Additional file 1 of Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma
Creators: name: Xue, Zhichao
name: Lui, Vivian Wai Yan
name: Li, Yongshu
name: Jia, Lin
name: You, Chanping
name: Li, Xin
name: Piao, Wenying
name: Yuan, Hui
name: Khong, Pek Lan
name: Lo, Kwok Wai
name: Cheung, Lydia Wai Ting
name: Lee, Victor Ho Fan
name: Lee, Anne Wing Mui
name: Tsao, Sai Wah
name: Tsang, Chi Man
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: Environmental Sciences not elsewhere classified
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Figure S1. IC50 values of palbociclib in different cancers as adapted from the Genomics of Drug Sensitivity in Cancer database [31] A. A mean Day-3 palbociclib IC50 of 35.7 μM was calculated from 770 cell lines corresponding to 32 types of cancer. The Day-3 IC50 of palbociclib was 49.2 μM in 26 head and neck cancer cell lines. B. Comparison of the Day-3 IC50 values of palbociclib for our NPC cell lines and those included in the databank. Figure S2. Quantification of protein expression in western blots of Fig. 1e. The grayscale value of each band in Fig. 1e was detected using MyImage software. The protein bands were normalized to the corresponding GAPDH level in each cell line and then compared with the normalized level in the control group. Figure S3. Cytostatic effect of palbociclib in three-dimensional cultures of NPC cell lines. Cells were allowed to form spheroids and treated with palbociclib at various doses. Three-dimensional spheroids of (A) C666–1 and (B) C17 NPC cells were grown in ultra-low attachment plates. The viability of spheroids was examined using the Cell Titer Glo assay. Dose–response curves were plotted for NPC spheroids after treatment with palbociclib for 3 and 5 days. Western blot analysis indicated the significant downregulation of RB (Ser780) phosphorylation and cyclin A expression in spheroids treated with palbociclib at concentrations greater than 0.2 μM. Figure S4. Expression of p16, RB, and cyclin D1 in NPC xenografts grown in mouse models. Although p16 was undetectable in all NPC models, RB, and cyclin D1 were detectable at various levels in all models. Figure S5. Comparison of the inhibitory effect of palbociclib on the growth of different xenografts in mice. The tumor growth curves (upper panel), final tumor volumes (lower left panel), and final tumor growth inhibition (lower right panel) of all xenografts in Fig. 2 are summarized to enable a comparison of drug efficacy. Figure S6. Enlarged IHC images of tissues as shown in Figure 2c. Figure S7. Analysis of Ki-67 expression cells in tumor sections. A. Whole-tumor sections were subjected to immunohistochemical analysis for Ki-67 and scanned using a Vectra Polaris imager. The images were imported into the Pheno-Chart 13 analysis program and divided into numerous 466 μm × 349 μm regions. B. Within each region, the software automatically compartmentalized each cell according to the nuclear stain. Blue spots represent the hematoxylin-stained nuclei, and brown spots represent the Ki-67–positive cells. C. After pooling all the data from each analyzed region, the percentage of Ki-67–positive cells was calculated as the number of positive DAB-stained cells / total number of tumor cells × 100%. Figure S8. Inhibition of NPC metastasis by palbociclib. Each NOD/SCID mouse was injected with 106 C666–1 cells via the tail vein, and treatment was initiated 10 days later. A. All control mice developed NPC metastases in the lungs; in contrast, only one of four mice in the palbociclib treatment group developed lung metastases. In addition, the metastatic nodules in the lungs of the control group were larger than those in the lungs of the affected treatment group mouse. B. Solid tumor nodules in the control group lung tissues are indicated by blue arrows. C. Hematoxylin-eosin (H&amp;E) staining of representative tumors from each treatment group. The tumor nodules among the air sacs in the lungs are indicated by blue arrows. D. Higher magnification of the H&amp;E-stained slides reveals the densely packed tumor cells next to the air sacs in the lung tissues of control group mice. Figure S9. Enlarged IHC images of tissues as shown in Fig. 4b. Figure S10. Suppression of metabolic activity in Xeno23 tumors after combined treatment with palbociclib and SAHA. Micro-PET/MRI scans of Xeno23-bearing mice reveal the suppressed metabolic activity in tumors treated with palbociclib + SAHA. The mice received vehicle or combined treatment for 17 days. Figure S11. Co-treatment with palbociclib and SAHA did not promote cell differentiation or apoptosis relative to palbociclib or SAHA monotherapy. NPC43 cells were treated with 15-μM palbociclib or 5-μM SAHA alone or in combination for 6 or 24 h. The levels of proteins related to cell cycle, differentiation, and apoptosis in the treated cells were examined by western blotting. Figure S12. Lack of prominent activation of the EBV lytic cycle in NPC cells subjected to palbociclib or SAHA monotherapy or combination treatment. RNAscope was used to detect the expression of BZLF1 (which encodes the early lytic protein, Zta) in C666–1 tumors isolated from mice in the control and treatment groups. Five random regions were selected from the slides corresponding to each treatment group. The numbers of cells that harbor positive signals for BZLF1 RNA were calculated using inForm analysis software. The numbers of positive BZLF1 hybridization signal were comparable between the tumors from all control and treatment groups. Fewer than 1% of all cells in the analyzed tumor areas were positive for BZLF1, indicating that EBV lytic reactivation was not a major factor in the cell death induced by the drug treatments. Figure S13. Assessment of autophagic flux and viability in NPC43 and C17 cells in response to palbociclib and SAHA monotherapy and combined treatment. A. Analysis of autophagic flux in NPC43 and C17 cells treated with palbociclib, SAHA, or palbociclib + SAHA and the autophagic flux inhibitor chloroquine (CQ, 25 μM) for 24 h. B. Bar chart of the fold changes in LC3-II in treated cells relative to the vehicle control as shown in Fig. A. C. Viability of NPC43 and C17 cells in response to the conditions described in A. Figure S14. Knockdown of the autophagy-related proteins BECN1 and ATG5 partially reversed the cytotoxic effects of palbociclib and SAHA in NPC cells. A. NPC43 cells were transfected with siRNAs specific for BECN1 or ATG5 and then treated with palbociclib and SAHA for 24 h. Cell viability was tested using a resazurin assay. B. Knockdown of ATG5 and beclin-1 protein expression after transfection with corresponding siRNAs was verified by western blotting. The normalized grayscale values corresponding to the protein band intensities are marked in red above the bands. Figure S15. Assessment of the autophagy levels in NPC43, C666–1 and C17 cell lines by visualizing the autophagic vacuoles with GFP-LC3II. The number of autophagic vacuole as indicated by the expression of GFP-LC3-II were counted and represented in the bar charts. Increased autophagic flux in cells treated by palbociclib (15 μM) and SAHA (5 μM) for 24 h was observed.