Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: Identification of candidates involved in the trafficking of MMP2. (A) Scheme of the MMP2 RUSH construct. SS-Flag-MMP2-HA-SBP-eGFP was used as a reporter. Fluorescence images show HeLa cells expressing MMP2-SBP-eGFP counterstained against TGN46 (red). Without biotin, MMP2 is retained in the ER (0 min). It reaches the Golgi 15 min after biotin addition and is sorted into vesicles (arrowheads) at 30 and 45 min, respectively. Scale bars, 5 µm. (B) MS strategy to identify MMP2 interacting partners in the Golgi. HeLa cells expressing MMP2-SBP-eGFP or SS-SBP-eGFP were incubated for 20 min with biotin to enrich reporter proteins at the Golgi. After GFP IP, samples were analyzed using MS ( n = 3). (C) Volcano plot highlights significantly enriched MMP2 interactors in pink. 42 sorting-related candidates were found, among them TIMP2, a known inhibitor of MMP2, and NUCB1. Two-sample t test, false discovery rate = 0.3, minimum fold change = 0.5. (D) Fluorescence images of HeLa cells labeled with endogenous NUCB1 (green) and GM130 or TGN46 (red). Scale bars, 5 µm; zoom, 2 µm. (E) HEK 293T cells expressing SS-MMP2-SBP-eGFP or SS-SBP-eGFP were processed for GFP IP and WB analysis. (F) Semiquantitative analysis of the normalized NUCB1 to GFP signal from two independent experiments. Significance: one-sample t test. (G) His-tag coIP of recombinant rNUCB1-His. Endogenous MMP2 from HeLa Golgi membranes coimmunoprecipitated with rNUCB1-His but not rGFP-His. (H) Semiquantitative analysis of the MMP2 signal from three independent experiments. Bars, mean ± SD. Paired t test: *, P < 0.05; ***, P < 0.001.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Construct, Fluorescence, Expressing, Incubation, Labeling, Recombinant

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2-eGFP secretion and evaluation of CRISPR NUCB1-KO clones . (A) HeLa cells stably expressing SS-MMP2-eGFP were seeded on glass slides and incubated at 37°C for 3 d to evaluate MMP2-eGFP secretion. After fixation, cells were incubated with GFP antibody and Alexa Fluor 594. Confocal fluorescence images show colocalization of MMP2-eGFP and GFP antibody of nonpermeabilized cells, evidencing secretion of MMP2-eGFP to the extracellular space. Scale bars, 10 µm; zoom bar, 2 µm. (B and C) NUCB1-KO cells were generated using the CRISPR-Cas9 system with three different gRNAs and selection of single colonies. After puromycin selection, three NUCB1-KO clones were identified by WB (B) and later confirmed by immunofluorescence (C). *, unspecific band; KO, HeLa NUCB1-KO cells; CN, HeLa control. Semiquantitative analysis shows normalized NUCB1-to-β-actin signal.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: CRISPR, Clone Assay, Stable Transfection, Expressing, Incubation, Fluorescence, Generated, Selection, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2 is partially sorted in LyzC-positive secretory vesicles. HeLa cells expressing MMP2-eGFP were immunolabeled with a-Rab5, a-Rab7, or Rab11 antibodies (red). MMP2-eGFP–expressing cells were cotransfected with mCherry (mCh)-lysosomes or LyzC-mCherry to label lysosomes or LyzC-positive secretory vesicles, respectively. Rab6-GFP or Rab8-GFP constructs were cotransfected with MMP2-tagRFP. Images were acquired by confocal microscopy. White arrowheads point to distinct vesicles; magenta arrowheads point to colocalizing vesicles. Bars, 10 µm; zoom, 2 µm.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Immunolabeling, Construct, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: Protein purification and evaluation of the direct interaction between MMP2 and NUCB1. (A) Coomassie-stained SDS-PAGE for the evaluation of His-tag purified recombinant NUCB1-His (rNUCB1-His). (B) Anti-NUCB1 WB analysis of the elution fraction shown in line 4 from A. (C) WB analysis of purified His-SUMO-MMP2 using MMP2 antibody. (D) Recombinant His-SUMO-MMP2 (rHS-MMP2) was bioconjugated with Cy3 via maleimide labeling and subsequently analyzed by AUC. The lowest panel shows peak of sedimentation of rHS-MMP2 at 4.705 S. (E) AUC profile of rHis-SUMO-MMP2-Cy3 and NUCB1-His. The lowest panel shows a peak at 3.189 S, indicating a change in the sedimentation velocity associated to a direct interaction of NUCB1 and MMP2. (F) Coomassie-stained SDS-PAGE of purified His-tagged NUCB1 Ca 2+ binding mutant (rNUCB1mEFh1+2). (G) WB analysis of the elution fraction shown in line 4 of F using NUCB1 antibody. (H) CD measurement of rNUCB1-His and rNUCB1mEFh1+2-His under presence or absence of 1 mM Ca 2+ . rNUCB1-mEF1+2 molar ellipticity is lower compared with rNUCB1-His. Evaluation of the CD spectra using CONTIN showed an increase in rNUCB1-His α-helicity upon Ca 2+ addition (from 0.385 to 0.413) that was not observed in rNUCB1-mEFh1+2 (from 0.256 to 0.147). Instead, an increase in β-sheet content (from 0.151 to 0.322) was observed. These findings are in accordance with the results described by .

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Protein Purification, Staining, SDS Page, Purification, Recombinant, Labeling, Sedimentation, Binding Assay, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: NUCB1-KO impairs the trafficking of MMP2. (A) Fluorescent images of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP with or without NUCB1-WT, counterstained against NUCB1 (red) and captured after 0, 15, 30, and 45 min of biotin incubation. Arrowheads, cytoplasmic vesicles. Scale bars, 5 µm. (B) Cytoplasmic vesicle counts as described in A are plotted as number of vesicles per cell ( n ≥ 90 cells, median ± IQR of two independent experiments; ***, P < 0.001; n.s., not significant). (C) Confocal microscopy images of HeLa or NUCB1-KO cells expressing LyzC-SBP-eGFP and counterstained against NUCB1 (red) after 0, 20, 40, and 60 min of biotin incubation. Arrowheads, cytoplasmic vesicles. Scale bars, 5 µm. (D) Cytoplasmic vesicle counts from C of two independent experiments ( n ≥ 42 cells, median ± IQR). (E) Secretion assay of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP or LyzC-SBP-EGFP and incubated with biotin for 45 or 60 min, respectively. WCL, whole-cell lysates. [SNs], 10×-concentrated supernatants. (F) Semiquantitative analysis from three independent experiments, one-sample t test. Bars, mean ± SD. (G) GFP-coIP of HeLa or NUCB1-KO cells expressing LyzC-eGFP, with or without NUCB1-WT. GFP-HA, negative control; CN, HeLa control; KO, NUCB1-KO. (H) Semiquantitative analysis of NUCB1 to GFP signal from three independent experiments. Bars, mean ± SD; paired t test.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Incubation, Confocal Microscopy, Negative Control

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2 IG trafficking is exclusively dependent on Golgi-localized NUCB1, which also impairs IG trafficking of MT1-MMP. (A) HeLa or NUCB1-KO cells expressing SS-SBP-MMP2-eGFP alone or with a cytosolic variant of NUCB1 lacking its SS (NUCB1-cyto) were fixed after 0, 15, 30, and 45 min of biotin incubation. Maximal Z-projection analysis of confocal microscopy images shows no differences in MMP2 trafficking of NUCB1-cyto transfected cells compared with NUCB1-KO cells (arrowheads). Scale bars, 10 µm. (B) Quantification of cytoplasmic MMP2 vesicles from cells in A. n > 18 cells; mean ± SD; two independent experiments. Significant differences with P < 0.05 were analyzed via nonparametric Kruskal–Wallis test with Dunn’s multiple comparison, **, P < 0.01. (C) mCherry-tagged MT1-MMP RUSH construct (SS-MT1-MMP-SBP-mCh). Cyto, cytosolic domain. (D) Confocal fluorescence images of HeLa or NUCB1-KO cells transfected with or without NUCB1-WT and fixed after 30, 60, and 90 min of biotin incubation. Arrowheads, cytoplasmic vesicles. Scale bars, 5 µm. (E) Quantification of cytoplasmic vesicles observed in A. n = 24 cells; two independent experiments; median ± IQR; ***, P < 0.001; n.s., non-significant. (F) Cell surface biotinylation assay coupled with streptavidin pull-down. HeLa or NUCB1-KO cells were untreated (time 0) or incubated with sulfo-NHS-Biotin for 90 min to label cell surface proteins, and then pulled down with Neutravidin beads. WB analysis shows a reduction in the amount of endogenous active MT1-MMP at the surface of NUCB1-KO cells compared with HeLa control. β-1 integrin was used as loading control. (G) Semiquantitative analysis of surface labeled active MT1-MMP from F represented as % of normalized MT1-MMP intensity to β-1 integrin in comparison to control (100%). n = 3 independent experiments; one-sample t test, **, P < 0.01. Bars, mean ± SD.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Variant Assay, Incubation, Confocal Microscopy, Transfection, Construct, Fluorescence, Cell Surface Biotinylation Assay, Labeling

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: NUCB1 does not affect MMP2 activation nor trafficking of other cargoes such as HRP and Cathepsin D. (A) Zymography assay of HeLa cells expressing SS-MMP2-SBP-eGFP. Untsf HeLa, Hela without transfection; [SN], 10×-concentrated supernatants; CN, HeLa control; KO, NUCB1-KO. (B) Semiquantitative analysis of experiment shown in A. n = 3 independent experiments; one-sample t test; n.s., nonsignificant. (C) Whole-cell lysates of HeLa and NUCB1-KO cells stably expressing SS-HRP-FLAG were analyzed by anti-FLAG, anti-NUCB1, and anti-β-actin WB. SS-HRP-FLAG is expressed in HeLa and NUCB1-KO cells to similar levels. (D) Cell culture supernatants of cells described in C were analyzed for HRP activity by chemiluminescence after 4-h secretion. BFA served as a positive control for perturbed secretion and was added for 1 h before HRP secretion analysis. No significant differences were observed between NUCB1-KO and HeLa control cells. *, P < 0.05. (E) HeLa or NUCB1-KO cells expressing SS-SBP-eGFP-Cathepsin D were fixed 20, 40, and 60 min after biotin addition. Representative maximum Z-projection images show Cathepsin D trafficking from Golgi to cytoplasmic vesicles (arrowheads). Scale bars, 10 µm. (F) Quantification of cytoplasmic Cathepsin D vesicles from cells shown in E. n > 30 HeLa and NUCB1-KO cells per time point; two independent experiments; mean ± SD. Statistical analysis was performed using a nonparametric Kruskal–Wallis test with Dunn’s multiple comparison test. No significant differences with P < 0.05 were detected.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Activation Assay, Zymography, Expressing, Transfection, Stable Transfection, Cell Culture, Activity Assay, Positive Control

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2 trafficking delay occurs at the cis-Golgi. (A) Fluorescence images of HeLa or NUCB1-KO cells transiently expressing SS-MMP2-SBP-eGFP, fixed at 2.5, 5, and 7.5 min after biotin addition, and counterstained against ERGIC53 (red). Scale bars, 5 µm. (B) Average PC per time point. (C) Colocalization of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP with GM130 (red) after 10, 15, 20, and 25 min of biotin incubation. Scale bars, 5 µm. (D) Average PC illustrates decreased colocalization at 10, 15, and 20 min after biotin addition. (E) Colocalization of SS-MMP2-SBP-eGFP with TGN46 (red) expressed in HeLa or NUCB1-KO cells at 20, 25, 30, 35, and 40 min after biotin addition. Scale bars, 5 µm. (F) Average PC shows that MMP2 is equally colocalizing with TGN46 in HeLa and NUCB1-KO cells upon arrival at the TGN. Error bars represent SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Fluorescence, Expressing, Incubation

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: MMP2 trafficking is exclusively delayed at the Golgi in living cells. (A) HeLa or NUCB1-KO cells expressing SS-SBP-MMP2-eGFP were analyzed by live-cell wide-field microscopy. Representative images of MMP2 trafficking after 0, 30, 35, and 40 min of biotin incubation. Images were acquired in 1-min frames for each analyzed cell. Arrowheads, cytoplasmic MMP2 vesicles. Scale bars, 10 µm. (B) Quantification of cytoplasmic MMP2 vesicles per frame from cells shown in A. n.s., nonsignificant. *, P < 0.05; **, P < 0.01. (C) Schematic representation of ER–Golgi cargo transport analysis, measured as normalized Golgi area over time in cells shown in A. (D) Normalized Golgi area for each time point (median ± IQR). A reduced Golgi compaction was observed in the time range 15–23 min in NUCB1-KO cells compared with HeLa control. *, P < 0.05. (E and F) HeLa or NUCB1-KO cells ( n = 11) expressing SS-SBP-MMP2-eGFP fixed without biotin addition and immunostained for ER exit site marker Sec16 (red). Scale bar, 10 µm; zoom, 2 µm. Retained MMP2 in the ER partially colocalized with Sec16 in both control and NUCB1-KO cells to the same extent (F). Magenta arrowheads, MMP2 structures that colocalized with ER exit sites; white arrowheads, ER exit sites. t test: P < 0.05.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Microscopy, Incubation, Marker

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: NUCB1 EFhs are essential for Golgi trafficking of MMP2. (A) Protein alignment of human NUCB1 (Q02818, aa 241–400), CaM (P0DP23), Calumenin (O43852), and Cab45 (Q9BRK5). Pink boxes, NUCB1 EFhs. (B) NUCB1 adapted PDB protein model (accession no. 1SNL ); NUCB1 EFhs, cyan; NUCB1-WT, EFhs with first and last amino acid of the domain in dark blue; NUCB1-mEFh1+2, amino acid substitutions E264Q and E316Q in pink. (C) CoIP of MMP2-eGFP transiently expressed in NUCB1-KO cells transfected with NUCB1-WT or NUCB1-mEFh1+2. n = 4 biological replicates. (D) Semiquantitative analysis of NUCB1 signal per sample normalized to the one of NUCB1-KO cells reexpressing NUCB1-WT. Bars, mean ± SD; one-sample t test. (E) Confocal fluorescence images of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP and cotransfected with or without NUCB1-WT or NUCB1-mEFh1+2. After 15, 30, and 45 min of biotin incubation, cells were fixed and costained with NUCB1 antibody (red). Scale bars, 5 µm. Arrowheads, cytoplasmic vesicles. (F) Quantification of cytoplasmic vesicles as in E from two independent experiments (median ± IQR), n ≥ 19 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Transfection, Fluorescence, Expressing, Incubation

Journal: The Journal of Cell Biology

Article Title: Nucleobindin-1 regulates ECM degradation by promoting intra-Golgi trafficking of MMPs

doi: 10.1083/jcb.201907058

Figure Lengend Snippet: NUCB1 depletion impairs ECM invasion and degradation in MDA-MB-231 cells. (A) Expression levels of NUCB1 after siRNA-mediated silencing ( n = 3 independent experiments: R1, R2, and R3). *, unspecific band. (B) Semiquantitative analysis of normalized NUCB1 signal from A in silenced cells compared with control. Bars, mean ± SD. (C) Quantitative PCR analysis of relative MMP2 expression in siRNA-treated MDA-MB-231 cells ( n = 3 independent experiments, one-sample t test). (D) Secretion assay of endogenous MMP2 in MDA-MB-231 cells. [SN], 20×-concentrated supernatant; WCL, whole cell lysates. (E) Semiquantitative analysis of three independent experiments. Bars, mean ± SD. Significance, one-sample t test. (F and G) Representative pictures of Matrigel-coated Transwell invasion (F) or gelatin degradation (G) experiments. Scale bars, 150 µm. (H and I) Quantification of the number of migrating cells (H) and degraded gelatin area (I). Both invasion and degradation were reduced in siNUCB1 cells. Data: median ± IQR; n = 3 independent experiments. Paired t test: *, P < 0.05; **, P < 0.01; n.s., not significant.

Article Snippet: The human MMP2 gene was amplified from a pCMV3-SP-Flag-MMP2 vector (Sino Biological) using 5′-CCC​AAG​CTT​ATG​CCA​CTG​CTG​CTC​TTG​CT-3′ as a forward (Fw) primer and 5′-TTT​TCC​TTT​TGC​GGC​CGC​TCA​AGC​GTA​ATC​TGG​AAC​ATC​GTA​TGG​GTA​GCA​GCC​TAG​CCA​GTC​GGA​TTT-3′ as a reverse (Rv) primer.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production

doi: 10.3389/fimmu.2025.1563845

Figure Lengend Snippet: Heterogenous expression of SLPI in steady-state conditions in murine macrophages. Comparison of Slpi transcript (A) and SLPI protein level (B) in different macrophage populations. Data obtained from publicly available dataset. (C) Representative SLPI expression in freshly isolated peritoneal, splenic, alveolar macrophages and BMDMs from WT (black line) and Slpi -/- mice (shaded histogram). (D) Fold change of geometrical MFI was compared between WT and Slpi -/- macrophages as in (C) Data pooled from n=2-7. WT vs Slpi -/- **p < 0.01, ***p < 0.001 by one-way ANOVA, Tuckey post hoc test. (E) qRT-PCR analysis of Slpi . mRNA expression by freshly isolated BMDMs and peritoneal macrophages. Data pooled from n=5. WT BMDM vs WT peritoneal macrophages **p < 0.01 by unpaired two-tailed t-test. (F) Volcano plot of differentially expressed genes (log2 fold change > 1 and p-adj < 0.05) between BMDMs and peritoneal macrophages with top 10 upregulated (red) and downregulated (blue) genes highlighted on the plot. (G) UMAP of CD11b + peritoneal cells. (H) Dot plot of 6 clusters from CD11b + peritoneal cells. (I) Violin plots with expression level of Slpi , Fn1 , Thbs1 , Prg4 , Lyz1 , F5 in 6 clusters from CD11b + peritoneal cells.

Article Snippet: The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBrightTM Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).

Techniques: Expressing, Comparison, Isolation, Quantitative RT-PCR, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production

doi: 10.3389/fimmu.2025.1563845

Figure Lengend Snippet: One sample analysis of cytokine proteome array. (A) The array images for supernatants of WT and Slpi -/- resident peritoneal macrophages stimulated with LPS (100 ng/ml) for 48h. Negative duplicate control spots are marked by red rectangle. Positive signal reference spots are marked by green rectangle. (B) Heatmap of relative levels (pixel density) of selected cytokines secreted by peritoneal macrophages.

Article Snippet: The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBrightTM Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production

doi: 10.3389/fimmu.2025.1563845

Figure Lengend Snippet: SLPI regulates MMP-9 secretion in resident peritoneal macrophages. (A) SLPI, IL-6 and TNF in supernatants of WT and Slpi -/- resident peritoneal macrophages incubated with LPS (100 ng/ml) for 24h.WT control vs WT LPS ** p< 0.01 by one-way ANOVA, Tuckey post hoc test. (B) IL-1α and IL-1β in supernatants of WT and Slpi -/- peritoneal macrophages incubated with LPS (100 ng/ml) for 3 h followed by addition of alum (100 µg/ml) or nigericin (10 µM). (C) Total cell and neutrophil count in WT and Slpi -/- mice injected i.p. with PBS or 100 μg alum for 24h. (D) Representative immunoblots of MMP-9 and SLPI in supernatants and lysates of WT and Slpi -/- peritoneal macrophages incubated with LPS (100 ng/ml) for 24h.Samples were resolved by SDS-PAGE and probed by Western blotting. β-actin was used as loading control. (E) Densitometry analysis of (D) WT LPS vs Slpi -/- LPS ***p < 0.001 by one-way ANOVA, Tukey post hoc test. (F) MMP-9 in supernatants of WT and Slpi -/- resident peritoneal macrophages incubated with LPS (100 ng/ml) for indicated times. WT LPS vs Slpi -/- LPS ***p < 0.001 by one-way ANOVA, Tukey post hoc test. (G) Representative zymography of equal volume of supernatants obtained from WT and Slpi -/- peritoneal macrophages incubated with LPS (100 ng/ml) for 24h.Representative of four separate experiments. (H) MMP-9 in supernatants of WT and Slpi -/- resident peritoneal macrophages incubated with DMSO or inhibitors: JNK-INH-8 (10 μM), SP600125 (20 μM), PD98059 (20 μM) or SB203580 (10 μM) for 1h followed by LPS treatment (100 ng/ml) for 24h.LPS vs LPS + inhibitor *p<0.05 by one-way ANOVA, Tukey post hoc test. (I) SLPI, IL-6 and MMP-9 in supernatants of WT and Slpi -/- resident peritoneal macrophages incubated with LPS (100 ng/ml) and IFN-γ (50 ng/ml) for 24h.Control vs IFN−γ *p<0.05, ***p<0.001 by multiple unpaired t-test. (A, B, F, H, I) Data are presented as the mean of three (A, B, F) or four (H, I) independent experiments. Error bars show means ± SEM. (C) Data represent 5 to 6 mice per experimental group pooled from two independent experiments. Error bars show means ± SEM.

Article Snippet: The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBrightTM Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).

Techniques: Incubation, Control, Injection, Western Blot, SDS Page, Zymography

Journal: Frontiers in Immunology

Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production

doi: 10.3389/fimmu.2025.1563845

Figure Lengend Snippet: SLPI regulates proinflammatory responses in recruited peritoneal macrophages. (A) Total cell and thioglycolate-elicited macrophages (TGMs) count in WT and Slpi -/- mice injected i.p. with saline or 4% thioglycolate (1 ml) for 96h. (B) SLPI, IL-6, TNF and MMP-9 in supernatants of WT and Slpi -/- TGMs incubated with LPS (100 ng/ml) for 24h.WT control vs WT LPS *p<0.05; WT LPS vs Slpi -/- LPS **p<0.01 by one-way ANOVA, Tukey post hoc test. (C) SLPI and MMP-9 in supernatants of WT and Slpi -/- TGMs incubated with various TLR ligands (PAM3CSK4, 10 ng/ml; zymosan, 1 µg/ml; HKLM, 10 7 cells; poly(I:C) HMW, 10 µg/ml; poly(I:C) LMW, 10 µg/ml; FLA, 1 µg/ml; FSL-1, 10 ng/ml; ssRNA, 2 µg/ml; ODN1826, 2 µg/ml) for 24h.WT TLR ligand vs Slpi -/- TLR ligand, ** p<0.01 by multiple unpaired t-test. (A) Data represent 4 to 6 mice per experimental group pooled from three independent experiments. Error bars show means ± SEM. (B, C) Data are presented as the mean of four independent experiments. Error bars show means ± SEM.

Article Snippet: The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBrightTM Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).

Techniques: Injection, Saline, Incubation, Control

Journal: Frontiers in Immunology

Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production

doi: 10.3389/fimmu.2025.1563845

Figure Lengend Snippet: The impact of SLPI on local response to LPS injection. (A) Total PEC number isolated from WT and Slpi -/- mice injected with PBS or LPS for 24h. (B) Peritoneal macrophage percentage and total number as in (A) . (C) Representative gating for resident macrophages in WT and Slpi -/- mice injected with PBS or LPS for 24h. (D) SLPI expression in resident peritoneal macrophages isolated from WT (black line) and Slpi -/- mice (shaded histogram) injected with PBS or LPS for 24h. (E) Fold change of geometrical MFI was compared between WT and Slpi -/- macrophages as in (D) . (F) MMP-9 in supernatants of WT and Slpi -/- total PEC or peritoneal adherent cells (macrophages) isolated from PBS or LPS injected mice and stimulated with LPS (100ng/ml) or HKLM (10 7 cells) for 24h.WT vs Slpi -/- **p<0.01 by multiple unpaired t-test. (A, B, E) Data represent 5 to 8 mice per experimental group pooled from three independent experiments. Error bars show means ± SEM. (F) Data represent 3 to 5 mice per experimental group pooled from two independent experiments. Error bars show means ± SEM.

Article Snippet: The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBrightTM Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).

Techniques: Injection, Isolation, Expressing

Journal: Frontiers in Immunology

Article Title: Endogenous SLPI contributes to the regulation of inflammatory responses in peritoneal macrophages by modulating MMP-9 production

doi: 10.3389/fimmu.2025.1563845

Figure Lengend Snippet: LPS induces changes in expression pattern of SLPI in selected immune cell populations in vivo . (A) Total blood CD45 + leukocyte number isolated from WT and Slpi -/- mice injected with PBS or LPS for 24h. (B) Representative gating for neutrophils (CD11b + Ly6G + SiglecF - ), eosinophils (CD11b + Ly6G - SiglecF + ) and monocytes (CD11b + Ly6C + Ly6G - ) as in (A) . (C) Percentage of neutrophils, eosinophils and monocytes isolated as in (A) . (D) Percentage of Ly6C hi , MHC II + F4/80 + and Ly6C low monocytes isolated as in (A) . (E) Representative gating of Ly6C hi (CD11b + Ly6C high ), MHC II + F4/80 + and Ly6C low monocytes isolated as in (A) . (F, G) SLPI expression in blood neutrophils isolated from WT (black line) and Slpi -/- mice (shaded histogram) injected with PBS or LPS for 24h.Data represented as mean ± SEM of fold change in geometrical MFI values (WT vs Slpi -/- ). (H) SLPI expression in blood monocytes represented as a fold change in geometrical MFI values (WT vs Slpi -/- ). (I) Representative histograms of SLPI expression in blood monocytes isolated from WT (black line) and Slpi -/- mice (shaded histogram) injected with PBS or LPS for 24h. (A, C, D, F, H) Data represent 7 to 8 mice per experimental group pooled from three independent experiments Error bars show means ± SEM. (A, C, D, F) Control vs LPS or WT vs Slpi -/- * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA, Tukey post hoc test. (H) WT Ly6C hi monocytes vs WT Ly6C low monocytes ****p<0.0001 by one-way ANOVA, Tukey post hoc test.

Article Snippet: The following antibodies were used: anti-CD3 PerCPCy5.5 (0.1 μg/100 μl; clone 17A2, Cat. No. 100218), anti-F4/80 Alexa Fluor 647 (0.25 μg/100 μl; clone BM8, Cat. No. 123122), anti-CD45 APCCy7 (0.2 μg/100 μl; clone 104; Cat. No. 109824), anti-Ly6C PE-Cy7 (0.005 μg/ml; clone HK1.4, Cat. No. 128017), anti-Ly6G FITC (0.25 μg/100 μl; clone 1A8, Cat. No. 127605) all from Biolegend (Amsterdam, The Netherlands); anti-CD11b BV650 (0.04 μg/100 μl; clone M1/70; Cat. No. 563402), anti-CD11c BV421 (0.2 μg/100 μl; clone HL3; Cat. No. 562782), anti-Ly6G BV711 (0.2 μg/100 μl; clone 1A8; Cat. No. 563979), anti-SiglecF PerCPCy5.5 (0.1 μg/100 μl; clone E50-2440; Cat. No. 565526) all from BD (Warsaw, Poland); anti-CD16/CD32 monoclonal antibodies (0.25 μg/100 μl; clone 93; Cat. No. 14-0161-85), eBioscience anti-c-kit FITC (0.25 μg/100 μl; clone 2B8; Cat. No. 11-1171-85), anti-CD11c PerCPCy5.5 (0.1 μg/100 μl; clone N418, Cat. No. 45-0114), anti-CD19 PerCPCy5.5 (0.1 μg/100 μl; clone 1D3, Cat. No. 45-0193-82), anti-MHC II Alexa Fluor 700 (0.02 μg/100 μl; clone M5/114.15.2, Cat. No. 56-5321-80), anti-SiglecF eFluor660 (0.04 μg/100 μl; clone 1RNM44N, Cat. No. 50-1702-82) all from Thermo Fisher Scientific (Life Technologies, Warsaw, Poland); anti-mouse SLPI – biotin (0.067 μg/100μl; Cat. No. BAF-1735), anti-mouse SLPI (2 μg/ml; Cat. No. AF1735), anti-mouse MMP9 (0.25 μg/ml; Cat. No. AF909-SP) all from R&D Systems (Bio-Techne, Warsaw, Poland); anti-β-actin (1:5000 dilution; clone AC15; Cat. No. A1978), rabbit anti-goat IgG antibody HRP conjugated (0.5 μg/ml; Cat. No. AP106P) all from Sigma-Aldrich (Merck Life Science, Poznan, Poland); StarBrightTM Blue 520 goat anti-mouse IgG (1:2500 dilution; Cat. No. 12005867, Bio-Rad, Warsaw, Poland).

Techniques: Expressing, In Vivo, Isolation, Injection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: SNCG protein is associated with β1 integrin and activates β1 integrin. a - b . Coimmunoprecipitation. Cell membrane proteins of HCT116 cells were collected and subjected to immunoprecipitated (IP) using anti-SNCG ( a ), anti-SNCG or anti-β1 integrin antibody ( b ). The IP proteins or total cell lysates were analyzed by Western blot. Normal IgG served as the negative control. c . Far-Western blot analysis. HCT116 cells were transfected with control siRNA (lane 1-2), and specific siRNA-β1-2 (lanes 3-4) for 48 h. Cells were treated without (lane 1, 3) or with 1 μmol/L rhSNCG (lane 2, 4). Cell lysates were subjected to SDS-PAGE and transferred to NC membrane. β1 integrin (prey protein) on the membrane is detected with SNCG (bait protein). More SNCG was associated with membrane β1 integrin in SNCG-treated cells than that in the control cells (lane 1, 2). Correspondingly, less SNCG were detected in β1 integrin knock-down cells than that in control cells (lane 2, 4). d - e , Effect of concentration and time treatment of SNCG on activated β1 integrin. HCT116 cells were stimulated with GST or GST-SNCG at various concentrations for 60 min ( d ) or at fixed concentration (1 μmol/L) for various times ( e ). Cell lysates were analyzed with the HUTS-21 mAb recognizing the activated form of β1 integrin. f , GST-SNCG treatment (1 μmol/L) upregulated activated β1 integrin subunit in HCT116 and SW480 cells. g , Colocalization of SNCG with F-actin. HCT116 cells grown on coverslips were transiently transfected with control siRNA or β1-specific siRNA-2. After 72 h, cells were treated with GST or GST-SNCG (1 μmol/L) for 60 min. Cells were fixed and stained with anti-SNCG (red) and FITC-Phalloidin (green). Colocalization of SNCG and F-actin was shown in yellow. Nuclei were counterstained with DAPI (blue). Scale bars, 5 μm

Article Snippet: Human CRC cell lines HT29, HCT116, DLD-1, RKO, CL187, LS 174T, SW480, and LOVO, were obtained from the American Type Culture Collection and cultured in RPMI-1640 (GBICO) with 10% fetal bovine serum (FBS) at 37°C under 5% CO 2 in air.

Techniques: Membrane, Immunoprecipitation, Western Blot, Negative Control, Far Western Blot, Transfection, Control, SDS Page, Knockdown, Concentration Assay, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: Integrin β1 is required for enhancement of SNCG on tumor cell migration and invasion. a - b , The functional blocking antibody for β1 integrin subunit (5, 10, 20 μg/mL) was added in the upper compartment of migration or invasion chambers stimulated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( a ) or 48 h for invasion ( b ). c - d , HCT116 cells were transfected with control siRNA, and β1-specific siRNA-2 for 48 h. then cells were treated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( c ) or 48 h for invasion ( d ). e - f , HCT116 cells were treated with PBS or 200 μmol/L RGD for 30 min and then treated with or without GST-SNCG (1 μmol/L) for 24 h for migration ( e ) or 48 h for invasion ( f ). Graphed data represent the mean ± SE from at least six 200-power field for each condition, two-sample t-test. g , HCT116 cells were transfected with control siRNA, and β1-specific siRNA-2 for 48 h. Then cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed by Western blot. h , HCT116 cells were treated with PBS or 200 μmol/L RGD for 30 min. Then cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed by Western blot

Article Snippet: Human CRC cell lines HT29, HCT116, DLD-1, RKO, CL187, LS 174T, SW480, and LOVO, were obtained from the American Type Culture Collection and cultured in RPMI-1640 (GBICO) with 10% fetal bovine serum (FBS) at 37°C under 5% CO 2 in air.

Techniques: Migration, Functional Assay, Blocking Assay, Transfection, Control, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: FAK is essential for SNCG-enhanced tumor cell migration and invasion. a - b , HCT116 cells were transfected with control siRNA and FAK-specific siRNA-2, -3 for 48 h. Then cells were treated with or without GST-SNCG (1 μmol/L) for migration ( a ) or invasion ( b ). c - d , HCT116 cells were treated with PBS, 50 μmol/L FAK inhibitor 14 for 30 min and then treated with or without GST-SNCG (1 μmol/L) for migration ( c ) or invasion ( d ). Graphed data represent the mean ± SE from at least six 200-power field for each condition, two-sample t-test. e - f , HCT116 cells were transfected with control siRNA (lane 1-2), and FAK-specific siRNA-2 (lane 3-4) and -3 (lane 5-6) for 72 h. cells were treated with or without GST-SNCG (1 μmol/L) for 30 min and cell lysates were analyzed for activated and total β1 integrin ( e ) or activated and total FAK ( f )

Article Snippet: Human CRC cell lines HT29, HCT116, DLD-1, RKO, CL187, LS 174T, SW480, and LOVO, were obtained from the American Type Culture Collection and cultured in RPMI-1640 (GBICO) with 10% fetal bovine serum (FBS) at 37°C under 5% CO 2 in air.

Techniques: Migration, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: SNCG is an indicator of adverse prognosis and positively correlates with activated β1 integrin, p-FAK (Y 397 ) in CRC tissues. a - b , Kaplan-Meier estimation of disease-free survival (DFS) for stage I-II ( a ) and III-IV ( b ) colorectal adenocarcinoma patients according to SNCG levels. c , Correlations of SNCG levels in CRC tissues with post-operative recurrence and status. d , Representative blots from three independent experiments were presented. Protein levels of SNCG, activated β1 integrin, and p-FAK (Y 397 ) in clinical colon cancer tissue samples were evaluated by Western blot analysis. In order to increase the reproducibility, HCT116 cell lysates were used in each blot as the internal control ( c ) to minimize the effect of band intensity variation. GAPDH was used as the loading control. e - g , Correlation between the relative protein levels of activated β1 integrin levels and p-FAK (Y 397 ) ( e ), SNCG and active β1 integrin ( f ), and SNCG and p-FAK (Y 397 ) ( g ) were plotted as a scatter plots

Article Snippet: Human CRC cell lines HT29, HCT116, DLD-1, RKO, CL187, LS 174T, SW480, and LOVO, were obtained from the American Type Culture Collection and cultured in RPMI-1640 (GBICO) with 10% fetal bovine serum (FBS) at 37°C under 5% CO 2 in air.

Techniques: Western Blot, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

doi: 10.1186/s13046-018-0783-6

Figure Lengend Snippet: Exogenously added SNCG remodels the microenvironment of tumor cells and increases MMP-2 activity by β1 integrin. a , Antibody array screening of CM from GST and GST-SNCG-treated HCT116 cells. b , CM (left panel) and whole cell lysates (right panel) from HCT116 and SW480 cells treated with or without GST-SNCG (1 μmol/L) were subjected to Western blot analysis. Representative blots from three independent experiments were presented. c - d , HCT116 cells were treated with diluent, 50 μmol/L MMP-2 inhibitor for 40 min, then 1 μmol/L GST or GST-SNCG was added in the cell medium for migration ( c ) or invasion ( d ) assay. Migrated or invaded cells were quantitated after 24 h or 48 h, respectively. Error bars, SE of three determinations. e - f , Western blot and gelatin zymography analysis. CM from HCT116 cells treated with GST or GST-SNCG (1 μmol/L) in β1 integrin knock-down ( e ) or RGD-treated cells ( f ) was analyzed by gelatin zymography with FBS as the positive control, and secreted protein levels were analyzed by Western blot

Article Snippet: Human CRC cell lines HT29, HCT116, DLD-1, RKO, CL187, LS 174T, SW480, and LOVO, were obtained from the American Type Culture Collection and cultured in RPMI-1640 (GBICO) with 10% fetal bovine serum (FBS) at 37°C under 5% CO 2 in air.

Techniques: Activity Assay, Ab Array, Western Blot, Migration, Zymography, Knockdown, Positive Control

Journal: Microbiology (Reading, England)

Article Title: Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

doi: 10.1099/mic.0.000537

Figure Lengend Snippet: Fig. 2. Western immunoblot detection of matrix metalloproteinase 9 (MMP-9) in mouse-passaged (MP) and (Ax) whole-cell lysates (WCL) and cytosolic fraction (Cyto) of N. fowleri trophozoites. Immunoblots were incubated with polyclonal rabbit anti-MMP-9 antibody (1 : 500) followed by HRP-conjugated goat anti-rabbit secondary antibody. (a) Immunoreactive bands of approximately 48 kDa were detected in WCL and additional bands of lower molecular weight were detected in both samples. (b) Immunoreactive bands were present in the cytosolic frac- tion of MP and Ax trophozoites.

Article Snippet: N. fowleri (ATCC 30894) was isolated from a fatal case of PAM that occurred in a 15-year-old human female in Richmond, Virginia.

Techniques: Western Blot, Incubation, Molecular Weight

Journal: Microbiology (Reading, England)

Article Title: Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

doi: 10.1099/mic.0.000537

Figure Lengend Snippet: Fig. 1. Western immunoblot detecting matrix metalloproteinase-2 (MMP-2) in mouse-passaged (MP) and (Ax) whole-cell lysates (WCL) and membrane fraction (Mem) of N. fowleri trophozoites. Immunoblots were incubated with polyclonal rabbit anti-MMP-2 antibody (1 : 500) followed by HRP-conjugated goat anti-rabbit secondary antibody. (a) Prominent bands of approximately 72 kDa were detected in homoge- nates from WCL, and additional bands at lower relative molecular weights were detected in both lysates. (b) MMP-2 immunoreactivity was shown to be confined to the membrane fraction and predominant in mouse-passaged amoebae. Immunoreactivity was not detected in the cytosolic fraction (data not shown).

Article Snippet: N. fowleri (ATCC 30894) was isolated from a fatal case of PAM that occurred in a 15-year-old human female in Richmond, Virginia.

Techniques: Western Blot, Membrane, Incubation

Journal: Microbiology (Reading, England)

Article Title: Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

doi: 10.1099/mic.0.000537

Figure Lengend Snippet: Fig. 4. Gelatin zymography of samples collected from the invasion assay performed with MP N. fowleri trophozoites, depicting media col- lected from top/upper chambers (‘T’) and bottom chambers (‘B’), where the inserts were coated with Matrigel (‘+’) at 6 h and 18 h. Media collected from invasion assays in the absence of Matrigel yielded no detectable protease activity.

Article Snippet: N. fowleri (ATCC 30894) was isolated from a fatal case of PAM that occurred in a 15-year-old human female in Richmond, Virginia.

Techniques: Zymography, Invasion Assay, Activity Assay

Journal: Microbiology (Reading, England)

Article Title: Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

doi: 10.1099/mic.0.000537

Figure Lengend Snippet: Fig. 3. Western immunoblot analysis for detection of matrix metallo- proteinase 14 (MMP-14) in mouse-passaged (MP) whole-cell lysates (WCL) of N. fowleri trophozoites. Immunoblots were incubated with polyclonal rabbit anti-MMP-14 antibody (1 : 1000) followed by HRP-con- jugated goat anti-rabbit secondary antibody. (a) A prominent band at approximately 80 kDa was detected in the MP sample, while a fainter band of the same molecular weight appeared in the Ax culture. Faint bands between 100 and 150 kDa were detected in both samples. (b) The same two immunoreactive bands were detected in the cytosolic fractions (Cyto), with higher levels apparent in the highly virulent MP sample.

Article Snippet: N. fowleri (ATCC 30894) was isolated from a fatal case of PAM that occurred in a 15-year-old human female in Richmond, Virginia.

Techniques: Western Blot, Incubation, Molecular Weight

Journal: Microbiology (Reading, England)

Article Title: Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

doi: 10.1099/mic.0.000537

Figure Lengend Snippet: Fig. 7. Scanning electron microscopy (SEM) of MP N. fowleri tropho- zoites invading through Matrigel after 2 h incubation. Bars, 10 µm.

Article Snippet: N. fowleri (ATCC 30894) was isolated from a fatal case of PAM that occurred in a 15-year-old human female in Richmond, Virginia.

Techniques: Electron Microscopy, Incubation

Journal: Microbiology (Reading, England)

Article Title: Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

doi: 10.1099/mic.0.000537

Figure Lengend Snippet: Fig. 6. Western immunoblot of media collected from invasion assays of MP N. fowleri. Secreted protein was separated by 10 % SDS- PAGE and transferred to nitrocellulose membranes, which were incubated with polyclonal rabbit anti-MMP-2 antibody (a), anti-MMP-9 (b) and anti-MMP-14 (c), followed by HRP-conjugated goat anti-rabbit antibody. T=medium from top chamber, B=medium from bottom chamber; the numerical subscripts designate the time at which medium was recovered; +=Matrigel was used as protein substrate.

Article Snippet: N. fowleri (ATCC 30894) was isolated from a fatal case of PAM that occurred in a 15-year-old human female in Richmond, Virginia.

Techniques: Western Blot, SDS Page, Incubation

Journal: Microbiology (Reading, England)

Article Title: Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

doi: 10.1099/mic.0.000537

Figure Lengend Snippet: Fig. 8. (a) Representative light microscopy images of the bottom chambers of invasion assays. Untreated control, vehicle control (DMSO) and 1,10-phenanthroline samples were incubated for 6 and 18 h. Scale bars are 100 µm. (b) Triplicate wells were counted to determine the invasive capability of MP N. fowleri trophozoites. Continuous variables were compared using a two-tailed, unpaired Stu- dent t-test with significance set at P<0.05.

Article Snippet: N. fowleri (ATCC 30894) was isolated from a fatal case of PAM that occurred in a 15-year-old human female in Richmond, Virginia.

Techniques: Light Microscopy, Control, Incubation, Two Tailed Test

Journal: The FEBS journal

Article Title: Membrane type-1 matrix metalloprotease-independent activation of pro-matrix metalloprotease-2 by proprotein convertases.

doi: 10.1111/j.1742-4658.2009.07335.x

Figure Lengend Snippet: Fig. 1. Cellular expression of MT-MMPs, TIMP-2 and integrin avb3. (A) Analysis of MT-MMP mRNA in various cell lines. The data represent semi-quantitative RT-PCR analysis of MT-MMP mRNA in HCT116, HEK293F, MCF-7, MDAH 2774, K-562, NCI-H460 and Hep G2 cells. PCR products (25 cycles) were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Arrowheads indicate PCR products of MT-MMPs or GAPDH. (B) Wes- tern blotting of conditioned medium with mouse anti-TIMP-2 IgG2a. The conditioned medium from the cells on 12-well plates was concentrated prior to SDS-PAGE. Arrow indicates TIMP-2 protein. (C) Flow cytometric analysis for cell surface expression of integrin avb3. The percentage changes in fluorescence intensity by the presence of integrin avb3 are shown. A sample lacking primary antibody was used as a control (n = 3 representative experi- ments).

Article Snippet: Cell culture, transfection, cell treatments and cell viability assay HEK293F, COS-1, HCT116 (human colorectal carcinoma) (ATCC no. CCL-247), MDAH 2774 (human ovarian adenocarcinoma) (ATCC no. CRL-10303), NCI-H460 (human lung carcinoma) (ATCC no. HTB-177) and HT-1080 (human fibrosarcoma) (ATCC no. CCL-121) cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, and MCF-7 (human breast adenocarcinoma) (ATCC no. HTB-22), K-562 (human chronic myelogenous leukemia) (ATCC no. CCL-243) and Hep G2 (human hepatocellular carcinoma) (ATCC no. HB-8065) cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum.

Techniques: Expressing, Quantitative RT-PCR, Agarose Gel Electrophoresis, Staining, SDS Page, Control

Journal: The FEBS journal

Article Title: Membrane type-1 matrix metalloprotease-independent activation of pro-matrix metalloprotease-2 by proprotein convertases.

doi: 10.1111/j.1742-4658.2009.07335.x

Figure Lengend Snippet: Fig. 2. MT-MMP-independent processing of pro-MMP-2 in various cell types. (A) Effect of metalloprotease inhibitors on the propeptide processing of MMP-2 in HEK293F, MDAH 2774 and MCF-7 cells. Cells were treated with GM6001 (20 lM) or TIMP-2 (5 lgÆmL–1) in 500 lL of 293 SFM-II medium on 24-well plates. After 14 h of incu- bation, MMP-2 was captured from 500 lL of the conditioned med- ium using gelatin–Sepharose according to the manufacturer’s recommendations (Amersham Biosciences) and eluted in 30 lL of SDS sample buffer, and the eluted sample was analyzed by zymog- raphy; 10 lL of the conditioned medium (pre-column sample, unconcentrated) was loaded on the first lane. Arrows indicate pro- and processed MMP-2 (MMP-2). (B) Effect of metalloprotease inhibitors on the processing of pro-MMP-2 in K-562, NCI-H460 and Hep G2 cells. Cells were treated as described above. MMP-2 was concentrated from 125 lL of the conditioned medium using gela- tin–Sepharose and the eluted sample was used for zymographic analysis. Conditioned medium from HT-1080 cells was used as a positive control for pro-MMP-2. (C) Validation of the metallopro- tease inhibitors to prevent MT1-MMP-mediated processing of pro-MMP-2. HT-1080 cells were treated with Con A (50 lgÆmL–1) in the presence and absence of the metalloprotease inhibitors for 14 h (n = 3 representative experiments).

Article Snippet: Cell culture, transfection, cell treatments and cell viability assay HEK293F, COS-1, HCT116 (human colorectal carcinoma) (ATCC no. CCL-247), MDAH 2774 (human ovarian adenocarcinoma) (ATCC no. CRL-10303), NCI-H460 (human lung carcinoma) (ATCC no. HTB-177) and HT-1080 (human fibrosarcoma) (ATCC no. CCL-121) cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, and MCF-7 (human breast adenocarcinoma) (ATCC no. HTB-22), K-562 (human chronic myelogenous leukemia) (ATCC no. CCL-243) and Hep G2 (human hepatocellular carcinoma) (ATCC no. HB-8065) cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum.

Techniques: Positive Control, Biomarker Discovery

Journal: The FEBS journal

Article Title: Membrane type-1 matrix metalloprotease-independent activation of pro-matrix metalloprotease-2 by proprotein convertases.

doi: 10.1111/j.1742-4658.2009.07335.x

Figure Lengend Snippet: Fig. 5. PC-dependent processing of pro-MMP-2. (A) Effect of various inhibitors on the propeptide processing of MMP-2 in HEK293F, MCF-7 and MDAH 2774 cells. Cells were treated with aprotinin (20 lgÆmL–1), chymostatin (10 lgÆmL–1), leupeptin (20 lgÆmL–1) and pepstatin (2 lM). After 8 h of incubation, the conditioned media were analyzed by zymography. Arrows indicate pro- and processed MMP-2 (MMP-2). (B) Protease activities of PCs in cell lysates from the cells indicated. (C) Inhibition of pro-MMP-2 processing by a PC inhibitor dec-RVKR-cmk in HEK293F, MCF-7 and MDAH 2774 cells. Cells were incubated with 0–100 lM of the inhibitor for 8 h, and the conditioned medium was analyzed by zymography. The bar graph shows the ratio of processed MMP-2 to pro-MMP-2 in the conditioned medium of cells treated with 100 lM of the PC inhibitor. Data are the means and standard deviations of n = 3 experiments. *P < 0.05 versus untreated control. (D) Inhibition of pro-MMP-2 processing by the PC inhibitor in K-562, NCI-H460 and Hep G2 cells. Cells were incubated with 100 lM of the inhibitor. Note that almost complete inhibition of pro-MMP- 2 processing is seen in the conditioned medium of the inhibitor-treated cells. (E) Zymograms of conditioned medium from cells expressing pro-MMP-2 or pro-MMP-2 R101A mutant with furin, PACE4 or PC5A (n = 3 representative experiments).

Article Snippet: Cell culture, transfection, cell treatments and cell viability assay HEK293F, COS-1, HCT116 (human colorectal carcinoma) (ATCC no. CCL-247), MDAH 2774 (human ovarian adenocarcinoma) (ATCC no. CRL-10303), NCI-H460 (human lung carcinoma) (ATCC no. HTB-177) and HT-1080 (human fibrosarcoma) (ATCC no. CCL-121) cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, and MCF-7 (human breast adenocarcinoma) (ATCC no. HTB-22), K-562 (human chronic myelogenous leukemia) (ATCC no. CCL-243) and Hep G2 (human hepatocellular carcinoma) (ATCC no. HB-8065) cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum.

Techniques: Incubation, Zymography, Inhibition, Control, Expressing, Mutagenesis

Journal: Journal of Cellular and Molecular Medicine

Article Title: Exogenous pulmonary surfactant prevents the development of intra‐abdominal adhesions in rats

doi: 10.1111/jcmm.12758

Figure Lengend Snippet: Expression and activity of MMP ‐9 in vivo . Gel zymography analysis showed that MMP ‐9 activity was significantly higher in GE ‐S versus GE animals ( P < 0.05) ( A ). In situ zymography analysis of MMP ‐9 expression showed a larger number of cells positive for gelatinase activity in GE ‐S versus GE animals. ( B ) Isolectin B4 ( IB 4) staining of the gastroenterostomy region revealed no significant difference in total number of macrophages ( C , arrows in the left‐hand column) between the GE and GE ‐S animals ( C , first‐ versus second‐row, and D ). The number of ED 1‐positive cells ( E , arrows in the left‐hand column) was significantly higher ( P < 0.001) in GE ‐S versus GE animals ( F ). No significant differences in number of cells double‐labelled for MMP ‐9/ IB 4 were observed between the GE ‐S and GE groups (third column in C ). Note that virtually all cells positive for ED 1 were MMP ‐9+ (third column in E ), scale bar: 200 μm.

Article Snippet: The expression and activity of MMP‐9 were investigated in all rats of groups L, LS, GE and GE‐S by SDS‐PAGE gelatin zymography using a 4% polyacrylamide stacking gel and a 10% polyacrylamide resolving gel (Bio‐Rad Co., Richmond, VA, USA) containing 1.5 mg/ml gelatin (BDH Laboratory Supplies, Poole, UK).

Techniques: Expressing, Activity Assay, In Vivo, Zymography, In Situ, Staining

Journal: PLoS ONE

Article Title: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

doi: 10.1371/journal.pone.0046526

Figure Lengend Snippet: ( A ) RNA from Huh7.5 or Huh7.5-FL cells was used in the SYBR green real-time PCR to analyze CTGF expression. ** P<0.001 versus Huh7.5 cells. ( B ) Conditioned medium from Huh7.5 or Huh7.5-FL cells incubated for various time periods was collected, concentrated and equal amounts of protein subjected to SDS-PAGE and analyzed for CTGF by Western blotting. Albumin was used as a internal control. ( C ) Huh7.5 or Huh7.5-FL cells were grown for 48 hours, after which the cells were fixed, permeabilized and treated with anti-CTGF followed by FITC-coupled secondary antibodies and examined using an Olympus FV1000 confocal microscope. HepG2 cells were transfected with JFH1 RNA and CTGF expression was analyzed by ( D ) Western blotting and ( E ) confocal microscopy respectively. Equal protein loading was verified using antibodies against GAPDH. Data represent mean ± SD of 3 independent experiments.

Article Snippet: The antibodies used in the study were HCV NS5B (Alexis Biochemicals, San Diego, CA), HCV Core (Abcam, Cambridge, MA), HCV NS4A, TGF-β1 (Chemicon, Temecula, CA), Phospho-Smad2, Phospho-Smad3, Smad2, Smad3, Phospho-P38, P-38, Phospho-JNK, JNK, vimentin and Slug (Cell Signaling, Danvers, MA), TGF-β receptor I, Phospho-ERK, ERK, CTGF, Procollagen I and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and α-SMA (Sigma, St. Louis, MO).

Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Expressing, Incubation, SDS Page, Western Blot, Control, Microscopy, Transfection, Confocal Microscopy

Journal: PLoS ONE

Article Title: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

doi: 10.1371/journal.pone.0046526

Figure Lengend Snippet: ( A ) Huh7.5 or Huh7.5-FL cells were incubated in conditioned medium (medium containing 0.5%FCS) for ninety-six hours and the cell lysates were blotted to examine α-SMA expression, vimentin and slug expression. Equal protein loading was verified using GAPDH antibody. The conditioned medium was used for the measurement of MMP-2 activity by zymography assay. ( B ) HepG2 cells were transfected with or without JFH-1RNA for different time points and cell lysates were blotted for α-Sma I protein. GAPDH was used as an internal control. ( C ) Lysates of Huh7.5 or Huh7.5-FL cells transfected with non targeting or CTGF shRNA for 48 hrs were blotted for CTGF, procollagen I or GAPDH. The bar graphs show the quantitative analysis of CTGF or procollagen I expression relative to that of GAPDH. * P≤0.05 versus Huh7.5-FL cells. Data represent mean ± SD of 3 independent experiments.

Article Snippet: The antibodies used in the study were HCV NS5B (Alexis Biochemicals, San Diego, CA), HCV Core (Abcam, Cambridge, MA), HCV NS4A, TGF-β1 (Chemicon, Temecula, CA), Phospho-Smad2, Phospho-Smad3, Smad2, Smad3, Phospho-P38, P-38, Phospho-JNK, JNK, vimentin and Slug (Cell Signaling, Danvers, MA), TGF-β receptor I, Phospho-ERK, ERK, CTGF, Procollagen I and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and α-SMA (Sigma, St. Louis, MO).

Techniques: Incubation, Expressing, Activity Assay, Zymography, Transfection, Control, shRNA

Journal: PLoS ONE

Article Title: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

doi: 10.1371/journal.pone.0046526

Figure Lengend Snippet: ( A ) Lysates of Huh7.5 or Huh7.5-FL cells transfected with non-targeting or TGF-β1 ShRNA for 48 hrs were blotted to determine expression of TGF-β1, CTGF or procollagen I (upper panel). Equal protein loading was determined using GAPDH antibody. The bar graph shows the quantitative analysis of TGF-β1, CTGF or procollagen I expression relative to that of GAPDH (lower panel). * P≤0.05 versus Huh7.5-FL cells. ( B ) Medium from Huh7.5 or Huh7.5-FL cells incubated for 48 hrs with anti-TGF-β1 or non-immune IgG was collected, concentrated and equal amounts of protein were used for Western blot analysis using CTGF antibody (upper panel). The bar graph shows the quantitative analysis of CTGF expression obtained by densitometry (lower panel). * P≤0.05 versus Huh7.5 cells. ( C ) Human hepatic stellate cells (LX2 cells) were co cultured with medium from Huh7.5 and Huh7.5-FL cells for 48 hrs and cell lysates were analyzed for procollagen I expression. GAPDH was used as an internal control. For all experiments, data represent mean ± SD of 3 independent experiments.

Article Snippet: The antibodies used in the study were HCV NS5B (Alexis Biochemicals, San Diego, CA), HCV Core (Abcam, Cambridge, MA), HCV NS4A, TGF-β1 (Chemicon, Temecula, CA), Phospho-Smad2, Phospho-Smad3, Smad2, Smad3, Phospho-P38, P-38, Phospho-JNK, JNK, vimentin and Slug (Cell Signaling, Danvers, MA), TGF-β receptor I, Phospho-ERK, ERK, CTGF, Procollagen I and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and α-SMA (Sigma, St. Louis, MO).

Techniques: Transfection, shRNA, Expressing, Incubation, Western Blot, Cell Culture, Control

Journal: PLoS ONE

Article Title: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

doi: 10.1371/journal.pone.0046526

Figure Lengend Snippet: Cell lysates from Huh7.5 or Huh7.5-FL cells were collected at different time points and blotted with anti-TGF-βRI ( A ) or phospho-Smad 2, phospho-Smad3 and total Smad2/3 antibodies ( C ). The bar graphs show the quantitative analyses of TGF-β RI and p-smad2 protein expression as obtained by densitometry. RNA from Huh7.5 or Huh7.5-FL cells was used in the reverse transcriptase PCR to analyze the TGF-β RI expression ( B ). ( D ) HepG2 cells were transfected with and without JFH-1 RNA. The cell lysates were collected at different time points and blotted for TGF-βRI. ( E ) Huh7.5 and Huh7.5-FL cells were transfected with different CTGF promoter/SEAP reporter constructs for 48 hrs. CTGF promoter activity was determined by measuring SEAP reporter expression. ** P<0.001 versus Huh7.5 cells; + P<0.05 versus Huh7.5 cells; and ## P<0.001 versus Huh7.5-FL cells. Data represent mean ± SD of 3 independent experiments.

Article Snippet: The antibodies used in the study were HCV NS5B (Alexis Biochemicals, San Diego, CA), HCV Core (Abcam, Cambridge, MA), HCV NS4A, TGF-β1 (Chemicon, Temecula, CA), Phospho-Smad2, Phospho-Smad3, Smad2, Smad3, Phospho-P38, P-38, Phospho-JNK, JNK, vimentin and Slug (Cell Signaling, Danvers, MA), TGF-β receptor I, Phospho-ERK, ERK, CTGF, Procollagen I and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and α-SMA (Sigma, St. Louis, MO).

Techniques: Expressing, Reverse Transcription, Transfection, Construct, Activity Assay

Journal: PLoS ONE

Article Title: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

doi: 10.1371/journal.pone.0046526

Figure Lengend Snippet: ( A ) Lysates from Huh7.5 or Huh7.5-FL cells collected at the indicated time points were blotted with Phospho-p38, Phospho-JNK, Phospho-ERK, p38, JNK and ERK antibodies. The bar graph shows the quantitative analysis of p38 activation relative to the total p38 production assessed by densitometry. * P≤0.05 versus Huh7.5 cells. ( B ) Huh7.5 or Huh7.5-FL cells were pretreated with p38 MAPkinase inhibitor (SB220025; 50 µM) for 36 hours, after which cells were lysed and blotted with antibodies to phospho-p38, p38, CTGF, Phospho-Smad2, or Smad2. The bar graph shows the quantitative analysis of the data obtained by densitometry (left panel). * P≤0.05 versus Huh7.5-FL cells. ( C ) Similarly HepG2 cells were transfected with and without JFH1 RNA and cells were treated with p38 MAPKinase inhibitor (SB220025) for 24 hrs ,after which the cell lysates were analyzed for activation of p38 and Smad2. Data represent mean ± SD of 3 independent experiments.

Article Snippet: The antibodies used in the study were HCV NS5B (Alexis Biochemicals, San Diego, CA), HCV Core (Abcam, Cambridge, MA), HCV NS4A, TGF-β1 (Chemicon, Temecula, CA), Phospho-Smad2, Phospho-Smad3, Smad2, Smad3, Phospho-P38, P-38, Phospho-JNK, JNK, vimentin and Slug (Cell Signaling, Danvers, MA), TGF-β receptor I, Phospho-ERK, ERK, CTGF, Procollagen I and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and α-SMA (Sigma, St. Louis, MO).

Techniques: Activation Assay, Transfection

Journal: PLoS ONE

Article Title: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor β 1 (TGF-β 1) Axis in Hepatitis C Virus-Expressing Hepatocytes

doi: 10.1371/journal.pone.0046526

Figure Lengend Snippet: We hypothesize that HCV infection in hepatocytes induces TGF-β1 expression. TGF-β1, in turn mediates an enhanced expression of profibrogenic cytokine CTGF through Smad phosphorylation and p38 MAP kinase activation. CTGF may further act in a paracrine manner on hepatic stellate cells (HSCs) or in an autocrine manner on hepatocytes and drive expression of fibrotic markers including collagen and α-Sma.

Article Snippet: The antibodies used in the study were HCV NS5B (Alexis Biochemicals, San Diego, CA), HCV Core (Abcam, Cambridge, MA), HCV NS4A, TGF-β1 (Chemicon, Temecula, CA), Phospho-Smad2, Phospho-Smad3, Smad2, Smad3, Phospho-P38, P-38, Phospho-JNK, JNK, vimentin and Slug (Cell Signaling, Danvers, MA), TGF-β receptor I, Phospho-ERK, ERK, CTGF, Procollagen I and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) and α-SMA (Sigma, St. Louis, MO).

Techniques: Infection, Expressing, Phospho-proteomics, Activation Assay

Journal: Scientific Reports

Article Title: Prolactin and its receptor as therapeutic targets in glioblastoma multiforme

doi: 10.1038/s41598-019-55860-x

Figure Lengend Snippet: Expression of prolactin receptor in glioblastoma multiforme cells. ( A ) A representative microphotograph shows PRLR expression in human U251-MG GBM cells, as assessed by immunofluorescence using a specific anti-human PRLR (green fluorescence). ( B ) A representative blot shows PRLR isoforms, as evaluated by WB in protein extracts from human (U251-MG, LN229), mouse (GL26) and rat (C6) GBM cells.

Article Snippet: Brain sections and cells were blocked with PBS- 10% goat serum for 1 h and incubation with antibody against human PRLR (sc-20992, Santa Cruz Biotechnology) was performed overnight in PBS− 1% goat serum.

Techniques: Expressing, Immunofluorescence, Fluorescence

Journal: Scientific Reports

Article Title: Prolactin and its receptor as therapeutic targets in glioblastoma multiforme

doi: 10.1038/s41598-019-55860-x

Figure Lengend Snippet: Effect of prolactin or its receptor blockade on proliferation, viability and chemoresistance of glioblastoma multiforme cells. ( A ) Human U251-MG and U373-MG GBM cells were incubated for 6 h with PRL (100 ng/ml) and proliferation was evaluated by BrdU incorporation ELISA. *p < 0.05 vs. respective control (Student’s t test). ( B ) The viability of U87-MG, U373-MG and GL26 cells was assessed 72 h after incubation with PRL by MTT assay. ( C ) U251-MG cells were incubated with PRL and cisplatin (5 µM) for 72 h. Cell viability was evaluated by MTT assay. *p < 0.05 vs. respective control without PRL, ^p < 0.05 vs. respective control without cisplatin (ANOVA). ( D ) U251-MG cells were incubated with PRL and TMZ (15 µM) for 72 h. Cell viability was evaluated by MTT assay. *p < 0.05 vs. respective control without PRL, ^p < 0.05 vs. respective control without TMZ (ANOVA). ( E ) Rat GBM cells (C6) were incubated with PRL and 16 h later they were treated with cisplatin (1 µM) for additional 24 h. Cells were then processed for the clonogenic assay. *p < 0.05 vs. respective control without PRL (ANOVA). ( F ) U251-MG and U373-MG GBM cells were incubated for 6 h with PRLR-A (∆1–9-G129R-hPRL, 2.5 µg/ml) and proliferation was evaluated by BrdU incorporation ELISA. *p < 0.05 vs. respective control (Student’s t test). ( G ) The viability of U87-MG, U373-MG and GL26 cells was assessed 72 h after incubation with PRLR-A by MTT assay. *p < 0.05 vs. respective control (Student’s t test). H) U251-MG cells were incubated with PRLR-A and cisplatin (5 µM) for 72 h. Cell viability was evalua t ed by MTT assay. *p < 0.05 vs. respective control without PRLR-A, ^p < 0.05 vs. respective control without cisplatin (ANOVA).

Article Snippet: Brain sections and cells were blocked with PBS- 10% goat serum for 1 h and incubation with antibody against human PRLR (sc-20992, Santa Cruz Biotechnology) was performed overnight in PBS− 1% goat serum.

Techniques: Incubation, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay, Control, MTT Assay, Clonogenic Assay

Journal: Scientific Reports

Article Title: Prolactin and its receptor as therapeutic targets in glioblastoma multiforme

doi: 10.1038/s41598-019-55860-x

Figure Lengend Snippet: Effect of the overexpression of prolactin, its receptor or the receptor antagonist on the response of glioblastoma multiforme cells to chemotherapy. ( A ) Human U251-MG GBM cells were transfected for 6 h with plasmids encoding human PRL (pPRL) or PRLR-A (pPRLR-A). 16 h later, they were incubated with cisplatin (5 µM) for 72 h. Cell viability was then evaluated by MTT assay. ( B ) Mouse GL26 GBM cells were transfected for 6 h with plasmids encoding the short (pSPRLR) or the long (pLPRLR) isoforms of the mouse PRLR. 16 h later, they were treated with cisplatin (2 µM) for 72 h. Cell viability was assessed by MTT. *p < 0.05 vs. respective control plasmid (pCTRL), ^p < 0.05 vs. respective control without cisplatin (ANOVA). C-D) Rat GBM cells (C6) were transfected for 6 h with plasmids encoding the ( C ) short (pSPRLR) or the ( D ) long (pLPRLR) isoforms of the rat PRLR. 16 h later, they were incubated with cisplatin for additional 24 h. Cells were then processed for the clonogenic assay. *p < 0.05 vs. respective pCTRL, ^p < 0.05 vs. respective control without cisplatin (ANOVA).

Article Snippet: Brain sections and cells were blocked with PBS- 10% goat serum for 1 h and incubation with antibody against human PRLR (sc-20992, Santa Cruz Biotechnology) was performed overnight in PBS− 1% goat serum.

Techniques: Over Expression, Transfection, Incubation, MTT Assay, Control, Plasmid Preparation, Clonogenic Assay

Journal: Scientific Reports

Article Title: Prolactin and its receptor as therapeutic targets in glioblastoma multiforme

doi: 10.1038/s41598-019-55860-x

Figure Lengend Snippet: Effect of prolactin receptor blockade on glioblastoma multiforme cell migration. ( A ) Rat (C6) and ( B ) human (LN229) GBM cells were cultured until confluence with PRLR-A (2.5 µg/ml). A scratch was performed in the monolayer and the scratch area was measured at different time points. *p < 0.05 vs. control (Non-lineal regression analysis). Each dot indicates the mean ± SEM of 2 wells. The graphs shown are representative of 3 experiments. ( C,D ) SDS-PAGE gelatine zymography of conditioned media from ( C ) rat and ( D ) human GBM cells incubated in the presence of PRL (100 ng/ml) for 48 h. Gels were stained with Coomassie blue and bands were analysed by densitometry with ImageJ software. Zymographic activity was expressed as percentage in relation to a standard internal sample that saturates at a density of 50%. *p < 0.05 (Student’s t test). Bars depict the mean ± SEM of 6 wells. The graphs shown are representative of 2 experiments. ( E,F ) Representative gels are shown.

Article Snippet: Brain sections and cells were blocked with PBS- 10% goat serum for 1 h and incubation with antibody against human PRLR (sc-20992, Santa Cruz Biotechnology) was performed overnight in PBS− 1% goat serum.

Techniques: Migration, Cell Culture, Control, SDS Page, Zymography, Incubation, Staining, Software, Activity Assay

Journal: Scientific Reports

Article Title: Gene silencing of TACE enhances plaque stability and improves vascular remodeling in a rabbit model of atherosclerosis

doi: 10.1038/srep17939

Figure Lengend Snippet: ( A ) Representative western blot showing of MMP2 expression; ( B ) Representative western blot showing of MMP9 expression; ( C ) Quantitative analysis of ( A ); ( D ) Quantitative analysis of ( B ); ( E ) Representative gelatin zymography showing MMP2 and MMP9 activities; ( F ) and ( G ) Quantitative analysis of ( E ). * P < 0.01 vs . Control group, # P < 0.01 vs . Mock group. All the gels have been run under the same experimental conditions. Black lines indicated the cropped gels and blots, and full-length blots/gels were presented in .

Article Snippet: Protein content was measured by a Bio-Rad protein assay, and SDS–polyacrylamide gel electrophoresis zymography was performed.

Techniques: Western Blot, Expressing, Zymography, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Protection of Retinal Ganglion Cells by Caspase Substrate-Binding Peptide IQACRG from N -Methyl- d -Aspartate Receptor-Mediated Excitotoxicity

doi: 10.1167/iovs.09-4102

Figure Lengend Snippet: Analysis of retinal MMP-9 gelatinase activity after excitotoxic insult. Rats received intravitreal injection of vehicle (10 nmol glycine alone) or NMDA (20 nmol) plus glycine along with either scrambled peptide or P-IQACRG (150 pmol). Six hours after injection, retinas were harvested for gelatin zymography to assess MMP activity. (A) MMP-2 and -9 in tissue homogenates were affinity-precipitated with gelatin beads and separated by SDS-PAGE on polymerized gelatin. Gelatin digestion indicated MMP activity and was visualized as a clear band on dye-stained gels. Conditioned medium from the fibrosarcoma cell line HT1080, which is known to contain gelatinase activity, served as a positive control. (B) Quantitative comparison of gelatin zymography by densitometric analysis (mean value of eyes treated with scrambled peptide set at 100%). Statistical analyses revealed that P-IQACRG inhibited NMDA-induced MMP-9 activity (*P < 0.01 by t-test). Values are mean ± SEM (n = 5–6 for each group).

Article Snippet: A human fibrosarcoma cell line, HT-1080 (American Type Culture Collection, Manassas, VA), and a rat mammary carcinoma cell line, BC1 48 (a kind gift from J.

Techniques: Activity Assay, Injection, Zymography, SDS Page, Staining, Positive Control, Comparison

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 1 – Effects of [D-Trp6]LHRH on basal and EGF-induced proliferation of A431 cells. Cells were treated with vehicle control, 100 nM [D-Trp6]LHRH and/or 10 nM EGF for 24–72 hr. At the indicated time, cell numbers of the cultures were determined using Coulter counter. Each point represents the mean ( SEM) of triplicate wells from 1 of 3 independent experiments, all of which gave similar results.

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Control

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 2 – Effects of [D-Trp6]LHRH on basal and EGF-induced cellular protein phosphorylation in A431 cells. (a) A representative autoradiogram of A431 cellular proteins after phosphorylation/dephosphorylation induced by factors. Cell lysates of 50 g proteins each were incubated with 100 nM [D-Trp6]LHRH and/or 100 nM EGF in the presence of 32P. Lanes: a, vehicle control (C); b, [D-Trp6]LHRH (L); c, EGF (E); d, EGF plus [D-Trp6]LHRH (EL). The reaction mixtures were subjected to SDS-PAGE and autoradiography. (b) Immunoblotting analysis of phosphotyrosine proteins in A431 cell lysates. The same procedure was used as in (a) except excluding [-32P]ATP as the kinase substrate, anti-phosphotyrosine antibody and enhanced chemiluminescence were used to detect phosphotyrosyl proteins. (c) The same blot (b) was stripped off primary and secondary antibodies, and reprobed with anti-EGFR antibody. Note that [D-Trp6]LHRH dramatically decreased the phosphor- ylation of most protein bands, including the 170 kDa EGFR band (a,b), while similar amounts of the EGFR band were observed in all lanes (c). Data shown are representatives of 3 separate experiments performed with duplicate samples of different cell preparations.

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Incubation, Control, SDS Page, Autoradiography, Western Blot

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 3 – Effects of [D-Trp6]LHRH on EGF-induced autophos- phorylation of EGFR in A431 cells. Cells were preincubated with 10 nM EGF for 20 min and then treated with vehicle, EGF and/or 100 nM [D-Trp6]LHRH for an additional 24 hr. Cells lysates were immuno- precipitated with EGFR antibodies and subsequently analyzed for EGFR and its tyrosine phosphorylation by immunoblotting as de- scribed in the Material and Methods section. Lanes: a, vehicle control (C); b, EGF (E); c, [D-Trp6]LHRH (L); d, EGF plus [D-Trp6]LHRH (EL). Quantitative changes of tyrosine phosphorylation of EGFR were estimated based on the ratio of the density of phosphotyrosyl-EGFR to the density of EGFR. The immunoblots shown are representatives of 3 separate experiments performed with duplicate samples of different cell preparations.

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 4 – Effects of EGF and [D-Trp6]LHRH on the phosphory- lation of serine and threonine residues of cellular proteins in A431 cells. The same reaction mixtures as described in Figure 2b were subjected to SDS-PAGE and immunoblotting analyses using anti- phosphoserine antibody (Anti-PS, a) and anti-phosphothreonine anti- body (Anti-PT, b). Lanes: a, vehicle control (C); b, EGF (E); c, [D-Trp6]LHRH (L); d, [D-Trp6]LHRH and EGF (EL). The immuno- blots shown are representatives of 3 separate experiments with dupli- cate samples of different cell preparations.

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: SDS Page, Western Blot, Control

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 5 – Effects of [D-Trp6]LHRH and EGF on the morphology of A431 cells. Cells were treated with 100 nM [D-Trp6]LHRH or 10 nM EGF for 48 hr and photographed under phase-contrast microscope. (a) Vehicle control; (b) EGF; (c) [D-Trp6]LHRH; (d) [D-Trp6]LHRH for 48 hr and then EGF alone for an additional 24 hr. Original magnification 200.

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Microscopy, Control

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 6 – Effects of [D-Trp6]LHRH and EGF on DNA integrity of A431 cells. Cells were treated with 100 nM [D-Trp6]LHRH or 10 nM EGF for 48 hr. Lanes: a, vehicle control (C); b, EGF (E); c, [D-Trp6]LHRH (L); d, [D-Trp6]LHRH for 48 hr and then EGF alone for an additional 24 hr (EL). At the end of the culture, approximately 2106 cells were harvested. DNA was isolated from each sample and subjected to electrophoresis in a 1.8% agarose gel. This ladder pattern of DNA fragments is a characteristic of apoptosis. The gel shown is a representative of 3 separate experiments performed in duplicate.

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Control, Isolation, Electrophoresis, Agarose Gel Electrophoresis

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 7 – Effects of [D-Trp6]LHRH and EGF on ICE protein expression and caspase activity in A431 cells. Cells were treated with 100 nM [D-Trp6]LHRH and/or 10 nM EGF for 48 hr. Cell lysates of 50 g each were subjected to immunoblotting analyses using anti-ICE protein antibody (a) and anti-PARP antibody recognizing N-terminal domain of PARP (b). The formation of PARP fragments is an indica- tion of casepase activity, the executioner of apoptosis. The lower arrowhead indicates the 25 kDa PARP fragment. Lanes: a, vehicle control (C); b, EGF (E); c, [D-Trp6]LHRH (L); d, [D-Trp6]LHRH for 48 hr and then EGF for an additional 24 hr. The immunoblots shown are representatives of three separate experiments performed in dupli- cate.

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Expressing, Activity Assay, Western Blot, Control

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 8 – Effects of [D-Trp6]LHRH and EGF on the secretion of gelatinases in A431 cells. Cells were treated with 100 nM [D-Trp6]LHRH and/or 10 nM EGF in serum free medium for 24 hr. The conditioned media were collected and normalized by cell numbers prior to gelatin zymography analysis. Representative zymograms in the absence (a) or presence (b) of a metalloproteinase inhibitor 5 mM 1,10-phenanthroline during substrate buffer incubation. Lanes: a, ve- hicle control (C); b, [D-Trp6]LHRH (L); c, EGF (E); d, EGF plus [D-Trp6]LHRH (EL). Trypsin, a serine protease (T; lane f), was not inhibited by 1,10-phenanthroline (lane e). (c) Immunoblotting analyses using anti-MMP-9 and MMP-2 antibodies indicated that the identity of 92 and 72kDa gelatinases were MMP-9 and MMP-2, respectively. The zymograms (a,b) and immunoblots (c) shown are representatives of 3 separate experiments performed in duplicate.

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Zymography, Incubation, Control, Western Blot

Journal: International journal of cancer

Article Title: Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

doi: 10.1002/ijc.10373

Figure Lengend Snippet: FIGURE 9 – Effects of [D-Trp6]LHRH and EGF on invasive activity of A431 cells. In vitro invasion assay was performed using 24-well Transwell units with 8 m porosity polycarbonate filter coated with EHS matrigel. A431 cells (2105 cells/0.4 ml) were placed in the upper compartment and treated with 100 nM [D-Trp6]LHRH and/or 10 nM EGF for 48 hr in the presence of serum. At the end of culture, filters of the Transwell units were fixed and stained with crystal violet. The numbers of cells that penetrated through the matrigel to the lower surface of the filter were determined under the microscope. (a) Rep- resentative photographs of the invaded cells: a, vehicle control; b, EGF; c, [D-Trp6]LHRH; d, [D-Trp6]LHRH plus EGF. (b) Each bar represents mean percentage ( SEM) of invaded cells relative to the control value expressed as 100%. Two independent experiments were performed in triplicate. Different lower-case letters (a–d) indicate significant differences among groups (p 0.05).

Article Snippet: Human epidermoid carcinoma A431 cell line was obtained from American Type Culture Collection (ATCC, Rockville, MD).

Techniques: Activity Assay, In Vitro, Invasion Assay, Staining, Microscopy, Control

Journal: Biology of reproduction

Article Title: Production of matrix metalloproteinase-9 in lipopolysaccharide-stimulated human amnion occurs through an autocrine and paracrine proinflammatory cytokine-dependent system.

doi: 10.1095/biolreprod.102.004721

Figure Lengend Snippet: FIG. 1. Representative zymographic analysis of matrix metalloprotein- ases secreted by human amniochorion explants throughout a 5-day in- cubation period. Sample media (0.5 mg protein/lane) from human fetal membranes were analyzed by gelatin zymography. MMP activities are visualized as white (clear) bands, corresponding to MMP-2 (62 kDa), proMMP-2 (72 kDa), and proMMP-9 (92 kDa).

Article Snippet: Zymography SDS-polyacrylamide gel electrophoresis was performed according to Laemmli [32] using a minigel apparatus (Bio-Rad, Richmond, CA).

Techniques: Zymography