Journal: Communications Biology

Article Title: Hypoxia-induced circPLOD2a/b promotes the aggressiveness of glioblastoma by suppressing XIRP1 through binding to HuR

doi: 10.1038/s42003-025-07503-3

Figure Lengend Snippet: A – D Proteins pulled down by biotin-labeled circPLOD2a ( A ) or circPLOD2b ( C ) antisense probes and corresponding scrambled control probes in hypoxic U87 cells were visualized by SDS-PAGE and silver staining. Unique peptides identified by mass spectrometry in ( B ) circPLOD2a ( n = 73) or ( D ) circPLOD2b ( n = 54) compared with corresponding control lanes. E , F Potential proteins interacting with ( E ) circPLOD2a and ( F ) circPLOD2b were verified by RNA pull-down assays with corresponding antisense probes in hypoxic U87 cells. Relative integrated density normalized to β-actin was marked above each band. G – J RIP assay confirms the interaction of circPLOD2a/b and linear PLOD2 with HuR. CircPLOD2a/b and linear PLOD2 were detected by RT-PCR ( G ). The immunoprecipitation products of anti-HuR compared with IgG were treated with or without RNase R. Quantitative enrichment analysis of circPLOD2a ( H ), circPLOD2b ( I ) and linear PLOD2 ( J ) were analyzed by qRT-PCR, n = 3. K Cellular localization of circPLOD2b (red) and HuR (green) were examined by dual RNA-FISH and IF in U87 cells treated with normoxic and hypoxic conditions. Scale bar: 25 μm. L Schematic diagram of wild type and various truncated HuR with Flag-tag. (M) The interaction of circPLOD2a/b with full-length or truncated HuR protein were detected by RIP assay in U87 cells. CircPLOD2a/b were detected by RT-PCR; full-length and truncated HuR proteins were detected by western blot using anti-Flag antibody. N CircPLOD2a and circPLOD2b were immunoprecipitated by anti-Flag beads in U87 cells transfected with plasmids carrying HuR- wildtype (HuR-wt) or truncated forms (HuR-△RRM1/2/3) and detected by qRT-PCR, n = 3. Data are shown as mean ± SEM (error bars) and analyzed using Student’s t -test ( H – J ) and ANOVA ( N ).

Article Snippet: The primary antibodies used for western blotting were anti-β-actin antibody (66009-1-Ig, Proteintech, 1:2000), anti-HIF1α antibody (ab179483, Abcam, 1:1000), anti-PLOD2 antibody (66342-1-Ig, Proteintech, 1:2000), anti-ALDA antibody (11217-1-AP, Proteintech, 1:2000), anti-BIP antibody (11587-1-AP, Proteintech, 1:2000), anti-DDX5 antibody(67025-1-Ig, Proteintech, 1:2000), anti-HuR antibody (11910-1-AP, Proteintech, 1:2000), anti-IGF2BP3 antibody (14642-1-AP, Proteintech, 1:2000), anti-E-cadherin antibody (20874-1-AP, Proteintech, 1:1000), anti-N-cadherin antibody (22018-1-AP, Proteintech, 1:1000), anti-Vimentin antibody (10366-1-AP, Proteintech, 1:1000), anti-Flag antibody (20543-1-AP, Proteintech, 1:2000), anti-XIRP1 antibody(PA5-48605, Invitrogen, 1:1000), HRP Goat anti-Rabbit antibody (AS014, Abclonal, 1:5000), and HRP Goat anti-Mouse antibody (SA00001-1, Proteintech, 1:5000).

Techniques: Labeling, Control, SDS Page, Silver Staining, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Quantitative RT-PCR, FLAG-tag, Western Blot, Transfection

Journal: Communications Biology

Article Title: Hypoxia-induced circPLOD2a/b promotes the aggressiveness of glioblastoma by suppressing XIRP1 through binding to HuR

doi: 10.1038/s42003-025-07503-3

Figure Lengend Snippet: A , B Relative expression of ( A ) circPLOD2a and ( B ) circPLOD2b were examined by qRT-PCR in clinical samples from patients with grade II-III ( n = 10) and grade IV ( n = 9) glioma. C Relative expression of XIRP1 in tissues from circPLOD2a-high and -low groups. The GBM tissues were divided into circPLOD2a-high ( n = 10) and -low ( n = 9) groups according to the expression of circPLOD2a. D Relative expression of XIRP1 in tissues from circPLOD2b-high and -low groups. The GBM tissues were divided into circPLOD2a-high ( n = 10) and -low ( n = 9) groups according to the expression of circPLOD2b. E Relative expression of XIRP1 in GBM and non-tumor brain tissues were analyzed from Rembrandt database. F Representative immunohistochemistry images of XIRP1 in a tissue microarray containing grade I ( n = 3), II ( n = 50), III ( n = 24) and IV ( n = 94) glioma. Scale bar: 100 μm. G , H Expression levels of XIRP1 in tissues from patients with different grade glioma were evaluated by ( G ) the percentage of cells with strong/moderate/weak intensity of XIRP1 staining and ( H ) histochemistry score (H-score) of each tissue on this microarray. I Kaplan–Meier survival curves of glioma patients in XIRP1-high and low groups. Glioma patients were categorized into XIRP1-high and -low group by the expression level of XIRP1 based on H-score. J Schematic illustration of HIF1α/circPLOD2a/b/HuR/XIRP1 axis. HIF1α induces the up-regulation of linear PLOD2 , circPLOD2a and circPLOD2b under hypoxia. CircPLOD2a/b suppress XIRP1 by competitively binding to HuR and enhance GBM cell aggressiveness. Data are shown as mean ± SEM (error bars) and analyzed using Student’s t -test ( A – E ), ANOVA ( H ) and Log-rank test ( I ).

Article Snippet: The primary antibodies used for western blotting were anti-β-actin antibody (66009-1-Ig, Proteintech, 1:2000), anti-HIF1α antibody (ab179483, Abcam, 1:1000), anti-PLOD2 antibody (66342-1-Ig, Proteintech, 1:2000), anti-ALDA antibody (11217-1-AP, Proteintech, 1:2000), anti-BIP antibody (11587-1-AP, Proteintech, 1:2000), anti-DDX5 antibody(67025-1-Ig, Proteintech, 1:2000), anti-HuR antibody (11910-1-AP, Proteintech, 1:2000), anti-IGF2BP3 antibody (14642-1-AP, Proteintech, 1:2000), anti-E-cadherin antibody (20874-1-AP, Proteintech, 1:1000), anti-N-cadherin antibody (22018-1-AP, Proteintech, 1:1000), anti-Vimentin antibody (10366-1-AP, Proteintech, 1:1000), anti-Flag antibody (20543-1-AP, Proteintech, 1:2000), anti-XIRP1 antibody(PA5-48605, Invitrogen, 1:1000), HRP Goat anti-Rabbit antibody (AS014, Abclonal, 1:5000), and HRP Goat anti-Mouse antibody (SA00001-1, Proteintech, 1:5000).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Microarray, Staining, Binding 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: 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