Journal: Scientific Reports

Article Title: PLOD2 promotes proliferation, migration and invasion of colorectal cancer cells via PI3K-AKT-GSK3β signaling pathway

doi: 10.1038/s41598-026-38593-6

Figure Lengend Snippet: High expression of PLOD2 in Colorectal Cancer. ( A ) Volcano plot showed the DEGs in GSE97689 . ( B ) GO enrichment of DEGs and the list of significant GO terms of the top 10. ( C ) KEGG pathways enrichment of DEGs. ( D ) Immunohistochemical detection of PLOD2 expression in colorectal cancer tissues and adjacent normal tissues (a. Strong PLOD2 staining in colorectal cancer tissues; b. Control showing weak PLOD2 staining in colorectal cancer tissues; c. Weak PLOD2 staining in normal tissues adjacent to the tumor; d. Negative control in normal tissues adjacent to the tumor. Images captured at 50× and 400× magnification, respectively.). ( E ) Expression levels of PLOD2 in colorectal cancer and normal tissues from the CPTAC database (z-values denote the standard deviation from the median sample for a given cancer type). ( F ) Correlation of PLOD2 expression with pathological staging of colon and rectal cancer tumors (a, Colon adenocarcinoma; b, Rectal adenocarcinoma.). ( G ) Kaplan-Meier analysis of overall survival in patients with high versus low PLOD2 expression.

Article Snippet: Antibodies used in this experiment were as follows, primary antibody: PLOD2 antibody (#21214-1-AP, Wuhan Proteintech Biotechnology Co., Ltd.), GAPDH antibody (5174,Cell Signaling Technology, Inc., USA), PI3K antibody (4257, Cell Signaling Technology, Inc., USA).Phosoho-PI3K antibody (4228, Cell Signaling Technology, Inc., USA).AKT antibody (4691, Cell Signaling Technology, Inc., USA), Phosoho-AKT antibody (13038, Cell Signaling Technology, Inc., USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Negative Control, Standard Deviation

Journal: Scientific Reports

Article Title: PLOD2 promotes proliferation, migration and invasion of colorectal cancer cells via PI3K-AKT-GSK3β signaling pathway

doi: 10.1038/s41598-026-38593-6

Figure Lengend Snippet: PLOD2 promotes cell proliferation, migration, and invasion in CRC cells. ( A ) Western blot and qPCR analysis of PLOD2 protein and mRNA expression in HCT116-PLOD2 and Caco-2-shPLOD2 cell lines, uncropped gel and blot images with edges marked are presented in Supplementary material. ( B ) Effect of PLOD2 on the proliferative capacity of colorectal cancer cells, assessed by CCK-8 assay. ( C ) Effect of PLOD2 on colony formation and proliferative ability of colorectal cancer cells, assessed by plate colony formation assay. ( D ) Effect of PLOD2 on the horizontal migration ability of colorectal cancer cells, assessed by cell Wound healing assay. ( E ) Effect of PLOD2 on the vertical migration ability of colorectal cancer cells, assessed by Transwell migration assay. ( F ) Effect of PLOD2 on the invasive ability of colorectal cancer cells, assessed by Transwell invasion assay.

Article Snippet: Antibodies used in this experiment were as follows, primary antibody: PLOD2 antibody (#21214-1-AP, Wuhan Proteintech Biotechnology Co., Ltd.), GAPDH antibody (5174,Cell Signaling Technology, Inc., USA), PI3K antibody (4257, Cell Signaling Technology, Inc., USA).Phosoho-PI3K antibody (4228, Cell Signaling Technology, Inc., USA).AKT antibody (4691, Cell Signaling Technology, Inc., USA), Phosoho-AKT antibody (13038, Cell Signaling Technology, Inc., USA).

Techniques: Migration, Western Blot, Expressing, CCK-8 Assay, Colony Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: Scientific Reports

Article Title: PLOD2 promotes proliferation, migration and invasion of colorectal cancer cells via PI3K-AKT-GSK3β signaling pathway

doi: 10.1038/s41598-026-38593-6

Figure Lengend Snippet: PLOD2 activates the PI3K-AKT-GSK3β signaling pathway in colorectal cancer cells. ( A ) KEGG enrichment analysis of PLOD2-interacting proteins and 500 expression-related genes. ( B ) Effects of PLOD2 overexpression and silencing on PI3K-AKT-GSK3β signaling pathway proteins and expression quantification, uncropped gel and blot images with edges marked are presented in Supplementary material. ( C ) Co-immunoprecipitation experiments for PLOD2 and PI3K, uncropped gel and blot images with edges marked are presented in Supplementary material.

Article Snippet: Antibodies used in this experiment were as follows, primary antibody: PLOD2 antibody (#21214-1-AP, Wuhan Proteintech Biotechnology Co., Ltd.), GAPDH antibody (5174,Cell Signaling Technology, Inc., USA), PI3K antibody (4257, Cell Signaling Technology, Inc., USA).Phosoho-PI3K antibody (4228, Cell Signaling Technology, Inc., USA).AKT antibody (4691, Cell Signaling Technology, Inc., USA), Phosoho-AKT antibody (13038, Cell Signaling Technology, Inc., USA).

Techniques: Expressing, Over Expression, Immunoprecipitation

Journal: Scientific Reports

Article Title: PLOD2 promotes proliferation, migration and invasion of colorectal cancer cells via PI3K-AKT-GSK3β signaling pathway

doi: 10.1038/s41598-026-38593-6

Figure Lengend Snippet: PLOD2 promotes CRC cell proliferation, migration, and invasion through the PI3K-AKT-GSK3β signaling pathway. ( A ) Expression of PI3K-AKT-GSK3β pathway-related proteins in colorectal cancer cells treated with the PI3K inhibitor (LY294002, 10 μM) or agonist (740Y-P, 20 μM) for 48 h, uncropped gel and blot images with edges marked are presented in Supplementary material. ( B ) CCK-8 assay assessing the proliferative capacity of colorectal cancer cells treated with PI3K inhibitors or agonists. ( C ) Colony formation assay evaluating the proliferation of colorectal cancer cells treated with PI3K inhibitors or agonists. ( D ) Wound healing assay evaluating the horizontal migration ability of colorectal cancer cells treated with PI3K inhibitors or agonists. ( E ) Transwell migration assay assessing the vertical migration ability of colorectal cancer cells treated with PI3K inhibitors or agonists. ( F ) Transwell invasion assay evaluating the invasive ability of colorectal cancer cells treated with PI3K inhibitors or agonists.

Article Snippet: Antibodies used in this experiment were as follows, primary antibody: PLOD2 antibody (#21214-1-AP, Wuhan Proteintech Biotechnology Co., Ltd.), GAPDH antibody (5174,Cell Signaling Technology, Inc., USA), PI3K antibody (4257, Cell Signaling Technology, Inc., USA).Phosoho-PI3K antibody (4228, Cell Signaling Technology, Inc., USA).AKT antibody (4691, Cell Signaling Technology, Inc., USA), Phosoho-AKT antibody (13038, Cell Signaling Technology, Inc., USA).

Techniques: Migration, Expressing, CCK-8 Assay, Colony Assay, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: Clinical features and PLOD2 protein expression in HCC patients

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Encapsulation

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: (A) Pan-cancer analysis of PLOD2 expression in different tumor types from The Cancer Genome Atlas (TCGA) database. (B) Relative mRNA levels of PLOD2 in HCC and normal tissues were determined based on the TCGA dataset. ** p <0.001. (C) Relative mRNA levels of PLOD2 were significantly increased in HCC tissues compared with adjacent normal tissues based on the TCGA dataset. * p <0.05. (D) mRNA expression of PLOD2 was significantly correlated with HCC patients’ individual cancer stages. * p <0.05, *** p <0.00. (E) mRNA expression of PLOD2 was positively correlated with tumor histologic grades of HCC. * p <0.05, *** p <0.001. (F, G) Representative IHC staining images showed that PLOD2 expression was significantly increased in HCC tissues based on the SYSUCC dataset (left). Protein level of PLOD2 were significantly increased in HCC tissues compared with paired adjacent liver tissues based on the SYSUCC dataset (Right). *** p <0.001. (H–I) PLOD2 mRNA expression was correlated with shorter overall and recurrence-free survival times in HCC patients based on the TCGA dataset; (J–K) Kaplan–Meier plots revealed that shorter overall and recurrence-free survival times were correlated with higher PLOD2 expression in the SYSUCC validation cohort. HCC, hepatocellular carcinoma; IHC, immunohistochemical; PLOD2, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2; SYSUCC, Sun Yat-sen Cancer Center; TCGA, The Cancer Genome Atlas.

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry, Biomarker Discovery, Immunohistochemical staining

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses for overall survival

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques:

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses for recurrence-free survival

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques:

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: (A) qPCR (top) and western blot (bottom) showing the expression of PLOD2 in MIHA and 5 HCC cell lines. (B) The efficiency of PLOD2 KD was measured by real-time PCR (top) and western blotting (bottom) assays. *** p <0.001. (C) KD of PLOD2 decreased Huh7 and MHCC-97H cell migration. *** p <0.001. (D) KD of PLOD2 decreased Huh7 and MHCC-97H cell invasion. ** p <0.01, *** p <0.001. (E) KD of PLOD2 impaired the scratch wound healing ability of Huh7 and MHCC-97H cells. (F) KD of PLOD2 inhibited Huh7 and MHCC-97H cell proliferation. ** p <0.01, *** p <0.001. (G, H) KD of PLOD2 significantly inhibited tumor size and growth. *** p <0.001. (I) KD of PLOD2 significantly inhibited tumor weight. *** p <0.001. (J) Representative IHC staining images showing that the expression of PLOD2 and Ki67 were significantly decreased in the KD groups. (K) KD of PLOD2 significantly suppressed the expression of Ki67 in a nude mouse model established by injection of Huh7 cells into the right flank ( n =6). *** p <0.001. (L) KD of PLOD2 significantly suppressed lung metastasis in a nude mouse model established by injection of Huh7 cells through the tail vein ( n =4). ** p <0.01. HCC, hepatocellular carcinoma; IHC, immunohistochemical; KD, knockdown; PLOD2, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2.

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Migration, Immunohistochemistry, Injection, Immunohistochemical staining, Knockdown

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: (A) KEGG pathway analysis shows the significantly affected signaling pathways in Huh7 cells. (B) GSEA showing the enrichment of Focal adhesion-related gene signatures in PLOD2 KD cells. (C) Relative mRNA levels of genes in the Focal adhesion signaling pathway in Huh7 and MHCC-97H cells. * p <0.05, *** p <0.001. (D) Western blot assays showing the relative levels of PLOD2 and BIRC3 after PLOD2 KD in Huh7 and MHCC-97H cells. (E, F) Representative IHC staining images show a positive correlation between PLOD2 and BIRC3 levels in human HCC samples ( n =200). (G) Correlation of PLOD2 and BIRC3 mRNAs in HCC samples from the TCGA database. BIRC3, Baculoviral IAP repeat containing 3; GSEA, Gene Set Enrichment Analysis; HCC, hepatocellular carcinoma; KD, knockdown; KEGG, Kyoto Encylopaedia of Genes and Genomes; PLOD2, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2; TCGA, The Cancer Genome Atlas.

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques: Protein-Protein interactions, Western Blot, Immunohistochemistry, Knockdown

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: (A) Western blots show relative levels of PLOD2 and BIRC3 after BIRC3 overexpression in PLOD2 KD HCC cells. (B) CCK-8 assays demonstrated that overexpression of BIRC3 increased cell proliferation in PLOD2 KD HCC cells. * p <0.05, *** p <0.001. (C) Transwell assays demonstrated that overexpression of BIRC3 increased cell migration in PLOD2 KD HCC cells. *** p <0.001. (D) Transwell assays demonstrated that overexpression of BIRC3 increased cell invasion in PLOD2 KD HCC cells. ** p <0.01, *** p <0.001. (E) Wound healing assays demonstrated that overexpression of BIRC3 increased cell migration in PLOD2 KD HCC cells. (F–G) KD of PLOD2 significantly suppressed the expression of PLOD2 and BIRC3 in a nude mouse model established by injection of Huh7 cells into the right flank ( n =6). ** p <0.01, *** p <0.001. BIRC3, Baculoviral IAP repeat containing 3; HCC, hepatocellular carcinoma; KD, knockdown; PLOD2, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2.

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques: Western Blot, Over Expression, CCK-8 Assay, Migration, Expressing, Injection, Knockdown

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: (A) Relative mRNA levels of PLOD2 in HCC and normal tissues were determined based on the TCGA dataset. *** p <0.001. (B) IRF5 mRNA expression was correlated with poor survival in HCC patients based on the TCGA dataset. (C, D) qPCR and western blot assays showing the relative levels of IRF5 and PLOD2 in the siIRF5 Huh7 and MHCC-97H cell lines. * p <0.05, *** p <0.001. (E) Representative IHC staining images showing a positive correlation between IRF5 and PLOD2 levels in human HCC samples ( n =200). (F) Correlation of IRF5 and PLOD2 mRNAs in HCC samples from the TCGA database. (G) Schematic view of the luciferase reporter constructs containing various sites of PLOD2. (H) KD of IRF5 reduced PLOD2 promoter activity. HCC cells were reversely transfected with NC or siIRF5 for 24 h, followed by transfection with the full-length PLOD2 promoter (−2,000/+99) for another 48 h, and then were subjected to luciferase activity assay. *** p <0.001. (I) KD of IRF5 decreased the luciferase activity of the site A mutant. HCC cells were reversely transfected with NC or siIRF5 for 24 h, followed by transfection with the site A mutant for another 48 h and then were subjected to luciferase activity assay. *** p <0.001. (J) The sequence of site A and the corresponding sequence of the mutated site are shown. (K) The sequence of site B and the corresponding sequence of the mutated site are shown. (L) KD of IRF5 did not change the luciferase activity of the site B mutant. HCC cells were reversely transfected with NC or siIRF5 for 24 h, followed by transfection with the site B mutant for another 48 h, and then were subjected to luciferase activity assay. HCC, hepatocellular carcinoma; IHC, immunohistochemical; IRF5, interferon regulatory factor 5; KD, knockdown; NC, Negative control; PLOD2, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2; TCGA, The Cancer Genome Atlas.

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques: Expressing, Western Blot, Immunohistochemistry, Luciferase, Construct, Activity Assay, Transfection, Mutagenesis, Sequencing, Immunohistochemical staining, Knockdown, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma

doi: 10.14218/JCTH.2022.00401

Figure Lengend Snippet: HCC, hepatocellular carcinoma; PLOD2, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2.

Article Snippet: Cell protein lysates were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA), and then incubated with mouse polyclonal antibody specific for PLOD2 (Origene, Rockville, USA), rabbit polyclonal antibody specific for IRF5 (Abcam, Shanghai, China) and rabbit polyclonal antibody specific for BIRC3 (Proteintech, Wuhan, China) at 4°C overnight.

Techniques:

Journal: bioRxiv

Article Title: Inflammation promotes tumor aggression by stimulating stromal cell-dependent collagen crosslinking and stromal stiffening

doi: 10.1101/2020.02.13.948141

Figure Lengend Snippet: ( a-c ) Gene expression analysis of LOX ( a ), LOXL2 ( b ), and PLOD2 ( c ) stratified by ER + (n = 1355), HER2 + (n = 127), and triple negative (TN; n = 299) subtypes. Gene expression is plotted as a scatter plot of mRNA z scores with the mean ± SEM. Statistical analysis was performed using one-way ANOVA for overall analysis and unpaired t-test was used for individual comparisons ( *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001). ( d ) Scatter plot of individual and mean values ± SEM comparing LOX (n = 47) and PLOD2 (n = 57) gene expression in microdissected epithelial and stromal compartments of human invasive breast carcinomas. Statistical analysis was performed using Mann-Whitney U test (****p < 0.0001). ( e ) Quantification of LOX and PLOD2 gene expression fold change from ( d ) in stromal cells relative to epithelial cells. ( f-g ) Restriction of the stromal/epithelial gene expression analysis in ( d ) and ( e ) to estrogen receptor (ER) negative and progesterone receptor (PR) negative samples. (LOX n = 11, PLOD2 n = 15). Statistical analysis was performed using Mann-Whitney U test (*p < 0.05, **p < 0.01).

Article Snippet: A primary mouse monoclonal Lysyl Hydroxylase 2 (LH2) antibody (Origene; Cat# TA803224, dilution 1:150) was used for the immunohistochemical staining.

Techniques: Gene Expression, MANN-WHITNEY

Journal: bioRxiv

Article Title: Inflammation promotes tumor aggression by stimulating stromal cell-dependent collagen crosslinking and stromal stiffening

doi: 10.1101/2020.02.13.948141

Figure Lengend Snippet: ( a ) Representative images of PyMT tumor tissue from 8 weeks of age mice treated with anti-CSF1 blocking antibody or IgG1 control. ( Top row ) IgG1 treated (n = 6) and anti-CSF1 treated (n = 5) PyMT tumor tissue stained for pan-cytokeratin (green) marking epithelial cells, F4/80 (white) marking tumor infiltrating macrophages, and DAPI marking nuclei (blue). ( Second row ) IgG1 treated (n = 6) and anti-CSF1 treated (n = 5) PyMT tumor tissue stained for tyrosine 397 phosphorylated focal adhesion kinase (red) indicative of mechanosignaling and DAPI marking nuclei (blue). ( Third row ) Polarized light images with brightfield inset of IgG1 treated (n = 6) and anti-CSF1 treated (n = 6) PyMT tumor tissue stained with picrosirius red to visualize fibrillar collagen. ( Bottom row ) Lox mRNA in situ hybridization in IgG1 treated (n = 6) and anti-CSF1 treated (n = 5) PyMT tumor tissue and DAPI marking nuclei (blue). Scale bar for all images is 100µm. ( b ) Quantification of the number of metastatic colonies in the lung tissues from IgG1 treated (n = 5) and anti-CSF1 treated (n = 6) mice at 11 weeks of age via PyMT IHC assessing 5 layers (5 micron section; 5 sections per layer; 50-100 microns steps). Statistical analysis was performed using unpaired t-test (*p < 0.05). ( c ) Quantification of fibrillar collagen by picrosirius red staining by percent area per field of view in 8 week old mice treated with anti-CSF1 blocking antibody or IgG1 control. The mean of 3-4 regions was calculated and plotted for each animal ± SEM. Statistical analysis was performed using unpaired t-test (p = 0.08). ( d ) Histogram showing the distribution of the top 10% of elastic modulus measurements by AFM microindentation in PyMT IgG1 treated (n =6) and anti-CSF1 treated (n = 4) tumors. Statistical analysis was performed using Mann-Whitney U test (****p < 0.0001). ( e ) Quantification of Lox mRNA signal by percent area of signal per field of view in 8 week old mice treated with anti-CSF1 blocking antibody (n = 5) or IgG1 control (n = 6). The mean of 5-6 regions was calculated and plotted for each animal ± SEM. Statistical analysis was performed using an unpaired t-test (*p < 0.05). ( f-i ) Scatter plots showing individual and mean values ± SEM of the levels of total hydroxyproline (collagen content) ( f ), total collagen crosslinks ( g ), HLCCs ( h ), and HLNL crosslinks ( i ) in 8 week old IgG1 treated and anti-CSF1 treated PyMT tumors. Quantity of crosslinks per tissue was calculated normalizing crosslinks to wet tissue weight. Values were plotted as log 2 transformed normalized peak areas as quantified from LC-MS data. Statistical analysis was performed using unpaired t-test (*p < 0.05). ( j ) Quantification of Tgfb1 gene expression by RT-qPCR in tumor cells, cancer-associated fibroblasts, and macrophages sorted out from PyMT tumors (n = 4). Gene expression was normalized to 18S. Statistical analysis was performed using Kruskal-Wallis one-way ANOVA for overall comparison and Mann-Whitney U test for individual comparisons (*p < 0.05, ***p < 0.001). ( k ) Representative images of PyMT tumor tissue from mice treated with IgG1 (n = 6) and anti-CSF1 (n = 5) stained for pan-cytokeratin (green) marking epithelial cells, SMAD2 pS465/467 (red), and DAPI marking nuclei (blue). ( l ) Scatter plot showing individual and mean values ± SEM of the mean nuclear intensity of pSMAD S465/467 in stromal cells of IgG1 treated (n = 6) and anti-CSF1 treated (n = 5) PyMT mice. The mean for each animal was calculated from 4-7 regions within the tumor. Statistical analysis was performed using unpaired t-test (p = 0.06). ( m-o ) Scatter plot depicting the Spearman correlation of CD163 gene expression with LOX ( m ), PLOD2 ( n ), and LOXL2 ( o ) in human breast tumors (n = 1904). ( p ) Representative IHC images of serial human breast tumor sections stained for CD68 ( top ) and pSMAD2 S465/467 ( bottom ) counterstained with hematoxylin to mark nuclei. ( q ) Scatter plot depicting the linear regression correlation of stromal pSMAD2 IHC staining with stromal CD68 IHC staining in human breast tumors (n = 10). ( r ) Scatter plot depicting the linear regression of CD14 + CD11b + HLA-DR + tumor associated macrophage infiltrate with tumor elastic modulus as measured by AFM microindentation in human breast tumors (n = 15).

Article Snippet: A primary mouse monoclonal Lysyl Hydroxylase 2 (LH2) antibody (Origene; Cat# TA803224, dilution 1:150) was used for the immunohistochemical staining.

Techniques: Blocking Assay, Control, Staining, In Situ Hybridization, MANN-WHITNEY, Transformation Assay, Liquid Chromatography with Mass Spectroscopy, Gene Expression, Quantitative RT-PCR, Comparison, Immunohistochemistry

Journal: bioRxiv

Article Title: Inflammation promotes tumor aggression by stimulating stromal cell-dependent collagen crosslinking and stromal stiffening

doi: 10.1101/2020.02.13.948141

Figure Lengend Snippet: ( a-b ) Kaplan-Meier plots showing overall survival for patients based on levels of LOX expression in epithelial cells (low n = 28, high n = 36) ( a ) or stromal cells (low n = 23, high n = 24) ( b ). The median level of expression was defined as the cutoff for low and high expression. ( c-d ) Kaplan-Meier plots showing overall survival for patients based on levels PLOD2 expression in epithelial cells (low n = 28, high n = 29) ( c ) or stromal cells (low n = 23, high n = 24) ( d ). The median level of expression was defined as the cutoff for low and high expression. ( e ) Schematic depicting the experimental timeline used to inhibit lysyl hydroxylase 2 in PyMT mice. ( f ) Representative polarized light images with brightfield insets of picrosirius red stained tumor tissue from PBS vehicle treated control (n = 8) and minoxidil treated (n = 7) PyMT mice. Scale bar is 100um. ( g ) Quantification of fibrillar collagen by picrosirius red staining by percent area per field of view. The mean was calculated and plotted for each animal ± SEM. ( h ) Histogram showing the distribution of the top 10% of elastic modulus measurements by AFM microindentation in PyMT control and Lox OX tumors. Statistical analysis was performed using Mann-Whitney U test (****p < 0.0001). ( i ) Scatter plot quantifying the area of lung sections occupied by metastases from vehicle treated (n = 9) and minoxidil treated (n = 8) mice at 13 weeks of age via H&E staining and assessing 4 layers (5 micron section; 5 sections per layer; 50-100 microns steps). Statistical analysis was performed using a two-tailed unpaired t-test (*p < 0.05). ( j ) Representative phase contrast images of sections from tissue microarrays (TMAs) of human breast cancers representing incident breast cancer cases collected and arrayed as 1-mm cores from each tumor. Sections were stained with Hematoxylin and Eosin (H&E; top) and lysyl hydroxylase two (LH2; bottom) via immunohistochemistry. ( k ) Bar graphs showing clinical correlation between lysyl hydroxylase two (LH2) score as a function of tumor grade ( see Table 1 for number of patients ). LH2 IHC staining was assessed with the semi-quantitative stromal specific H-score from 0 to 300. The lowest tertile of LH2 H-scores was defined as H-scores between 0 and less or equal to 120, the intermediate H-score to above 120 and equal or less than 230, and the highest stromal LH2 score as above 230. For tumor grade and LH2 H score, statistical analysis was performed using a linear-by-linear association (*** P<0.0001 ). ( l ) Kaplan-Meier curves indicating cumulative breast cancer specific survival (BCSS) based on stromal LH2 H score assessed in breast cancer patients up to 10 years after diagnosis (LH2 low n = 175, intermediate n = 188, high n = 146). ( m ) BCSS curves by stromal LH2 H score including only axillary lymph node negative patients (LH2 low n = 116, intermediate n = 116, high n = 90). ( n ) BCSS curves by stromal LH2 H score including only axillary lymph node positive patients (LH2 low n = 44, intermediate n =63, high n = 54). For Kaplan-Meier curves, statistical analyses were performed by LogRank test.

Article Snippet: A primary mouse monoclonal Lysyl Hydroxylase 2 (LH2) antibody (Origene; Cat# TA803224, dilution 1:150) was used for the immunohistochemical staining.

Techniques: Expressing, Staining, Control, MANN-WHITNEY, Two Tailed Test, Immunohistochemistry, Biomarker Discovery

Journal: Breast Cancer Research and Treatment

Article Title: Differential response of luminal and basal breast cancer cells to acute and chronic hypoxia

doi: 10.1007/s10549-023-06863-w

Figure Lengend Snippet: Chronic hypoxia affects metabolism in luminal breast cancer cells. A, B Heatmap of genes involved in glycolysis (A) and cytoskeleton (B) pathways in 24 h (acute) and 5 days (chronic) hypoxia in MCF-7 and HCC1143 cells. NA, not available; ****padj < 0.0001; ***padj < 0.001; **padj < 0.01; *padj < 0.05; ns, not significant. White color means that genes were filtered out in targeted RNA sequencing data after filtering by DESeq2 package in R software. C Lactate levels (mM) in 6 cell lines under normoxia normalized to the OD value determined in SRB assay. D, E Lactate levels measured in three luminal (MCF-7, T47D, BT474) (D) and 3 basal A (HCC1143, SUM149PT, HCC1806) (E) breast cancer cell lines under acute normoxia/ hypoxia and chronic normoxia/ hypoxia normalized to the OD value determined in SRB assay. F CA9 RNA expression level under hypoxia in luminal and basal A cell lines detected by qRT-PCR. Log 2 (2^(-ΔΔCT)) was calculated by normalizing to normoxia in each cell line. Error bars indicate SD for triplicate measurements. *** p < 0.001; ** p < 0.01; * p < 0.05. G GAPDH and PLOD2 protein expression detected by Western blot. B-actin serves as loading control. H Quantification of GADPH signal normalized to B-actin with Image J. Error bars indicate SD for triplicate measurements. ** p < 0.01; * p < 0.05; ns, not significant

Article Snippet: Membranes were blocked with 5% BSA and incubated with Carbonic Anhydrase IX (CA9) antibody (5649S; Cell Signaling Technology, Danvers, MA, USA), PLOD2 antibody (MAB4445; R&D Systems, Minneapolis, MN, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (sc-32233; Santa Cruz, Dallas, TX, USA), or β-actin antibody (sc-47778; Santa Cruz) overnight at 4 °C.

Techniques: RNA Sequencing, Software, Sulforhodamine B Assay, RNA Expression, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Breast Cancer Research and Treatment

Article Title: Differential response of luminal and basal breast cancer cells to acute and chronic hypoxia

doi: 10.1007/s10549-023-06863-w

Figure Lengend Snippet: Chronic hypoxia affects cell migration in basal breast cancer cells. A, B Cell tracking of MCF-7 and HCC1143 cell lines in acute (24 h) and chronic hypoxia (5 days) (A) , and calculated migration speed (B) . Error bars indicate SD for triplicate measurements. **** p < 0.0001; ** p < 0.01; ns, not significant. The pixels of acute hypoxia are 20 and of chronic hypoxia are 50 for cell tracking. C PLOD2 mRNA expression level detected by qRT-PCR in MCF-7 (two biological replicates) and HCC1143 cells (three biological replicates) comparing chronic hypoxia to normoxia. Error bars indicate SD for triplicate measurements. ** p < 0.01. D, E PLOD2 mRNA expression level in MCF-7 (D) and HCC1143 (E) cells under chronic hypoxia in presence of PLOD2 siRNA or control “kinasepool” siRNA (siKP). Average and SD from two biological replicates are shown. F Lactate levels measured for the indicated cell lines under chronic hypoxia normalized to the OD value determined in SRB assay. G, H Migration speed (G) and cell tracking (20 pixels) (H) analyzed for the indicated cell lines under chronic hypoxia. ns, not significant. Error bars indicate SD for triplicate measurements. ns, not significant

Article Snippet: Membranes were blocked with 5% BSA and incubated with Carbonic Anhydrase IX (CA9) antibody (5649S; Cell Signaling Technology, Danvers, MA, USA), PLOD2 antibody (MAB4445; R&D Systems, Minneapolis, MN, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (sc-32233; Santa Cruz, Dallas, TX, USA), or β-actin antibody (sc-47778; Santa Cruz) overnight at 4 °C.

Techniques: Migration, Cell Tracking Assay, Expressing, Quantitative RT-PCR, Control, Sulforhodamine B Assay

Journal: Bjc Reports

Article Title: Targeting PLOD2 suppresses invasion and metastatic potential in radiorecurrent prostate cancer

doi: 10.1038/s44276-024-00085-3

Figure Lengend Snippet: a Representative western blot of PLOD2 protein levels in DU145-PAR and DU145-CF cells; β-actin used as loading control. b Kaplan-Meier plot of biochemical relapse (BCR)-free survival rate for ICGC PRAD-CA patients with PLOD2 amplification versus neutral+deletion copy number alteration ( n = 380). c Comparison of PLOD2 RNA expression in localized prostate tumor samples ( n = 65) and metastatic castrate-resistant prostate cancer (mCRPC) samples ( n = 25) derived from metastatic sites of 4 patients (**** P < 0.0001). Data retrieved from the Gene Expression Omnibus (GEO) dataset GSE6919. d Comparison of PLOD2 RNA expression in benign, localized, and mCRPC patients ( n = 1321; One-way ANOVA test between subsets: F-value = 11.147, P < 0.001). Data derived from the Prostate Cancer Transcriptome Atlas (PCTA).

Article Snippet: To knockdown PLOD2 expression in the experimental cell lines, cells were seeded in 6-well plates and transfected with scramble control (CAT#: SR30004; Origene Inc., USA) or PLOD2 siRNA (CAT#: SR321350; Origene Inc., USA) using siTran 2.0 Transfection Reagent (CAT#: TT320001; Origene Inc., USA).

Techniques: Western Blot, Control, Amplification, Comparison, RNA Expression, Derivative Assay, Gene Expression

Journal: Bjc Reports

Article Title: Targeting PLOD2 suppresses invasion and metastatic potential in radiorecurrent prostate cancer

doi: 10.1038/s44276-024-00085-3

Figure Lengend Snippet: Matrigel-coated transwell invasion assays were conducted with numerous PCa cell lines transfected with scramble control (siCON) or PLOD2 (siPLOD2) siRNA: ( a ) DU145-CF; ( b ) DU145-PAR; ( c ) PC3; ( d ) 22RV1; ( e ) 107-I7 primary line. Transwell migration assays were repeated for each cell line: ( f ) DU145-CF; ( g ) DU145-PAR; ( h ) PC3; ( i ) 22RV1; ( j ) 107-I7 primary line. Means, standard deviations, and statistical significance of biological replicates are shown (ns = not significant, * P < 0.05, ** P < 0.01).

Article Snippet: To knockdown PLOD2 expression in the experimental cell lines, cells were seeded in 6-well plates and transfected with scramble control (CAT#: SR30004; Origene Inc., USA) or PLOD2 siRNA (CAT#: SR321350; Origene Inc., USA) using siTran 2.0 Transfection Reagent (CAT#: TT320001; Origene Inc., USA).

Techniques: Transfection, Control, Migration

Journal: Bjc Reports

Article Title: Targeting PLOD2 suppresses invasion and metastatic potential in radiorecurrent prostate cancer

doi: 10.1038/s44276-024-00085-3

Figure Lengend Snippet: a Representative images of ROIs on CAM embryos injected with scramble control siRNA-treated DU145-CF cells (siCON) or PLOD2 siRNA-treated DU145-CF cells (siPLOD2), at time = 0 h (intravascular cells) and time = 24 h (extravasated cells); Bright green foci: GFP-labeled DU145-CF cells; Dark green background: CAM stroma; Black: CAM blood vessels. Images were taken with a fluorescence microscope. b Extravasation efficiency of DU145-CF cells transfected with scramble control siRNA (siCON) or PLOD2 siRNA (siPLOD2), calculated from surviving embryos 24 h post-cell injection. Means, standard deviations, and statistical significance of biological replicates are shown (** P < 0.01).

Article Snippet: To knockdown PLOD2 expression in the experimental cell lines, cells were seeded in 6-well plates and transfected with scramble control (CAT#: SR30004; Origene Inc., USA) or PLOD2 siRNA (CAT#: SR321350; Origene Inc., USA) using siTran 2.0 Transfection Reagent (CAT#: TT320001; Origene Inc., USA).

Techniques: Injection, Control, Labeling, Fluorescence, Microscopy, Transfection

Journal: Bjc Reports

Article Title: Targeting PLOD2 suppresses invasion and metastatic potential in radiorecurrent prostate cancer

doi: 10.1038/s44276-024-00085-3

Figure Lengend Snippet: a Spearman correlation test of PLOD2 and HIF1α RNA expression in the PCa cohort of TCGA ( n = 333). b Western blot of HIF1α and PLOD2 protein levels in DU145-PAR (left) and DU145-CF (right) cell lysates. c Western blot of HIF1α and PLOD2 protein levels in DU145-CF cell lysates under normoxic (left) and hypoxic (right) conditions. For hypoxic conditions, DU145-CF cells were left incubated at 1% O 2 for 16 h. Western blots of HIF1α and PLOD2 protein levels in DU145-PAR cells ( d ) and DU145-CF cells ( e ) treated with H 2 O control or 20 μM of PX-478 (PX) for 24 h. β-actin was used in all western blots as a loading control.

Article Snippet: To knockdown PLOD2 expression in the experimental cell lines, cells were seeded in 6-well plates and transfected with scramble control (CAT#: SR30004; Origene Inc., USA) or PLOD2 siRNA (CAT#: SR321350; Origene Inc., USA) using siTran 2.0 Transfection Reagent (CAT#: TT320001; Origene Inc., USA).

Techniques: RNA Expression, Western Blot, Incubation, Control

Journal: Bjc Reports

Article Title: Targeting PLOD2 suppresses invasion and metastatic potential in radiorecurrent prostate cancer

doi: 10.1038/s44276-024-00085-3

Figure Lengend Snippet: a Heatmap of significantly dysregulated genes in DU145-CF cells transfected with PLOD2 siRNA (siPLOD2) compared to scramble control (siCON), after false discovery rate (FDR) correction (adjusted p-value < 0.05 and |log2(FoldChange)| > 0). PLOD2 ( b ) and LNCSRLR ( c ) RNA expression in DU145-CF cells transfected with PLOD2 siRNA, validated by qRT-PCR (normalized to GAPDH). d Spearman correlation test of PLOD2 and LNCSRLR RNA expression in ICGC PRAD-CA patients ( n = 140). e LNCSRLR knockdown with siRNA (siLNCSRLR) validated by qRT-PCR (normalized to GAPDH). f Matrigel-coated transwell invasion assay of DU145-CF cells treated with scramble control or LNCSRLR siRNA. g qRT-PCR measuring LNCSRLR RNA expression in DU145-CF cells treated with H 2 O control or 20 μM of PX-478 (PX) for 24 h. Means, standard deviations, and statistical significance of biological replicates are shown (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: To knockdown PLOD2 expression in the experimental cell lines, cells were seeded in 6-well plates and transfected with scramble control (CAT#: SR30004; Origene Inc., USA) or PLOD2 siRNA (CAT#: SR321350; Origene Inc., USA) using siTran 2.0 Transfection Reagent (CAT#: TT320001; Origene Inc., USA).

Techniques: Transfection, Control, RNA Expression, Quantitative RT-PCR, Knockdown, Transwell Invasion Assay