Additional file 1 of Superbinder based phosphoproteomic landscape revealed PRKCD_pY313 mediates the activation of Src and p38 MAPK to promote TNBC progression
Creators: name: Deng, Yujiao
name: Hou, Zhanwu
name: Li, Yizhen
name: Yi, Ming
name: Wu, Ying
name: Zheng, Yi
name: Yang, Fei
name: Zhong, Guansheng
name: Hao, Qian
name: Zhai, Zhen
name: Wang, Meng
name: Ma, Xiaobin
name: Kang, Huafeng
name: Ji, Fanpu
name: Dong, Chenfang
name: Liu, Huadong
name: Dai, Zhijun
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Plant Biology
subject: FOS: Biological sciences

description: Additional file 1: Table S1. PRKCD knockout-sgRNA sequences. Table S2. PRKCD knockdown shRNA primers. Table S3. PRKCD overexpression and Y313F mutation amplification primers. Table S4. Realtime RT-PCR primers. Table S5. The primary antibodies used in western blot analysis. Fig. S1. Functional enrichment analysis of significant serine/threonine peptides in breast cancer tissues. (A) Barplot of Gene Ontology analysis of significant serine/threonine peptides peptides related genes. (B) Barplot of top 10 KEGG pathways of significant serine/threonine peptides peptides related genes. KEGG, Kyoto Encyclopedia of Genes and Genomes. Fig. S2. Personalized kinase activity map of patient with Luminal A subtype of breast cancer. Each protein frame is divided into four small squares, each of which represents one pY, pS or pT site. The color represents the log10(Ca/N) value, red represents the upregulation of this site, and the darker the color, the higher the upregulation ratio. Blue indicates downregulation, darker color indicates higher downregulation, and gray indicates no such site. The color of the border of each protein bar represents the sum of the log10(Ca/N) values of all sites of the protein. The thickness of the lines between the proteins indicates a test score ranging from 1 to 10, and the color of the lines ranging from gray to red indicates a test score ranging from 0 to 1. Ca/N, ratio of expression levels in breast cancer tumors to that in normal breast tissue. Fig. S3. Personalized kinase activity map of patient with Luminal B subtype of breast cancer. Each protein frame is divided into four small squares, each of which represents one pY, pS or pT site. The color represents the log10(Ca/N) value, red represents the upregulation of this site, and the darker the color, the higher the upregulation ratio. Blue indicates downregulation, darker color indicates higher downregulation, and gray indicates no such site. The color of the border of each protein bar represents the sum of the log10(Ca/N) values of all sites of the protein. The thickness of the lines between the proteins indicates a test score ranging from 1 to 10, and the color of the lines ranging from gray to red indicates a test score ranging from 0 to 1. Ca/N, ratio of expression levels in breast cancer tumors to that in normal breast tissue. Fig. S4. Personalized kinase activity map of patient with HER2 positive subtype of breast cancer. Each protein frame is divided into four small squares, each of which represents one pY, pS or pT site. The color represents the log10(Ca/N) value, red represents the upregulation of this site, and the darker the color, the higher the upregulation ratio. Blue indicates downregulation, darker color indicates higher downregulation, and gray indicates no such site. The color of the border of each protein bar represents the sum of the log10(Ca/N) values of all sites of the protein. The thickness of the lines between the proteins indicates a test score ranging from 1 to 10, and the color of the lines ranging from gray to red indicates a test score ranging from 0 to 1. Ca/N, ratio of expression levels in breast cancer tumors to that in normal breast tissue. Fig. S5. Top five Gene Ontology-biological process pathways of significant pY/pS/pT peptides related genes in breast tissues. Fig. S6. Top five Gene Ontology-cellular component pathways of significant pY/pS/pT peptides related genes in breast tissues. Fig. S7. Top five Gene Ontology-molecular function pathways of significant pY/pS/pT peptides related genes in breast tissues. Fig. S8. Phosphorylated tyrosine/serine/threonine related genes in pathway of proteoglycans in cancer. The KEGG pathway enrichment results of genes from our mass spectrum data in pathway of proteoglycans in cancer. All colored rectangles represent genes corresponding to phosphorylated polypeptide proteins enriched in our mass spectrometry analysis. Red represents increased phosphorylation of this protein in tumor tissue, and green represents decreased phosphorylation of this protein in tumor tissue. This pathway includes hyaluronan, chondroitin sulfate/dermatan sulfate proteoglycan, keratan sulfate proteoglycan, and heparan sulfate proteoglycans. Fig. S9. Phosphorylated tyrosine/serine/threonine related genes in pathway of focal adhesion. The KEGG pathway enrichment results of genes from our mass spectrum data in pathway of focal adhesion. All colored rectangles represent genes corresponding to phosphorylated polypeptide proteins enriched in our mass spectrometry analysis. Red represents increased phosphorylation of this protein in tumor tissue, and green represents decreased phosphorylation of this protein in tumor tissue. This pathway includes ECM-receptor interaction, cytokine-cytokine receptor interaction, which could affect the cell motility, cell proliferation and cell survival. Fig. S10. Phosphorylated tyrosine/serine/threonine related genes in ErbB signaling pathway. The KEGG pathway enrichment results of genes from our mass spectrum data in ErbB signaling pathway. All colored rectangles represent genes corresponding to phosphorylated polypeptide proteins enriched in our mass spectrometry analysis. Red represents increased phosphorylation of this protein in tumor tissue, and green represents decreased phosphorylation of this protein in tumor tissue. This pathway includes ERBB1, ERBB2, ERBB3, and ERBB4, which could affect the proliferation of pancreatic cancer and non-small cell lung cancer, the differentiation of glioma and endometrial cancer. Fig. S11. Phosphorylated tyrosine/serine/threonine related genes in pathway of tight junction. The KEGG pathway enrichment results of genes from our mass spectrum data in pathway of tight junction. All colored rectangles represent genes corresponding to phosphorylated polypeptide proteins enriched in our mass spectrometry analysis. Red represents increased phosphorylation of this protein in tumor tissue, and green represents decreased phosphorylation of this protein in tumor tissue. This pathway includes claudin, occluding, JAM, bves, which could affect the cell polarity, cell proliferation, cell survival, cell differentiation, cell migration. Fig. S12. Phosphorylated tyrosine/serine/threonine related genes in pathway of adherens junction. The KEGG pathway enrichment results of genes from our mass spectrum data in pathway of adherens junction. All colored rectangles represent genes corresponding to phosphorylated polypeptide proteins enriched in our mass spectrometry analysis. Red represents increased phosphorylation of this protein in tumor tissue, and green represents decreased phosphorylation of this protein in tumor tissue. This pathway includes nectin (strong adhesion), cadherin (weak adhesion) et al. Fig. S13. Phosphorylated tyrosine/serine/threonine related genes in MAPK signaling pathway. The KEGG pathway enrichment results of genes from our mass spectrum data in MAPK signaling pathway. All colored rectangles represent genes corresponding to phosphorylated polypeptide proteins enriched in our mass spectrometry analysis. Red represents increased phosphorylation of this protein in tumor tissue, and green represents decreased phosphorylation of this protein in tumor tissue. This pathway includes classical MAP kinase pathway, JNK and p38 MAP kinase pathway, and ERK5 pathway, which could affect the cell proliferation, cell cycle, cell differentiation, and cell inflammation. Fig. S14. Survival analysis of patients with breast cancer in different PRKCD level. (A) Kaplan-Meier plot of disease specific survival of patients with breast cancer in different PRKCD level (DATASET: GSE3494-GPL96). (B) Kaplan-Meier plot of distant metastasis free survival of patients with breast cancer in different PRKCD level (DATASET: GSE9195). HR, Hazard Ratio. Fig. S15. PRKCD_pY313 promotes malignant biological behaviors of triple-negative breast cancer cells. (A) The knockdown and knockout rate of PRKCD and the PRKCD_pY313 level in MDA-MB-231 stable transfected cell lines. (B) Apoptosis rate of stable transfected MDA-MB-231 and BT549 cell lines with different PRKCD_pY313 level, detected by flow cytometry. (C) The colony formation rate of MDA-MB-231 and BT549 cell lines with different PRKCD_pY313 level. (D) The wound healing assay of stable MDA-MB-231 and BT549 cell lines with different PRKCD_pY313 level. (E) Invasion assay of stable transfected MDA-MB-231 and BT549 cell lines with different PRKCD_pY313 level. OE, overexpression; KD, knockdown. KO, knockout. Mean ± SD, n = 3 per group, * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, **** P &lt; 0.0001. Fig. S16. Expression levels of proteins involved in apoptosis and invasion and metastasis of MDA-MB-231 and BT549 cell lines. (A) Quantification of Fig. 5F, apoptotic proteins in MDA-MB-231 cell lines with overexpressed PRKCD_pY313 or PRKCD_Y313F. (B) Quantification of Fig. 5F, apoptotic proteins in MDA-MB-231 cell lines with low PRKCD_pY313 level. (C) Quantification of Fig. 5F, apoptotic proteins in BT549 cell lines with overexpressed PRKCD_pY313 or PRKCD_Y313F. (D) Quantification of Fig. 5F, proteins that mark metastatic capacity in MDA-MB-231 cell lines with overexpressed PRKCD_pY313 or PRKCD_Y313F level. (E) Quantification of Fig. 5F, Proteins that mark metastatic capacity in MDA-MB-231 cell lines with low PRKCD_pY313 level. (F) Quantification of Fig. 5F, Proteins that mark metastatic capacity in BT549 cell lines with overexpressed PRKCD_pY313 or PRKCD_Y313F level. Mean ± SD, n = 3 per group, * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, **** P &lt; 0.0001. Fig. S17. Proteins and phosphorylated levels of PRKCD-related kinases in MDA-MB-231 and BT549 cell lines with different PRKCD_pY313 level. (A) Quantification of Fig. 7A. (B) Quantification of Fig. 7B.(C) Quantification of Fig. 7C. Mean ± SD, n = 3 per group, * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, **** P &lt; 0.0001. Fig. S18. Proteins and phosphorylated levels of PRKCD-related kinases in MDA-MB-231 and BT549 cell lines with dasatinib treatment. (A) Quantification of Src_pY419/Src level in MDA-MB-231 cell lines in Fig. 7D. (B) Quantification of kinases’ activity in MDA-MB-231 cell lines in Fig. 7D. (C) Quantification of Src_pY419/Src level in BT549 cell lines in Fig. 7E. (D) Quantification of kinases’ activity in BT549 cell lines in Fig. 7E. Mean ± SD, n = 3 per group, * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, **** P &lt; 0.0001. Fig. S19. The MTT assay of stable MDA-MB-231 and BT549 cells treated with dasatinib. (A) The cell relative viability of MDA-MB-231 cell with dasatinib treatment and IC50 (Half maximal inhibitory concentration). (B) The cell relative viability of BT549 cell with dasatinib treatment and IC50. (C) The cell relative viability of MDA-MB-231 cell with 0.01 μM dasatinib treatment. (D) The cell relative viability of BT549 cell with 0.01 μM dasatinib treatment. (E) The cell relative viability of MDA-MB-231 cell with 0.025 μM dasatinib treatment. (F) The cell relative viability of BT549 cell with 0.025 μM dasatinib treatment. (G) The cell relative viability of MDA-MB-231 cell with 0.05 μM dasatinib treatment. (H) The cell relative viability of BT549 cell with 0.05 μM dasatinib treatment. (I) The cell relative viability of MDA-MB-231 cell with 0.1 μM dasatinib treatment. (J) The cell relative viability of BT549 cell with 0.1 μM dasatinib treatment. (K) The cell relative viability of MDA-MB-231 cell with 0.2 μM dasatinib treatment. (L) The cell relative viability of BT549 cell with 0.2 μM dasatinib treatment. Mean ± SD, n = 3 per group, * P &lt; 0.05, ** P &lt; 0.01, *** P &lt; 0.001, **** P &lt; 0.0001.