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: Cellular and Molecular Immunology

Article Title: The role of transcriptional factor D-site-binding protein in circadian CCL2 gene expression in anti-Thy1 nephritis

doi: 10.1038/s41423-018-0020-4

Figure Lengend Snippet: DBP regulated CCL2 expression via the target gene TRIM55. a siDBP inhibited TRIM55 mRNA expression, but not the expression of six other genes. b DBP bound to the TRIM55 promoter, as revealed by chromatin immunoprecipitation sequencing (ChIP)-PCR. c DBP bound to the TRIM55 promoter (-2984 bp), as revealed by the dual luciferase assay. d The TRIM55 promoter was divided into three subregions. e The dual luciferase assay indicated that DBP specifically bound to the -2116–2984-bp region. f, g: siTRIM55-1 and siTRIM55-2 significantly inhibited TNF-α and CCL2 expression at both the mRNA and protein levels in MCs. h The rescue assay showed that DBP-mediated CCL2 upregulation was blocked by si-TRIM55-1. si-TRIM55-1 and si-TRIM55-2, two siRNAs targeting TRIM55; Lv-Flag, lentivirus control; Lv-Flag-DBP: DBP-expressing lentivirus. *p < 0.05, **p < 0.01; n = 3. All assays were repeated at least three times

Article Snippet: DBP lentiviruses ( ≥ 1 × 10 9 TU/mL) expressing the Flag peptide (Lv-Flag-DBP and control Lv-Flag) were prepared by GenePharma.

Techniques: Expressing, ChIP-sequencing, Luciferase, Rescue Assay, Control

Journal: Arthritis Research & Therapy

Article Title: Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration

doi: 10.1186/s13075-014-0491-3

Figure Lengend Snippet: Decrease of HDAC4 protein determined by western blotting in human aging and OA cartilage specimens. (A) Safranin O staining was performed to show degeneration changes in the cartilage from a 61-year-old OA patient undergoing total knee arthroplasty, and the lack thereof in normal cartilage from a 60-year-old male patient and 23-year-old male patient undergoing amputation because of trauma. (B) Western blotting showed decreased expression of HDAC4 in human aging and OA cartilage. β-actin and Coomassie Blue staining was used to confirm equal loading of total proteins from cartilage samples. (C) Semi-quality by densitometry showed that the mean concentration of HDAC4 was 31% and 65% in the cartilage of OA patients (n = 6) and the 40 to 60 age group (n = 6), respectively, compared with the cartilage of the 20 to 40 age group (n = 6). * P represents that the difference was statistically significant ( P <0.05). HDAC4, histone deacetylase 4; OA, osteoarthritis.

Article Snippet: The next day, the first passage cells were transfected with HDAC4 small interfering RNA (siRNA) (100 nM), scrambled siRNA control (100 nM) (Cell Signaling Technology, Danvers, MA, USA), or HDAC4-flag or control vector (2.5 μg) using the GeneMute siRNA transfection reagent (SignaGen Laboratories, Gaithersburg, MD, USA).

Techniques: Western Blot, Staining, Expressing, Concentration Assay, Histone Deacetylase Assay

Journal: Arthritis Research & Therapy

Article Title: Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration

doi: 10.1186/s13075-014-0491-3

Figure Lengend Snippet: Increased expression of RUNX2 associated with decreased HDAC4 in OA cartilage. (A-a) X-ray film showed no radiographic changes in the normal controls, whereas there was joint space narrowing and cartilage damage in the OA patients. (A-b) The tibial plateaus from normal control and OA patients were shown (A-c) . Safranin O staining was performed to show degeneration in the cartilage from OA patients and the lack thereof in normal control. (B-a, B-b) Immunofluorescent histochemistry was used to show HDAC4 staining (blue: nucleus; red: HDAC4). HDAC4-positive cells (red) were significantly less in OA cartilage than the normal control. The upper boxed region indicates the magnified area shown at the boxed region below. HDAC4-positive cells were decreased to 17% of total cells in OA cartilage compared with 90% in normal control. (C-a) Immunohistochemistry was performed to show expression of Runx2 in OA cartilage and normal control. The boxed region indicates the magnified area shown at the right side. (C-b) The mRNA levels of Runx2 in OA cartilages (n = 3) and normal controls (n = 6) were tested by real-time PCR. ( * P <0.05) (C-c,C-d) Western blotting showed expression of Runx2 increased in OA samples compared with the normal control. Semi-quality by densitometry showed the mean concentration of Runx2 in the cartilage of OA patient (n = 3) compared with the cartilage of normal controls (40- to 60-year- olds) (n = 3) ( * P <0.05). HDAC4, histone deacetylase 4; OA, osteoarthritis; Runx2, runt-related transcription factor-2.

Article Snippet: The next day, the first passage cells were transfected with HDAC4 small interfering RNA (siRNA) (100 nM), scrambled siRNA control (100 nM) (Cell Signaling Technology, Danvers, MA, USA), or HDAC4-flag or control vector (2.5 μg) using the GeneMute siRNA transfection reagent (SignaGen Laboratories, Gaithersburg, MD, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay, Histone Deacetylase Assay

Journal: Arthritis Research & Therapy

Article Title: Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration

doi: 10.1186/s13075-014-0491-3

Figure Lengend Snippet: HDAC4 inhibits Runx2 and MMP-13 expression in human OA chondrocytes. (A) Human OA chondrocyte phenotype was confirmed by immunocytochemistry. The cells were positive for type II, but not for type I collagen. (B) Transfection efficiency of HDAC4 in OA chondrocytes was validated by western blotting. (C) Real-time PCR results indicated that overexpression of HDAC4 downregulated Runx2. The experiments were performed in triplicate. Data are presented as means ± SD. (D) HDAC4 inhibited Runx2 promoter activity in the human chondrocyte cell line (C28/12) in a dose-dependent manner. Conversely, inhibition of HDAC by TSA drug enhanced Runx2 promoter activity in a dose-dependent manner. * = P <0.05 versus control OA culture; # = P <0.05 versus the group of Runx2 promoter transfection. (E) HDAC4 inhibited MMP-13 promoter activity in the human chondrocyte cell line (C28/12) in a dose-dependent manner. Conversely, inhibition of HDAC by TSA drug enhanced MMP-13 promoter activity in a dose-dependent manner. All transfections were performed in triplicate. * = P <0.05 versus control OA culture; # = P <0.05 versus the group of Runx2 promoter transfection. Data are presented as means ± SD. Values represent the mean ± SD from three independent experiments. HDAC4, histone deacetylase 4; MMP, matrix metalloprotease; OA, osteoarthritis; Runx2, runt-related transcription factor-2; SD, standard deviation; TSA, trichostatin A.

Article Snippet: The next day, the first passage cells were transfected with HDAC4 small interfering RNA (siRNA) (100 nM), scrambled siRNA control (100 nM) (Cell Signaling Technology, Danvers, MA, USA), or HDAC4-flag or control vector (2.5 μg) using the GeneMute siRNA transfection reagent (SignaGen Laboratories, Gaithersburg, MD, USA).

Techniques: Expressing, Immunocytochemistry, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Activity Assay, Inhibition, Histone Deacetylase Assay, Standard Deviation

Journal: Arthritis Research & Therapy

Article Title: Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration

doi: 10.1186/s13075-014-0491-3

Figure Lengend Snippet: HDAC4 downregulates OA-related gene expressions in human OA chondrocytes. Human OA chondrocytes were transfected with HDAC4-flag or HDAC4 siRNA. All transfections were performed in triplicate. Data are presented as means ± SD. * P <0.05. (A) The mRNA of HDAC4, Runx2, MMP1, MMP3, MMP-13, type X collagen, Ihh, type II collagen, IL-1 beta, Cox2, iNos, ADAMTS-4 and -5 was tested by real-time PCR after HDAC4 or control vector transfection. (B) The mRNA of HDAC4, Runx2, MMP1, MMP3, MMP-13, type X collagen, Ihh, type II collagen, IL-1 beta, Cox2, iNos, ADAMTS-4 and -5 was tested by real-time PCR after HDAC4 siRNA or scrambled siRNA control transfection. HDAC4, histone deacetylase 4; MMP, matrix metalloprotease; OA, osteoarthritis; Runx2, runt-related transcription factor-2; SD, standard deviation; siRNA, small interfering RNA; TSA, trichostatin A.

Article Snippet: The next day, the first passage cells were transfected with HDAC4 small interfering RNA (siRNA) (100 nM), scrambled siRNA control (100 nM) (Cell Signaling Technology, Danvers, MA, USA), or HDAC4-flag or control vector (2.5 μg) using the GeneMute siRNA transfection reagent (SignaGen Laboratories, Gaithersburg, MD, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Histone Deacetylase Assay, Standard Deviation, Small Interfering RNA

Journal: Arthritis Research & Therapy

Article Title: Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration

doi: 10.1186/s13075-014-0491-3

Figure Lengend Snippet: HDAC4 partially blocks the effect of IL-1 beta on expression of catabolic events in human OA chondrocytes. Human OA chondrocytes were transfected with HDAC4 or control constructs for 48 h, then the cells were stimulated with or without IL-1 beta (5 ng/ ul) for 24 h. All transfections were performed in triplicate. * = P <0.05 versus the group of HDAC4 + IL-1 beta, # = P <0.05 versus the group of control vercor, ^ = P <0.05 versus the group of control vector + IL-1 beta. Real-time PCR showed that IL-1 beta significantly increased the mRNA levels of MMP-1, MMP3, MMP-13, iNOS, Cox2, ADAMTS-4 and -5 in the OA chondrocytes, respectively compared to those untreated controls. In addition, the mRNA levels of aggrecan, and type II collagen significantly decreased after stimulation with IL-1 beta ( P <0.05). Overexpression of HDAC4 partly blocked the upregulation of MMP-1, MMP3, MMP-13, iNOS, Cox2, ADAMTS-4, and -5 induced by IL-1 beta. In the meanwhile, the mRNA levels of aggrecan, and type II collagen significantly increased in those cells transfected by HDAC4 ( P <0.05) (n = 3). HDAC4, histone deacetylase 4; IL, interleukin; MMP, matrix metalloprotease; OA, osteoarthritis; Runx2, runt-related transcription factor-2.

Article Snippet: The next day, the first passage cells were transfected with HDAC4 small interfering RNA (siRNA) (100 nM), scrambled siRNA control (100 nM) (Cell Signaling Technology, Danvers, MA, USA), or HDAC4-flag or control vector (2.5 μg) using the GeneMute siRNA transfection reagent (SignaGen Laboratories, Gaithersburg, MD, USA).

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Real-time Polymerase Chain Reaction, Over Expression, Histone Deacetylase Assay