days and followed by 0.5 µM Palbociclib for another 7 days (lower) for 2 months. The emergence of the drug-resistant cell were shown in crystal violet staining.