Journal: Nature materials

Article Title: Tumor-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression

doi: 10.1038/s41563-020-00849-5

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 two-tailed unpaired t-test without adjustment for multiple comparisons was used for individual comparisons. ( a ) Overall ****p < 0.0001, ER+-HER2+ ***p = 0.0001, ER+-TN ****p < 0.0001, HER2+-TN ***p = 0.0008. ( b ) Overall p = 0.7984, ER+-HER2+ p = 0.5435, ER+-TN p = 0.7198, HER2+-TN p = 0.7505. ( c ) Overall ****p < 0.0001, ER+-HER2+ **p = 0.01, ER+-TN ****p < 0.0001, HER2+-TN ****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 two-tailed Mann-Whitney U test ****p < 0.0001. ( e ) Scatter plot of individual and mean values ± SEM 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 two-tailed Mann-Whitney U test **p = 0.002, *p = 0.0204. ( h ) Bar plot showing distribution of epithelial LOX staining intensity among human patient tumors stratified by molecular subtype. ( i ) Bar plot showing distribution of stromal LOX staining intensity among human patient tumors stratified by molecular subtype. LOX scoring of Biomax BC081116c TMA was performed by a researcher (ER+ n = 43, HER2+ n = 38, TN n = 18 epithelial, 19 stromal). ( j ) Bar plot showing distribution of epithelial LH2 staining intensity among human patient tumors stratified by molecular subtype. ( k ) Bar plot showing distribution of stromal LH2 H Score among human patient tumors stratified by molecular subtype. LH2 scoring of patient samples from the MDCS cohort was performed by a pathologist. See and for patient information. All n values represent biologically independent human tissue specimens.

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, Two Tailed Test, MANN-WHITNEY, Staining

Journal: Nature materials

Article Title: Tumor-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression

doi: 10.1038/s41563-020-00849-5

Figure Lengend Snippet: ( a ) Neoplastic epithelial LOX staining of the Biomax BC081116c TMA was assessed by a researcher with an intensity score and stratified as negative/low, moderate, or high. ( b ) Stromal LOX staining of the Biomax BC081116c TMA was assessed by a researcher with an intensity score and stratified as negative/low, moderate, or high. Scale bar is 500 μm. ( c ) Tumor samples from incident breast cancer cases were collected, and a tissue microarray (TMA) including two 1-mm cores from each tumor was constructed. Neoplastic epithelial LH2 staining was assessed by a pathologist with semi-quantitative intensity score and stratified as negative, low, or moderate/high. Scale bars are 500 μm. See for patient information. ( d ) Clinical correlation of neoplastic epithelial LH2 intensity score with tumor grades. ( e ) Kaplan-Meier curves indicating cumulative breast cancer specific survival (BCSS) based on epithelial LH2 intensity assessed in breast cancer patients up to 10 years after diagnosis (LH2 negative n = 271, weak n = 112, moderate/high n = 77). ( f ) BCSS curves by epithelial LH2 intensity including only axillary lymph node negative patients (LH2 negative n = 175, weak n = 67, moderate/high n = 50). ( g ) BCSS curves by epithelial LH2 intensity including only axillary lymph node positive patients (LH2 negative n = 84, weak n = 42, moderate/high n = 26). For tumor grade and LH2 intensity score, statistical analysis was performed using a linear-by-linear association (***p < 0.001). For Kaplan-Meier curves, statistical analyses were using LogRank test ( e ) *p = 0.025 ( f ) *p = 0.038 ( g ) p = 0.165.

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

Techniques: Staining, Microarray, Construct, Biomarker Discovery

Journal: Nature materials

Article Title: Tumor-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression

doi: 10.1038/s41563-020-00849-5

Figure Lengend Snippet: ( a ) Representative images of PyMT tumors from 8-week-old mice treated with anti-CSF1 blocking antibody or IgG1 control. IgG1 treated (n = 6) and anti-CSF1 treated (n = 5) PyMT tumors stained for pan-cytokeratin (green), F4/80 (white) and DAPI (blue). Polarized light images with brightfield inset of IgG1 treated (n = 6) and anti-CSF1 treated (n = 6) PyMT tumors stained with picrosirius red (PS). IgG1 treated (n = 6) and anti-CSF1 treated (n = 5) PyMT tumors stained pY397 FAK (red) and DAPI (blue). Scale bars are 100 μm. ( b ) Scatter plot with individual values and mean ± SEM of metastatic colonies in lung tissues from 11-week-old IgG1 treated (n = 5) and anti-CSF1 treated (n = 6) mice via PyMT IHC. Statistical analysis was performed using two-tailed unpaired t-test *p = 0.0363. ( c ) Scatter plot with individual values and mean ± SEM of PS staining by percent area per field of view in 8-week-old mice treated with anti-CSF1 blocking antibody (n = 6) or IgG1 control (n = 6). Statistical analysis was performed using two-tailed unpaired t-test p = 0.08. ( d ) Histogram of the top 10% of elastic modulus measurements by AFM microindentation in PyMT IgG1 treated (n = 7) and anti-CSF1 treated (n = 5) tumors. Statistical analysis was performed using two-tailed Mann-Whitney U test ****p < 0.0001. ( e ) Representative images of Lox and Plod2 mRNA in situ hybridization in IgG1 treated (n = 6) and anti-CSF1 treated (n = 5) PyMT tumors and DAPI (blue). Scale bars are all 100μm. ( f-g ) Scatter plot with individual values and mean ± SEM of stromal Lox ( f ) or stromal Plod2 ( g ) mRNA in situ hybridization mean stromal intensity in 8-week-old mice treated with anti-CSF1 (n = 5) or IgG1 control (n = 6). Statistical analyses were performed using two-tailed Mann-Whitney U test ( f ) *p = 0.0381 and ( g ) *p = 0.0303. ( h ) Scatter plot showing individual and mean values ± SEM of total hydroxyproline (collagen content) in tumors from IgG1 (n = 6) and anti-CSF1 (n = 6) treated mice. ( i-n ) Scatter plots showing individual and mean values ± SEM of the levels of total collagen crosslinks ( i ), LNL ( j ), HLNL ( k ), DHLNL ( l ), Pyr ( m ), and total HLCCS ( n ) in 8-week-old IgG1 treated (n = 9 i,k,l,n or 10 j,m ) and anti-CSF1 (n = 11) treated PyMT tumors. Quantity of crosslinks per tissue was calculated normalizing crosslinks to dry tissue weight. Values were plotted as normalized peak areas as quantified from LC-MS data. Statistical analyses ( h-n ) were performed using two-tailed unpaired t-test ( h ) *p = 0.0447 ( i ) *p = 0.0174 ( j ) *p = 0.0359 ( k ) *p = 0.0235 ( l ) *p = 0.0311 ( n ) *p = 0.0366. ( o ) Scatter plot with individual values and mean ± SEM of Tgfb1 gene expression by RT-qPCR in tumor cells, cancer-associated fibroblasts, and macrophages sorted from PyMT tumors (n = 4). Statistical analysis was performed using Kruskal-Wallis one-way ANOVA for overall comparison and two-tailed Mann-Whitney U test for individual comparisons. Overall ***p = 0.0002, Tumor-CAF *p = 0.0286, Tumor-TAM *p = 0.0286, CAF-TAM *p = 0.0286. ( p ) Representative images of PyMT tumors from mice treated with IgG1 (n = 6) and anti-CSF1 (n = 5) stained for pan-cytokeratin (green), pS465/467 SMAD2 (red), and DAPI (blue). Scale bar is 100 μm. ( q ) Scatter plot showing individual and mean values ± SEM of the mean nuclear intensity of stromal pS465/467 SMAD2 of IgG1 treated (n = 6) and anti-CSF1 treated (n = 5) PyMT mice. Statistical analysis was performed using two-tailed unpaired t-test (p = 0.06). For ( a-q ) all n values represent biologically independent mouse tissue specimens. ( r ) Representative IHC images of serial human breast tumor sections stained for pS465/467 SMAD2 and CD68 and counterstained with hematoxylin. Scale bar is 100 μm. ( s ) Scatter plot with linear regression correlation of stromal pS465/467 SMAD2 IHC staining with stromal CD68 IHC staining in human breast tumors (n = 10, *p = 0.0248). ( t ) Scatter plot depicting the linear regression of CD14 + CD11b + HLA-DR + tumor-associated macrophage (as %CD45+ by flow cytometry) with mean elastic modulus as measured by AFM microindentation in human breast tumors (n = 15, **p = 0.0025). For ( r-t ) all n values represent biologically independent human tissue specimens.

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, Two Tailed Test, MANN-WHITNEY, In Situ Hybridization, Liquid Chromatography with Mass Spectroscopy, Gene Expression, Quantitative RT-PCR, Comparison, Immunohistochemistry, Flow Cytometry

Journal: Nature materials

Article Title: Tumor-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression

doi: 10.1038/s41563-020-00849-5

Figure Lengend Snippet: ( a ) Scatter plot showing individual values and mean ± SEM of the number of F4-80 positive cells per field of view in IgG1 treated (n = 6) or anti-CSF1 treated (n = 4) PyMT tumors from 8-week-old mice. ( b-c ) Scatter plots showing individual values and mean ± SEM of the percentage of F4/80 positive cells expressing markers MHCII and CD86 ( b ) and markers CD163 and Relmα ( c ) in IgG1 treated (n = 6) or anti-CSF1 treated (n = 4-5) PyMT tumors from 8-week-old mice. ( d-g ) Scatter plots showing individual values and mean ± SEM of the number of cells per field of view (FOV) of other non-macrophage immune populations CD11b + F4-80 − ( d ), MHCII hi F4-80 − ( e ), CD45 + CD4 + ( f ), and CD45 + CD8 + ( g ) in IgG1 (n = 4 e,g ; n = 5 d,f ) and anti-CSF1 (n = 6) treated mice. Statistical analysis ( a-g ) was performed using two-tailed Mann-Whitney U test ( a ) *p = 0.019 ( b ) MHCII hi p = 0.7190, CD86 + p = 0.4286 ( c ) CD163 + *p = 0.0303, Relmα + p = 0.0381 ( d ) p = 0.9719 ( e ) p = 0.1524 ( f ) p = 0.3074 ( g ) p = 1.000. ( h ) Heat map of selected differentially expressed genes from macrophages sorted from 8-week and 11-week PyMT tumors. ( i ) Representative images from 8-week-old IgG1 treated and anti-CSF1 treated PyMT mice stained for cytokeratin 8/18 (green), PDGFRα (magenta), vimentin (yellow), and DAPI (blue). Scale bar is 100 μm. ( j ) Scatter plot showing mean ± SEM of fibroblasts (vimentin + and PDGFRα + ) per field of view in 8-week-old IgG1 treated (n = 6) and anti-CSF1 (n = 5) treated PyMT mice. All n for ( a-j ) values represent biologically independent mouse tissue specimens. ( k-m ) Scatter plot depicting the Spearman correlation of CD163 gene expression with LOX ( k ), PLOD2 ( l ), and LOXL2 (*p = 0.0143) ( m ) in human breast tumors (n = 1904 biologically independent human tissue specimens).

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, Two Tailed Test, MANN-WHITNEY, Staining, Gene Expression

Journal: Nature materials

Article Title: Tumor-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression

doi: 10.1038/s41563-020-00849-5

Figure Lengend Snippet: ( a-b ) Kaplan-Meier plots showing overall survival for patients based on LOX expression in epithelial cells (low n = 28, high n = 36) ( a ) or stromal cells (low n = 23, high n = 24) ( b ). ( c-d ) Kaplan-Meier plots showing overall survival for patients based on PLOD2 expression in epithelial cells (low n = 28, high n = 29) ( c ) or stromal cells (low n = 23, high n = 24) ( d ). For ( a-d ) the median expression was defined as the cutoff for low and high expression. ( e ) Representative phase contrast images from tissue microarrays (TMAs) of human breast cancers. Sections were stained with H&E and lysyl hydroxylase two (LH2) via IHC. ( f ) Bar graphs showing clinical correlation between stromal LH2 H score and tumor grade (see for number of patients). LH2 IHC staining was assessed by a pathologist 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 moderate 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). ( g ) 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, moderate n = 188, high n = 146). ( h ) BCSS curves by stromal LH2 H score including only axillary lymph node negative patients (LH2 low n = 116, moderate n = 116, high n = 90). ( i ) BCSS curves by stromal LH2 H score including only axillary lymph node positive patients (LH2 low n = 44, moderate n =63, high n = 54). For Kaplan-Meier curves, statistical analyses were performed by LogRank test ( g ) **p = 0.008, ( i ) *p = 0.026. All n values represent biologically independent human tissue specimens.

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, Immunohistochemistry, Biomarker Discovery

Journal: Nature materials

Article Title: Tumor-associated macrophages drive stromal cell-dependent collagen crosslinking and stiffening to promote breast cancer aggression

doi: 10.1038/s41563-020-00849-5

Figure Lengend Snippet: ( a ) Log2 scaled data graphs showing relative PLOD2 gene expression levels in patients stratified by estrogen and epidermal growth factor receptor two (ER/HER2) status ( ER+ HER2− n=314; ER− or + /HER2+ n=73; ER−/HER2− n=133 ). Whiskers denote minima and maxima, box denotes 25 th and 75 th percentiles and median. Statistical analysis was performed to compare PLOD2 expression levels among subtypes using Kruskal-Wallis one-way ANOVA (****p < 0.0001) and two-tailed Mann-Whitney U test without adjustment for multiple comparisons for individual comparisons (****p < 0.0001). ( b ) Line graphs showing distant metastasis-free survival (DMFS) for patients with estrogen receptor positive and epidermal growth factor receptor two negative breast tumors (ER+/− HER2−; low n=157 & high=157 ). ( c ) Line graph of DMFS for patients with estrogen receptor negative or negative and epidermal growth factor receptor two positive breast tumors (ER− or +/HER2+; low n=36 & high=37 ). ( d ) Line graph of DMFS for patients with estrogen receptor negative epidermal growth factor receptor negative breast tumors (ER− HER2−; low n=66 & high=67 ). Statistical analyses were performed to compare PLOD2 and DMFS for each subtype or Log-rank (Mantel-Cox) test ( b ) p = 0.8051 ( c ) *p = 0.0144 ( d ) p = **0.0050. ( e-g ) Kaplan-Meier curves indicating the probability of relapse-free survival assessed in ER+/PR+ ( e ), HER2+ ( f ), and triple negative ( g ) breast cancer patients up to 16 years after diagnosis. A correlation between PLOD2 (gene encoding LH2) expression and RFS has been determined using an online tool ( http://kmplot.com/analysis/ ) as described in the methods. Top and bottom panels represent two distinct Affymetrix PLOD2 probes (202619 and 202620) from the same database. For Kaplan-Meier curves, statistical analyses were performed using 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: Gene Expression, Expressing, Two Tailed Test, MANN-WHITNEY, 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: 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, 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: 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: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: Schematic representation of genetic constructs CMV—CMV promoter: ( A ) wt PLOD2 ( LH2a ) expression plasmid, ( B ) wt PLOD2 ( LH2b ) expression plasmid, and ( C ) mutant Thr629Ala allele PLOD2 expression plasmid.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Construct, Expressing, Plasmid Preparation, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: Transfection efficiency of a plasmid with a GFP reporter. Flow cytometry analysis of transfection efficacy of HEK293 ( left ) and DF2 ( right ) cells in 24 h ( A ). Visualization of HEK293 ( left ) and DF2 ( right ) cells in 48 h using the EVOS FL Auto Imaging System ( B ). Green – PLOD2 fused to GFP. Scale bar—200 µm.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: Confocal microscopy images of HEK293 cells transfected with three different constructs, including GFP-LH2a ( B ), GFP-LH2b ( C ), and GFP-LH2b with mutation Thr629Ala ( D ) and wild-type without constructs ( A ). In Figure ( D ), the arrow shows a change in the morphology of the cell with expression of the LH2b (Thr629Ala). The left column (green) shows the images of GFP-tagged protein PLOD2, the middle column (red) shows the images of ER, and the right column shows the overlaid images of the left and middle columns. Scale bar—25 µm.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Confocal Microscopy, Transfection, Construct, Mutagenesis, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: List of primers used in this study.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Sequencing, Amplification, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Cellular and Molecular Effects of the Bruck Syndrome-Associated Mutation in the PLOD2 Gene

doi: 10.3390/ijms252413379

Figure Lengend Snippet: Cloning scheme.

Article Snippet: DF2 cells were additionally stained with antibodies to the PLOD2 protein (1:50, PAE257Hu01, Cloud-Clone Corp., Katy, TX, USA) and Alexa-Fluor594 (1:400, ab150080, Abcam, Cambridge, UK) to confirm transfection.

Techniques: Clone Assay, Construct