Supplementary Data from Stabilized Peptide HDAC Inhibitors Derived from HDAC1 Substrate H3K56 for the Treatment of Cancer Stem–Like Cells <i>In Vivo</i>
Authors: Wang Dongyuan , Li Wenjun , Zhao Rongtong , Chen Longjian , Liu Na , Tian Yuan , Zhao Hui , Xie Mingsheng , Lu Fei , Fang Qi , Liang Wei , Yin Feng , Li Zigang 
Published: March 2023

<p>Figure S1ï¼šHDAC distribution in HeLa cells. Figure S2ï¼šEnzyme activity of peptides in SIRT1. Figure S3: CD spectra of peptide 16cyc-HxA, 16lin-HxA, 13cyc-HxA and 13lin-HxA. Figure S4: Enzyme activity of peptides in HDAC1, 3, 6, 8. Figure S5: Cellular uptake in cancer stem-like cells treated with peptide inhibitors for 4 hours. Figure S6: The transfection efficiency of peptides in cancer cells concluded from cell flow cytometry assays Figure S7: Cellular uptake in HeLa cells treated with different peptides for 4 hours. Figure S8: Cellular uptake in A549 cells treated with different peptides for 4 hours Figure S9: Time tracking of peptide cell stability in PA-1 cells treated with 10 Î¼M FITC-labeled peptides Figure S10: The comparison of cellular permeability and anti-proliferation effect in PA-1 cells treated with 16cyc-HxA and the scramble peptides Scr-1 and Scr-2. Figure S11: Anti-proliferation effect of different small inhibitors in cancer stem like cells and normal cells. Figure S12: LDH release from PA-1 cells for 4 hours and 12 hours. Figure S13: Hemolysis assays were performed to assess the erythrocyte toxicity of peptide inhibitors. Figure S14: The peptide binding affinity towards these endogenous HDAC isoforms using SA pull down assays. Figure S15: HDACs expression affected by peptide inhibitors in cancer stem-like cells and HDAC activity in A549 cells treated with peptide inhibitors. Figure S16: The irreversible inhibition of HDACs in PA-1 cell lines for 48 hours' treatment. Figure S17: The validation of HDAC inactivation using HDAC selective small inhibitors as positive controls. Figure S18: The quantifying of the different protein levels from Fig 4C. Figure S19: Peptide inhibitors could alter the recruitment of HDAC1 and LSD1 to Sox2 gene promoters by ChIP experiment in PA-1 cells. Figure S20: Apoptotic cells stained with FITC-Annexin V/PI (propidium iodide) were measured by flow cytometry. Figure S21ï¼šThe western blot assays were performed to confirm the caspase-3 dependent apoptosis in PA-1 cells Figure S22ï¼šCell cycle distribution of flow cytometry analysis treated with different peptides. Figure S23: The expression of cell cycle regulatory proteins in PA-1 cells treated with different peptide inhibitors. Figure S24: Transcriptome analysis of PA-1 cells treated with different inhibitors Figure S25: 2D diagram to show the number of genes with change of expression level and gene ontology analysis in PA-1 cells treated with different peptides. Figure S26: The relative genes were selected for RT-PCR assays to valid the results of RNA-microarray analysis. Figure S27ï¼šThe protein levels of EGFR and WNT5A1 in PA-1 cells treatment with peptide inhibitors Figure S28: The cage-wheel exercise assay was performed to study the motor learning ability of mice. Figure S29: The antitumor activities of peptides and SAHA in a NTERA-2 xenograft animal model Figure S30: The body distribution of Cy-5 labeled peptides 16cyc-HxA, 16lin-HxA and PBS at different time points after intratumor injections. Figure S31: Hematoxylin and eosin (H&E) staining of organs and tumor sections collected from different groups of mice 3 weeks after treatment Figure S32: Immunohistochemistry analysis of HDAC1 and HDAC6 and their related protein level in tumor tissue slides. Figure S33: The proliferative index was tested by doing KI67 staining on PA-1 tumor tissues. Table S1: Peptides Characterization. Table S2: Primer sequence of genes for RT-PCR analysis Table S3: Sequences of primers used for ChIP assays Table S4: Cancer related genes affect by HDAC inhibitor 16cyc-HxA in PA-1 cells.</p>