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. 2020 Aug 25;11:4249. doi: 10.1038/s41467-020-17996-7

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a BrdU proliferation assay was performed on PDAC cells treated by free PAL/HCQ mixtures at various combination molar ratios. Treatment of each concentration was tested in triplicate by a 96-well BrdU proliferation assay (n = 3). Combination index (CI) were calculated for each combination ratio via the CompuSyn software. CI < 1 indicated a synergistic effect and CI ≤ 0.5 indicated a strong level of synergy. The detailed in vitro screening data in five cell lines are provided online (Supplementary Fig. 2). PANC-1 cells were incubated with PAL/HCQ combination or single drug at equivalent concentration (PAL/HCQ = 1:5, PAL = 2.5 μM, HCQ = 12.5 μM) for 72 h and subjected to the following assays to elaborate the associated biological events. b Crystal violet staining of PANC-1 cells visualized the synergistic anti-proliferative effect by PAL/HCQ pair. c Immunofluorescence staining of Ki67 proliferative marker (red), followed by quantification of Ki67⁺cells in six randomly selected ROIs (n = 6). Free PAL/HCQ combination displayed significantly enhanced anti-proliferative effect compared to PAL mono-treatment (p = 0.0027). Scale bar: 20 μm. d Immunofluorescence staining of phospho-Rb (pRb) suggested that PAL/HCQ combination effectively reduced the pRb expression, which was the hallmark for CDK4/6i-mediated growth arrest. The experiment was repeated twice. Scale bar: 20 μm. e GFP-RFP-LC3B dual reporter assay to determine the blockade on autophagosome–lysosome fusion. Six randomly selected ROIs were used in the analysis (n = 6). The abundance of RFP-LC3B puncta was significantly enhanced over GFP puncta in PAL-treated cells (p = 2.45 × 10⁻⁸), demonstrating the accumulation of autolysosomes. HCQ in combination with PAL prevented the autophagosome–lysosome fusion. Scale bar: 20 μm. f The generation of reactive oxygen species (ROS) were stained by CellROX reagent, and the mean intracellular ROS level was analyzed via flow cytometry. Data represent mean ± SD, and the statistical difference was evaluated by one-way ANOVA followed by Tukey’s post hoc test (*p < 0.05). Source data are provided as a Source data file.