Publicly-available databases
The microarray datasets GSE72094, GSE68465 and GSE32863 were retrieved from the Gene Expression Omnibus datasets (https://www.ncbi.nlm.nih.gov/geo/). The GSE32863 dataset was applied to analyze the mRNA expression of HERPUD1 in LUAD tissues as compared to the adjacent normal tissues. GSE72094 and GSE68465 datasets were applied to construct an ER stress status classifier, and Kaplan-Meier (KM) curve to explore the prognostic value of HERPUD1.
The TCGA database (https://cancergenome.nih.gov/) was used to get the RNA-sequencing data and the clinical features of the LUAD patients. The number of fragments per kilobase of transcript per million mapped reads (FPKM) was used to determine the level of gene expression.
UCSC Xena (https://xenabrowser.net/) (Goldman et al., 2020) is an online database based on TCGA gene expression data containing genomic, mutation information and phenotypic characteristics of cancer patients..
The Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/index.html) (Tang et al., 2017a) was used to analyze the differential expression of HERPUD1 in tumors and normal samples and to explore the prognostic value of HERPUD1 level based on TCGA data.
The LinkedOmics (http://www.linkedomics.org/login.php) database was used to identify the genes interacting with HERPUD1 (Vasaikar et al., 2018).
The Tumor Immune Single Cell Hub (TISCH, http://tisch.comp-genomics.org) (Sun et al., 2021) and Tumor immune estimation resource (TIMER) (https://cistrome.shinyapps.io/timer/) (Li et al., 2020) were used to explore HERPUD1 immunophenotypic characteristics.
Construction of ER stress status classifier
The gene expression data were preprocessed and normalized by removing probes without corresponding genes and averaging the expression of probes corresponding to the duplicated gene. The ER stress-related gene set (GO RESPONSE TO ENDOPLASMIC RETICULUM STRESS) was downloaded from the Molecular Signature Database (MSigDB. v2023.1.Hs) (http://www.broad.mit.edu/gsea/msigdb/) (Liberzon et al., 2011) that contained 256 ER stress-related genes (Supplementary Table 1). Functional enrichment analysis of the ER stress-related gene set was performed the ssGSEA analysis using GSVA function in R (Hanzelmann et al., 2013b). The GSVA results were obtained employing k-means clustering and heatmap plotting by “pheatmap” package in R. Moreover, the optimal clusters were further verified to perform consensus clustering by “ConsensusClusterPlus” package in R using k-means algorithm with the Euclidean distance (pItem = 0.8, reps = 1000) (Wilkerson and Hayes, 2010), and the principal component analysis (PCA) was applied by “stats” package in R to better verify the variations between clusters.
The expression of HERPUD1 in lung cancer compared to normal lung tissue
We utilized the GEPIA (http://gepia.cancer-pku.cn/index.html) database to investigate the expression of HERPUD1 in the TCGA dataset (Tang et al., 2017b). Furthermore, we employed the GSE32863 dataset to assess the mRNA expression of HERPUD1 in both lung cancer patients and their corresponding normal adjacent individuals. Moreover, we analyzed HERPUD1 expression in tumor tissues and adjacent normal tissues obtained from 17 diagnosed lung cancer patients at Renmin Hospital of Wuhan University. This study has been approved by the Ethics Committee of the Medical School of Wuhan University (Wuhan, China).
Immunohistochemistry
The tissue sample sections were dewaxed using xylene twice for 15 minutes each, followed by rehydration with gradient ethanol. After rinsing with PBS buffer three times for 3 minutes each, the antigen was retrieved by boiling the slides in citrate buffer (Wuhan Servicebio Technology, G1202) for 15 minutes at a high temperature. Once cooled to room temperature, 50µL of peroxidase blocker was added to each tissue section. Subsequently, the tissues were treated with a blocking solution containing 5% BSA for 15 minutes at normal temperature. Next, the tissue microarray slides were incubated overnight at 4℃ with the primary antibody, anti-HERPUD1, at a dilution of 1:150. After incubation, the slides were washed with PBS and incubated with a secondary antibody (Wuhan Servicebio Technology, G1215) for 30 minutes at 37℃. Finally, the tissues were reacted with 3,3-diaminobenzidine and counterstained with hematoxylin. To evaluate the immunohistochemical results, the histochemistry score (H-score) was calculated using the formula: H-score = (percentage of cells of weak intensity × 1) + (percentage of cells of moderate intensity × 2) + (percentage of cells of strong intensity × 3). The LUAD tissue microarray HEsoS045Ly01 comprised 30 LUAD carcinoma tissues and 30 paired non-cancerous lung tissues (Xinchao, China). Antibodies; HERPUD1 (Abclonal, A14097).
Cell culture
The human lung cancer cell lines A549 and PC9, along with the human normal lung cell line BEAS-2B, were obtained from the American Type Culture Collection (ATCC) in Manassas, VA. A549 and BEAS-2B cells were cultured in RPMI-DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin from Biological Industries, Israel, and Beyotime Biotechnology, China, respectively. These cells were maintained at 37°C in a humidified atmosphere with 5% CO2. The PC9 cell line was cultured similarly, using RPMI-1640 medium with the same supplements. For lentiviral transfection, HEK293T cells were obtained from ATCC and cultured in DMEM supplemented with 10% FBS.
Construction of HERPUD1 overexpression stable lung cancer cell lines
The HERPUD1 expression construct pLenti/CMV-HERPUD1-GFP-Puro and control vector pLenti/CMV-GFP-Puro were purchased from the Public Protein/Plasmid Library Company. The plasmids were co-transfected into HEK293T cells with the packaging plasmids psPAX2 and pMD2.G, Lentivirus was produced in HEK293T cells using PEI-MAX according to the instruction. Supernatants containing the lentivirus were collected. For transduction, 3×105 target lung cancer cells, A549 and PC9, were incubated with 0.5mL lentiviral supernatant containing polybrene (8µg/mL) (Santa cruz, Germany) in 1.5mL RPMI-1640 or DMEM medium. The stable cell lines expressing HERPUD1 were generated. After 48 hours, the cells were selected in the culture medium containing puromycin. Finally, the overexpression efficiency was validated by RT-qPCR and western blotting.
Real-time quantitative PCR (RT-qPCR)
Total RNA was extracted from patients’ tissue samples or LUAD cell lines, and RT-qPCR analysis was performed. Reverse transcription was facilitated using HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, China) following the manufacturer's protocol. For quantitative RT-PCR analysis, the ChamQ SYBR qPCR Master Mix (Vazyme, China) was used in conjunction with the state-of-the-art Quant Studio 6 Flex Real-Time PCR System from Life Technologies, USA. To ensure result accuracy, the average Ct value of the target gene was normalized using the reference gene GAPDH. Gene expression quantification was performed using the 2^(−ΔΔCt) method. For detailed information on the primer sequences used in the RT-qPCR experiments, please refer to Supplementary Table 2.
Western blot
Cells were washed three times with ice-cold PBS (Servicebio Technology, China) and lysed with RIPA (Beyotime Biotechnology, China) supplemented with protease inhibitor PMSF and phosphatase inhibitors (Beyotime Biotechnology, China) on ice. The protein concentrations were measured using the bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, China). The protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies overnight at 4°C. After the overnight incubation with the primary antibodies, membranes were washed with TBS-T buffer (Monad, China) for 6 times, 5min each time and incubated with HRP-labeled secondary antibody (Proteintech, China) at room temperature for 2h. The proteins were exposed using an ultra-high sensitivity ECL kit (MedChemExpress, USA). GAPDH was used as a loading control. Antibodies; HERPUD1 (Abcam, ab150424), JAK2 (Cell Signaling Technology, O60674), p-JAK2 (ABclonal, AP0917), STAT3 (Abclonal, A19566), p-STAT3 (Abclonal, AP0705), CHOP (Abclonal, A11346), GRP78 (Abclonal, A0241), IRE1 (Abclonal, A21021), E-cadherin (Proteintech, 20874-1-AP), N-cadherin (Proteintech, 22018-1-AP), Vimentin (Proteintech, 10366-1-AP), GAPDH (ABclonal, AC002), Cyclin D1 (ABclonal, AC19038).
Immunofluorescence assay
After transfection with either the control vector or OE-HERPUD1 plasmid, A549 cells were plated in a 6-well culture plate with adhesive cover slides (50 × 104 cells per well). The plate was then kept in a 37°C, 5% CO2 incubator for 24 hours. After the incubation period, the cells on the cover slides were fixed using 4% paraformaldehyde for 30 minutes. A tissue pen was used to draw a circle around the evenly distributed cells to prevent the antibody from running away. The cells within the marked circle were covered with 5% BSA at room temperature for 30 minutes. Subsequently, the primary antibody for HERPUD1 at a dilution of 1:150 was added in PBS and incubated overnight at 4°C. Following the incubation with the primary antibody, the corresponding secondary antibody with a species-specific label was added to the cover slide and incubated at room temperature for 50 minutes. DAPI dye solution was added and incubated for 10 minutes at room temperature in the dark to visualize cell nuclei. After the section was slightly dried, a self-fluorescence quenching agent was added to the labeled circle for 5 minutes. Finally, the slides were sealed with anti-fluorescence quenching tablets and observed under a fluorescence microscope for imaging and analysis.
Cell counting kit-8 assay
The CCK-8 assay kit from Dojindo Molecular Technologies, Japan, was utilized to assess cell proliferation following the manufacturer's instructions. A549 and PC9 cells were transfected with either the control vector or the OE-HERPUD1 plasmid and cultured in 96-well plates. Each well was seeded with 2 × 103 cells and allowed to attach overnight. At different time intervals (0, 24, 48, 72, and 96 hours), 10µL of the CCK-8 solution was added to each well, followed by a 2-hour incubation at 37°C. The absorbance of each well was then measured at an optical density of 450 nm (OD 450 nm) using a spectrophotometer.
Clone formation assay
After transfection with the control vector or OE-HERPUD1 plasmid, A549 and PC9 cells were seeded into a 6-well culture plate at a density of 800 cells per well. The plate was then placed in a 37°C, 5% CO2 incubator for three weeks to allow cell growth and colony formation. After the three-week incubation period, the cell colonies were fixed using 4% paraformaldehyde for 30 minutes. Subsequently, the colonies were stained with crystal violet for 15 minutes to visualize and distinguish them. Finally, the colonies were observed and photographed under a microscope for further analysis.
Migration and invasion assays
For migration assays, a transwell chamber with an 8 µm pore size (Corning, USA) was used. In the upper chambers, 3 × 104 cells were plated and cultured in 100µL RPMI-1640 medium with 1% FBS. In contrast, the lower chambers contained 600µL RPMI-1640 medium supplemented with 10% FBS. After 24 hours, cells on the upper side of the chamber were removed, while those on the lower side were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet for 15 minutes, and photographed under a microscope. Cell counts were performed in five randomly selected fields under a light microscope. For the invasion assay, transwell membranes were coated with a diluted Matrigel (BD Biosciences) at a 1:10 ratio with RPMI-1640 medium. Then, 3 × 104 cells per chamber were resuspended in 100µL RPMI-1640 medium and seeded into the upper chamber of a 24-well chemotaxis chamber with polycarbonate filters (8µm pore) (Corning Incorporated, Glendale, AZ, USA). In the lower chamber, 600µL RPMI-1640 supplemented with 10% FBS was added. After 24 hours, cells were treated and counted similarly to the migration assay.
Immune infiltration analysis
RNA-sequencing expression profiles and corresponding clinical information for LUAD were downloaded from the TCGA dataset. To assess immune score, we used immuneeconv (Sturm et al., 2020), which is software package in R program that integrates six latest algorithms, including TIMER, XCell, MCP-counter, CIBERSORT, EPIC and QuanTIseq.
CIBERSORT, one of the algorithms integrated into "immuneeconv," employs a deconvolution algorithm to characterize the composition of immune cells. This method is validated using a leukocyte gene signature matrix comprising 22 human immune subsets and a total of 547 genes. To evaluate the immune score in lung cancer samples exhibiting high or low HERPUD1 expression, we downloaded the microarray dataset and the corresponding clinical information for LUAD from GSE72094. Subsequently, we employed the "immuneeconv" R package to perform the analysis and assess the immune score in these samples.
Cytokine determinations
Cell supernatant samples from vector or OE-HERPUD1 group were centrifuged for 10 minutes, the supernatant was taken, and diluent was used for 4-fold dilution. Finally, the diluted 50 µL sample was taken for testing. Bio-Plex Pro Human Cytokine Screening 40-plex Panel (Bio-Rad Laboratories, Hercules, CA) was used to screen 40 cytokine and chemokine cell signaling molecules in complex extracellular events and signaling pathways. The panel covered a biologically relevant collection of adaptive mononuclear/macrophage chemotactic-related proteins (CCL7, CCL8, CCL15, CCL2, and MIF), M2 polarization-related factors (IL-1β, IL-4), neutrophil chemotactic (CXCL2, CXCL5, CXCL6, IL-8), dendritic cells (DCs) chemotactic-related proteins (CCL19, CCL20, and CCL21), Natural killer (NK) cell related cytokines (CXCL9, CXCL10), Th2 cell-related cytokines (CCL1, CCL11, CCL26) (Table 4). After the samples and standards were detected by the Luminex 200 detector (Luminex Corporation, Austin, TX), the fluorescence obtained was automatically calculated and optimized by the software to form an output file in Excel format. Representative assay working ranges, assay sensitivity.
Table 4
Additional information of cytokines for Luminex assay
Cytokines | Name in Bio-Plex Pro Human Chemokine Panel | Description |
CCL21 | 6Ckine/CCL21 (12) | C-C Motif Chemokine Ligand 21; Secondary Lymphoid Tissue Chemokine |
CXCL5 | ENA-78/CXCL5 (73) | C-X-C Motif Chemokine Ligand 5; Epithelial-Derived Neutrophil-Activating Protein 78 |
CCL22 | MDC/CCL22 (29) | Macrophage-Derived Chemokine; C-C Motif Chemokine 22 |
MIF | MIF (35) | Macrophage Migration Inhibitory Factor |
CCL15 | MIP-18/CCL15 (66) | C-C Motif Chemokine Ligand 15; Macrophage Inflammatory Protein 1delta |
CXCL12 | SDF-1a + b/CXCL12 (22) | C-X-C Motif Chemokine Ligand 12; Stromal Cell-Derived Factor 1 |
CCL25 | TECK/CCL25 (46) | C-C Motif Chemokine Ligand 25; Thymus Expressed Chemokine |
CCL27 | CTACK/CCL27 (72) | C-C Motif Chemokine Ligand 27; Cutaneous T-Cell Attracting Chemokine |
CCL24 | Eotaxin-2/CCL24 (30) | C-C Motif Chemokine Ligand 24; Myeloid Progenitor Inhibitory Factor 2 |
CXCL1 | Gro-a/CXCL1 (61) | C-X-C Motif Chemokine Ligand 1; Growth-Regulated Alpha Protein |
CXCL2 | Gro-b/CXCL2 (78) | C-X-C Motif Chemokine 2; Growth-Regulated Protein Beta |
IL-16 | IL-16 (27) | Interleukin 16; Lymphocyte Chemoattractant Factor |
IL-4 | IL-4 (52) | Interleukin 4 |
CXCL10 | IP-10/CXCL10 (48) | C-X-C Motif Chemokine Ligand 10; Protein 10 From Interferon (Gamma) |
CCL2 | MCP-1/CCL2 (53) | C-C Motif Chemokine Ligand 2; Monocyte Chemoattractant Protein-1 |
CCL7 | MCP-3/CCL7 (26) | C-C Motif Chemokine Ligand 7; Monocyte Chemoattractant Protein 3 |
CCL19 | MIP-3b/CCL19 (76) | C-C Motif Chemokine Ligand 19; Macrophage Inflammatory Protein 3 Beta |
CCL23 | MPIF-1/CCL23 (37) | C-C Motif Chemokine Ligand 23; Myeloid Progenitor Inhibitory Factor 1 |
CXCL16 | SCYB16/CXCL16 (64) | C-X-C Motif Chemokine Ligand 16; Scavenger Receptor For Phosphatidylserine |
CXCL13 | BCA-1/CXCL13 (74) | C-X-C Motif Chemokine Ligand 13; B-Cell-Attracting Chemokine 1 |
CCL26 | Eotaxin-3/CCL26 (65) | C-C Motif Chemokine Ligand 26; Macrophage Inflammatory Protein 4 |
CCL11 | Eotaxin/CCL11 (43) | C-C Motif Chemokine Ligand 11; |
CX3CL1 | Fractalkine/CX3CL1 (77) | C-X3-C Motif Chemokine Ligand 1; |
CXCL6 | GCP-2/CXCL6 (15) | C-X-C Motif Chemokine Ligand 6; Granulocyte Chemotactic Protein 2 |
GM-CSF | GM-CSF (34) | Granulocyte-Macrophage Colony-Stimulating Factor |
CCL1 | I-309/CCL1 (20) | C-C Motif Chemokine Ligand 1; T Lymphocyte-Secreted Protein I-309 |
CXCL11 | I-TAC/CXCL11 (25) | C-X-C Motif Chemokine Ligand 11; Interferon-Inducible T-Cell Alpha Chemoattractant |
IFN-g | IFN-g (21) | Interferon Gamma |
IL-10 | IL-10 (56) | Interleukin 10 |
IL-1b | IL-1b (39) | Interleukin 1 Beta |
IL-2 | IL-2 (38) | Interleukin 2; T Cell Growth Factor |
IL-6 | IL-6 (19) | Interleukin 6 |
IL-8 | IL-8/CXCL8 (54) | C-X-C Motif Chemokine Ligand 8; Interleukin 8 |
CCL8 | MCP-2/CCL8 (57) | C-C Motif Chemokine Ligand 8; Monocyte Chemoattractant Protein 2 |
CCL13 | MCP-4/CCL13 (28) | C-C Motif Chemokine Ligand 13; Monocyte Chemoattractant Protein 4 |
CXCL9 | MIG/CXCL9 (14) | C-X-C Motif Chemokine Ligand 9; Monokine Induced By Gamma Interferon |
CCL3 | MIP-1a/CCL3 (55) | C-C Motif Chemokine Ligand 3; Macrophage Inflammatory Protein 1-Alpha |
CCL20 | MIP-3a/CCL20 (62) | C-C Motif Chemokine Ligand 20; Macrophage Inflammatory Protein 3 Alpha |
CCL17 | TARC/CCL17 (67) | C-C Motif Chemokine Ligand 17; Thymus And Activation-Regulated Chemokine |
TNF-a | TNF-a (36) | Tumor Necrosis Factor-Alpha |
scRNA-seq data processing
GSE171145 dataset comprises annotated cell types from each sample in lung cancer. Diverse immune cell types were identified using the Seurat analysis package (Butler et al., 2018) in GSE171145. The t-distributed stochastic neighbor embedding (t-SNE) technique enabled the recognition of T cells (both CD4 and CD8), B cells, myeloid cells, mast cells, and NK cells. CD8 T cells, CD4 T cells, and myeloid cells were classified and recognized based on previously reported markers.
Immunotherapy response and chemotherapy
Tumor Immune Dysfunction and Exclusion (TIDE) (http://tide.dfci.harvard.edu/) (Fu et al., 2020), is a computational tool designed to model two key mechanisms of tumor immune escape. These mechanisms include the dysfunction of tumor-infiltrating cytotoxic T lymphocytes (CTL) and the rejection of CTL by immunosuppressive factors (Liu et al., 2021). Notably, higher TIDE scores are indicative of poorer clinical efficacy when it comes to immune checkpoint inhibitors. In our investigation of the clinic performance of chemotherapy agents in patients, we determined the half-inhibitory concentration (IC50) values of commonly used drugs. To accomplish this, we utilized the "pRRophetic" package (Geeleher et al., 2014) and based our computations on the GDSC (https://www.cancerrxgene.org/) database. Table 5 displays the chemotherapy agents that are associated with HERPUD1.
Table 5
Chemotherapeutic agents related to HERPUD1
Drug name | statistic | diff | p-value | p-adj |
XMD8-85 | -8.69882 | 0.267608 | 6.96E-17 | 1.08E-14 |
I-BET-762 | -8.66486 | 0.416653 | 9.08E-17 | 1.08E-14 |
TG101348 | -8.56233 | 0.371686 | 1.88E-16 | 1.48E-14 |
KIN001-260 | -8.47854 | 0.275825 | 3.48E-16 | 2.06E-14 |
TL-1-85 | -8.35 | 0.378475 | 8.97E-16 | 4.25E-14 |
TGX221 | -7.95418 | 0.249437 | 1.55E-14 | 6.12E-13 |
PFI-1 | -7.91157 | 0.271229 | 2.13E-14 | 7.21E-13 |
NG-25 | -7.62361 | 0.310037 | 1.54E-13 | 4.55E-12 |
Rapamycin | -7.55645 | 0.180105 | 2.67E-13 | 6.98E-12 |
BIX02189 | -7.52799 | 0.261193 | 2.95E-13 | 6.98E-12 |
THZ-2-102-1 | -7.38965 | 0.537685 | 7.71E-13 | 1.61E-11 |
CGP-60474 | -7.38579 | 0.205497 | 8.14E-13 | 1.61E-11 |
VNLG/124 | -7.16215 | 0.2063 | 3.39E-12 | 6.17E-11 |
JW-7-24-1 | -7.14401 | 0.331052 | 3.83E-12 | 6.24E-11 |
XMD11-85h | -7.13858 | 0.145943 | 3.95E-12 | 6.24E-11 |
Temsirolimus | -7.12272 | 0.376738 | 4.44E-12 | 6.57E-11 |
PIK-93 | -7.05798 | 0.394885 | 6.63E-12 | 9.25E-11 |
Sunitinib | -6.94298 | 0.245007 | 1.45E-11 | 1.91E-10 |
GSK2126458 | -6.86707 | 0.320145 | 2.25E-11 | 2.81E-10 |
THZ-2-49 | -6.75939 | 0.426377 | 4.53E-11 | 5.37E-10 |
DMOG | -6.72764 | 0.280943 | 5.56E-11 | 6.01E-10 |
BHG712 | -6.72242 | 0.294182 | 5.58E-11 | 6.01E-10 |
AP-24534 | -6.67844 | 0.295656 | 7.31E-11 | 7.53E-10 |
KIN001-244 | -6.65767 | 0.19384 | 8.34E-11 | 8.24E-10 |
YM201636 | -6.63746 | 0.194789 | 9.51E-11 | 8.92E-10 |
TPCA-1 | -6.63236 | 0.332604 | 9.78E-11 | 8.92E-10 |
XMD8-92 | -6.63061 | 0.227027 | 1.04E-10 | 9.12E-10 |
BAY 61-3606 | -6.59469 | 0.234954 | 1.26E-10 | 1.07E-09 |
Roscovitine | -6.57189 | 0.101894 | 1.46E-10 | 1.20E-09 |
CAL-101 | -6.49929 | 0.224307 | 2.19E-10 | 1.73E-09 |
AZD8055 | -6.43309 | 0.225233 | 3.30E-10 | 2.52E-09 |
PHA-665752 | -6.38307 | 0.066153 | 4.42E-10 | 3.27E-09 |
17-AAG | 6.36565 | -0.43598 | 5.12E-10 | 3.68E-09 |
Cyclopamine | -6.34279 | 0.09789 | 5.61E-10 | 3.91E-09 |
ZSTK474 | -6.29599 | 0.279987 | 7.48E-10 | 5.06E-09 |
CP466722 | -6.10886 | 0.227677 | 2.27E-09 | 1.49E-08 |
AC220 | -6.05627 | 0.148679 | 3.01E-09 | 1.93E-08 |
XMD14-99 | -5.92294 | 0.15966 | 6.42E-09 | 4.00E-08 |
Y-39983 | -5.86708 | 0.242445 | 8.73E-09 | 5.31E-08 |
KIN001-236 | -5.71931 | 0.162382 | 1.98E-08 | 1.17E-07 |
CAY10603 | -5.67759 | 0.275524 | 2.50E-08 | 1.45E-07 |
SB 505124 | -5.66989 | 0.146592 | 2.60E-08 | 1.47E-07 |
SNX-2112 | -5.6224 | 0.426261 | 3.39E-08 | 1.87E-07 |
Tamoxifen | -5.61274 | 0.119457 | 3.54E-08 | 1.91E-07 |
BX-912 | -5.57764 | 0.33264 | 4.28E-08 | 2.25E-07 |
CEP-701 | -5.57136 | 0.35388 | 4.46E-08 | 2.30E-07 |
Parthenolide | -5.55628 | 0.128205 | 4.78E-08 | 2.41E-07 |
JW-7-52-1 | -5.51487 | 0.030224 | 6.51E-08 | 3.21E-07 |
BEZ235 | -5.42727 | 0.194769 | 9.55E-08 | 4.62E-07 |
Crizotinib | -5.39375 | 0.132337 | 1.14E-07 | 5.39E-07 |
QL-XII-61 | -5.33688 | 0.168281 | 1.52E-07 | 7.06E-07 |
UNC1215 | -5.31656 | 0.086297 | 1.70E-07 | 7.74E-07 |
OSI-027 | -5.31063 | 0.373708 | 1.76E-07 | 7.85E-07 |
Masitinib | -5.29961 | 0.191631 | 1.86E-07 | 8.16E-07 |
Sorafenib | -5.27084 | 0.075299 | 2.23E-07 | 9.44E-07 |
S-Trityl-L-cysteine | -5.2635 | 0.112852 | 2.23E-07 | 9.44E-07 |
TL-2-105 | -5.22058 | 0.148153 | 2.79E-07 | 1.16E-06 |
Linifanib | -5.20472 | 0.134392 | 2.99E-07 | 1.22E-06 |
Ruxolitinib | -5.18258 | 0.117468 | 3.36E-07 | 1.35E-06 |
MK-2206 | -5.15486 | 0.17427 | 3.89E-07 | 1.54E-06 |
Zibotentan | -5.13321 | 0.082224 | 4.30E-07 | 1.67E-06 |
KIN001-270 | -5.11499 | 0.150668 | 4.71E-07 | 1.80E-06 |
PD-173074 | -5.10566 | 0.119808 | 4.92E-07 | 1.85E-06 |
Phenformin | -5.09424 | 0.189648 | 5.24E-07 | 1.94E-06 |
FR-180204 | -5.07323 | 0.093385 | 5.82E-07 | 2.12E-06 |
Tubastatin A | -5.00867 | 0.180525 | 7.97E-07 | 2.86E-06 |
HG-6-64-1 | -4.99426 | 0.267217 | 8.60E-07 | 3.04E-06 |
XL-184 | -4.98986 | 0.169816 | 8.73E-07 | 3.04E-06 |
XMD15-27 | -4.87017 | 0.128203 | 1.56E-06 | 5.36E-06 |
Dabrafenib | -4.81898 | 0.239311 | 2.01E-06 | 6.80E-06 |
FK866 | -4.78208 | 0.537876 | 2.38E-06 | 7.90E-06 |
KIN001-102 | -4.77983 | 0.192739 | 2.43E-06 | 7.90E-06 |
PI-103 | -4.77821 | 0.409516 | 2.43E-06 | 7.90E-06 |
Pazopanib | -4.64434 | 0.110404 | 4.52E-06 | 1.43E-05 |
Tivozanib | -4.64375 | 0.115827 | 4.54E-06 | 1.43E-05 |
AR-42 | -4.63811 | 0.21637 | 4.65E-06 | 1.45E-05 |
QL-XI-92 | -4.59085 | 0.201084 | 5.78E-06 | 1.78E-05 |
ZG-10 | -4.58854 | 0.141458 | 5.87E-06 | 1.78E-05 |
QL-VIII-58 | -4.58676 | 0.157934 | 5.94E-06 | 1.78E-05 |
piperlongumine | -4.56061 | 0.131879 | 6.65E-06 | 1.97E-05 |
ATRA | -4.51574 | 0.146018 | 8.12E-06 | 2.38E-05 |
Z-LLNle-CHO | -4.45467 | 0.143038 | 1.09E-05 | 3.14E-05 |
Genentech Cpd 10 | -4.44963 | 0.181757 | 1.10E-05 | 3.14E-05 |
IPA-3 | -4.44125 | 0.204349 | 1.14E-05 | 3.21E-05 |
QL-X-138 | -4.42695 | 0.23133 | 1.21E-05 | 3.38E-05 |
Belinostat | -4.4246 | 0.215584 | 1.23E-05 | 3.38E-05 |
Foretinib | -4.41901 | 0.206329 | 1.25E-05 | 3.41E-05 |
AMG-706 | -4.40882 | 0.085354 | 1.31E-05 | 3.50E-05 |
MG-132 | -4.40869 | 0.110171 | 1.32E-05 | 3.50E-05 |
CUDC-101 | -4.35436 | 0.211356 | 1.68E-05 | 4.42E-05 |
CP724714 | 4.316311 | -0.10306 | 1.98E-05 | 5.17E-05 |
FH535 | 4.308601 | -0.13657 | 2.04E-05 | 5.27E-05 |
AZD7762 | -4.29636 | 0.297268 | 2.15E-05 | 5.48E-05 |
VX-702 | -4.28413 | 0.108586 | 2.26E-05 | 5.70E-05 |
Epothilone B | 4.28171 | -0.20395 | 2.32E-05 | 5.79E-05 |
Bortezomib | -4.25329 | 0.161234 | 2.59E-05 | 6.39E-05 |
NU-7441 | -4.2327 | 0.10968 | 2.82E-05 | 6.89E-05 |
PHA-793887 | -4.12788 | 0.240099 | 4.39E-05 | 0.000106 |
STF-62247 | -4.02379 | 0.114397 | 6.77E-05 | 0.000162 |
QL-XII-47 | -3.98954 | 0.203073 | 7.76E-05 | 0.000184 |
FMK | -3.95081 | 0.088652 | 9.12E-05 | 0.000214 |
OSI-930 | -3.93216 | 0.116997 | 9.80E-05 | 0.000228 |
XMD13-2 | -3.91943 | 0.190213 | 0.000103 | 0.000238 |
LFM-A13 | -3.84294 | 0.061058 | 0.00014 | 0.000318 |
GSK429286A | -3.81823 | 0.133459 | 0.000154 | 0.000347 |
NPK76-II-72-1 | -3.79299 | 0.189619 | 0.00017 | 0.000377 |
WZ3105 | -3.7914 | 0.171999 | 0.000171 | 0.000377 |
Bexarotene | -3.7917 | 0.083126 | 0.000172 | 0.000377 |
Nilotinib | -3.70321 | 0.114819 | 0.000241 | 0.000525 |
CMK | -3.673 | 0.079829 | 0.00027 | 0.000581 |
T0901317 | -3.65281 | 0.110842 | 0.000292 | 0.000623 |
5-Fluorouracil | -3.60505 | 0.213781 | 0.000348 | 0.000737 |
AT-7519 | -3.60145 | 0.214313 | 0.000353 | 0.000738 |
CCT007093 | -3.5999 | 0.0676 | 0.000355 | 0.000738 |
KIN001-266 | -3.58786 | 0.078573 | 0.000372 | 0.000766 |
CH5424802 | -3.57441 | 0.095485 | 0.000391 | 0.000798 |
A-770041 | -3.5708 | 0.133507 | 0.000397 | 0.000804 |
Salubrinal | -3.4415 | 0.04288 | 0.000636 | 0.001277 |
BIRB 0796 | -3.36005 | 0.046181 | 0.00085 | 0.001693 |
Methotrexate | -3.2827 | 0.180021 | 0.001111 | 0.002194 |
Imatinib | -3.25454 | 0.037431 | 0.001226 | 0.002401 |
JNJ-26854165 | -3.24831 | 0.100345 | 0.001253 | 0.002435 |
MLN4924 | -3.16513 | 0.036618 | 0.001663 | 0.003204 |
KIN001-135 | -3.15429 | 0.029654 | 0.001722 | 0.003291 |
Vinorelbine | 3.134123 | -0.11269 | 0.001844 | 0.003496 |
EX-527 | -3.12346 | 0.068108 | 0.001909 | 0.003592 |
LAQ824 | -3.11005 | 0.078015 | 0.001994 | 0.003722 |
(5Z)-7-Oxozeaenol | -3.08266 | 0.188026 | 0.002182 | 0.00404 |
AS605240 | -3.06587 | 0.119463 | 0.002308 | 0.00424 |
HG-5-88-01 | -3.02874 | 0.101653 | 0.002607 | 0.004752 |
SGC0946 | -2.99926 | 0.047182 | 0.002865 | 0.005184 |
GSK690693 | -2.97478 | 0.136664 | 0.003102 | 0.00557 |
MPS-1-IN-1 | -2.90301 | 0.1229 | 0.003887 | 0.006926 |
Pyrimethamine | 2.865056 | -0.102 | 0.004381 | 0.007748 |
BX-795 | -2.84887 | 0.122735 | 0.004595 | 0.00805 |
Tipifarnib | 2.848553 | -0.08652 | 0.004619 | 0.00805 |
GSK-650394 | 2.834389 | -0.10247 | 0.004828 | 0.008351 |
Lapatinib | 2.803797 | -0.05029 | 0.005313 | 0.009124 |
MS-275 | -2.74478 | 0.07168 | 0.006316 | 0.010769 |
AKT inhibitor VIII | -2.73352 | 0.051398 | 0.006533 | 0.011059 |
BMS345541 | -2.71602 | 0.078512 | 0.006876 | 0.011558 |
Nutlin-3a (-) | -2.71212 | 0.131774 | 0.00696 | 0.011616 |
IOX2 | -2.7081 | 0.043724 | 0.007053 | 0.011689 |
Temozolomide | -2.69549 | 0.080134 | 0.007306 | 0.012024 |
Vorinostat | -2.68354 | 0.117531 | 0.007571 | 0.012374 |
CX-5461 | -2.66998 | 0.145973 | 0.007871 | 0.012776 |
Dasatinib | -2.60268 | 0.181641 | 0.009569 | 0.015428 |
WH-4-023 | -2.59558 | 0.132139 | 0.009765 | 0.015624 |
RO-3306 | -2.59376 | 0.011796 | 0.009823 | 0.015624 |
AZ628 | -2.58192 | 0.123229 | 0.010165 | 0.016061 |
Lenalidomide | -2.49735 | 0.069088 | 0.012887 | 0.020226 |
Veliparib | -2.4781 | 0.05129 | 0.013608 | 0.021217 |
EKB-569 | -2.4714 | 0.113677 | 0.013849 | 0.021452 |
Shikonin | -2.45448 | 0.071018 | 0.014524 | 0.022352 |
PF-4708671 | -2.42302 | 0.03372 | 0.01582 | 0.024189 |
Docetaxel | 2.348243 | -0.14417 | 0.01934 | 0.029382 |
ZM-447439 | 2.323013 | -0.07843 | 0.020681 | 0.031219 |
YK 4-279 | -2.30743 | 0.089201 | 0.021516 | 0.032274 |
FTI-277 | -2.18582 | 0.044513 | 0.029408 | 0.043834 |
BI-2536 | 2.182288 | -0.0697 | 0.029624 | 0.043881 |
Saracatinib | -2.14752 | 0.051202 | 0.032313 | 0.047567 |
TAK-715 | -2.13323 | 0.076159 | 0.033477 | 0.048976 |
BMS-509744 | -2.09829 | 0.029301 | 0.036473 | 0.053032 |
SL 0101-1 | -2.06045 | 0.038595 | 0.039968 | 0.057759 |
Cisplatin | -2.04476 | 0.08733 | 0.041489 | 0.059593 |
Doxorubicin | 1.979839 | -0.07537 | 0.048367 | 0.069054 |
Midostaurin | -1.97489 | 0.065767 | 0.048931 | 0.069441 |
Ispinesib Mesylate | -1.97089 | 0.116258 | 0.049405 | 0.069697 |
Statistical analysis
For the statistical analysis, we employed GraphPad Prism version 8.0. To assess different types of data, we utilized the paired and unpaired t-tests, as well as a one-way analysis of variance (ANOVA). In all instances, we considered statistical significance to be achieved when the P-value was less than 0.05.