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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Identification of osthole binding proteins. a Chemical structure of osthole intermediate and Biotin-labeled osthole. b Viability of MDA-MB-231 cells exposed to biotin-labeled osthole as determined by MTT assay. c A schematic illustrating the steps for identifying Bio-osthole binding to proteins fabricated on a microarray. d Representative image of an experimental microarray [Blue = negative control, red = positive control, yellow = positive spot]. e Magnified image of Bio-osthole binding to recombinant STAT3 protein spot on the microarray Signal to noise ratio (SNR) value is shown

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Binding Assay, Labeling, MTT Assay, Microarray, Negative Control, Positive Control, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Osthole inhibits STAT3 phosphorylation in TNBC cells. a MDA-MB-231 and BT-549 cells were treated with 200 μM osthole for the indicated times, and levels of P-STAT3 were determined by Western blot analysis. GAPDH and STAT3 were used as internal control. b MDA-MB-231 and BT-549 cells were exposed to osthole at indicated concentrations for 24 h or 12 h, respectively. P-STAT3 levels were determined by immunoblotting. GAPDH and STAT3 were used as internal control. c Cells were pretreated with 200 μM osthole for 24 h (MDA-MB-231) or 12 h (BT-549) and then stimulated with IL-6 (50 ng/mL) for 30 mins. STAT3 phosphorylation was determined by western blot. d Immunofluorescence staining of cells showing distribution of P-STAT3 (red) in MDA-MB-231 cells. DAPI was used as counter stain. e MDA-MB-231 cells were pretreated with osthole for 24 h before exposure to IL-6 (50 ng/mL) for 30 min. Nuclear extracts were subjected to P-STAT3 and STAT3 immunoblotting. Lamin B was used as loading control

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: STAT3 overexpression rescued osthole-mediated cytotoxic effects in MDA-MB-231 cells. a Representative western blot showing levels of STAT3 and P-STAT3 in MDA-MB-231 cells following transfection with STAT3 plasmid [Control plasmid = control vehicle vector, STAT3 = STAT3 plasmid]. b Quantification of STAT3 protein levels from panel A. [* P < 0.05 compared to control plasmid (V)]. c STAT3 overexpressing cells and vector control transfected cells were exposed to 200 μM osthole for 48 h, and apoptotic cells were determined by Annexin V/PI staining. d Quantification of annexin V/PI staining showing the percentage of apoptotic cells from panel C [*** P < 0.001]. (E) STAT3 overexpressing cells and vector control transfected cells were exposed to 200 μM osthole for 36 h, and representative histograms about G2/M cell cycle phase in cells were determined by flow cytometric analysis [* P < 0.05]

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Osthole inhibits MDA-MB-231 xenograft growth in vivo. a Tumor volume in vehicle- and osthole-treated mice. MDA-MB-231 cells were injected in the flanks of mice and tumors were allowed to develop for approximately 8 d (50–150 mm 3 ). Mice bearing MDA-MB-231 xenografts received osthole at 100 or 200 mg/kg interperitoneally [* P < 0.05]. b Images of resected tumor tissues at day 48. Two mice (one in 100 mg/kg osthole group and the other in 200 mg/kg osthole group did not have a visible tumor after 48 d treatment. c Tumor weights determined on day 48 [* P < 0.05 compared to vehicle control]. d Western blot analysis of P-STAT3 levels in resected tumor specimens. GAPDH was used as loading control. e Immunohistochemical staining of tumor sections for cell proliferation marker Ki-67, apoptosis markers Bcl-2 and cleaved caspase-3, and cell cycle markers MDM2, and CDC2. Representative images are shown

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: In Vivo, Injection, Western Blot, Immunohistochemical staining, Staining, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic β cells.

doi: 10.1073/pnas.1323066111

Figure Lengend Snippet: Fig. 2. Insulin signaling regulates CPE translation initiation. (A) Western blotting for proinsulin, PC1/3, CPE, and tubulin (loading control) in control or βIRKO cell lines with or without exogenous insulin stimulation (10 nM) for 12 h. (B) Western blotting for CPE or tubulin in control or βIRKO cell lines after pulse labeling with modified methionine and then harvesting at the in- dicated time points. (C) Western blotting for IR, CPE, PC1/3, PC2, or tubulin (loading control) in control or IR knockdown (KD) β cells harvested 24, 32, or 44 h after IR shRNA containing lentiviral infection. (D, Left) Quantitative PCR (qPCR) for mRNA of ER stress markers (BiP, total XBP-1, spliced XBP-1, or CHOP) in fluorescence-activated cell-sorted β cells from control (open bar) or βIRKO (gray bar) mice (n = 4). (D, Right) Western blotting for BiP, CHOP, or tubulin (loading control) in islets isolated from control or βIRKO mice and in control or βIRKO cell lines (n = 3). (E, Left) qPCR for mRNA of ER stress markers (BiP, total XBP-1, spliced XBP-1, and CHOP) in control, βIRKO, or βIRKO proinsulin KD cell lines (n = 4 per group). A.U., arbitrary units. (E, Right) Western blotting for p-IRE1α, total IRE1α, BiP, CHOP, and tubulin (loading control) in control, βIRKO, or βIRKO proinsulin KD cell lines (n = 3 per group). (F, Left) Translational profiling of control or βIRKO cells. Profiles through the gradient were measured by A254. Positions of the 40S and 80S subunits, 80S monosome, and polyribosomes are indicated. Data shown are from representative experiments performed on three occasions. (F, Right) Polysome/monosome (P/M) ratios for the translational profiles of control or βIRKO cells (n = 3). (G) Real-time qPCR for insulin, CPE, PC1/3, or PC2 mRNA extracted from pooled monosome or polysome fractions of control or βIRKO cells (n = 3). Data are mean ± SEM. *P < 0.05 (vs. control); **P < 0.01 (vs. control); #P < 0.05 (vs. βIRKO); ##P < 0.01 (vs. βIRKO).

Article Snippet: Human CPE was subcloned from pCMV6XL5 (Origene) into pCDH-CMV-MSC-EF1-GreenPuro lentiviral vector (System Biosciences).

Techniques: Western Blot, Control, Labeling, Modification, Knockdown, shRNA, Infection, Real-time Polymerase Chain Reaction, Fluorescence, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic β cells.

doi: 10.1073/pnas.1323066111

Figure Lengend Snippet: Fig. 3. Insulin signaling regulates the translation initiation complex to modulate CPE translation. (A) Western blotting for eIF4G1, eIF4G2, eIF4A1, polyadenylate-binding protein 1 (PABP1), eIF4E, eIF4B, or tubulin (loading control) in control or βIRKO cell lines under basal conditions (n = 3). (B) Western blotting for eIF4G1, eIF4G2, or tubulin (loading control) in control or IR KD β cells harvested 24, 32, or 44 h after IR shRNA containing lentiviral infection. (C) Representative Western blotting for eIF4G1 or tubulin (loading control) in islets (pooled) isolated from control or HFD-fed C57BL/6J mice (n = 6). (D) Representative Western blotting for eIF4G1 or tubulin (loading control) in islets isolated from controls or patients with T2D (n = 5). (E) Western blotting for CPE, eIF4G1, and tubulin (loading control) in control, βIRKO, or βIRKO eIF4G1-reexpressing cell lines. (F) Western blotting for CPE, eIF4G1, and tubulin (loading control) (Left) and proinsulin ELISA (Right) in islets isolated from lean control or obese ob/ob mice with or without eIF4G1 reexpression. (G) Length, percentage of GC, dG of CPE, or PC1/3 5′ UTR was analyzed with UNAFold. (H, Left) Western blotting for PC1/3, V5, CPE, or tubulin (loading control) in control, V5 tag-PC1/3 with CPE 5′ UTR, or V5-tag PC1/3 with CPE in IR KD cells. (H, Right) Western blotting for CPE, FLAG, PC1/3, and tubulin (loading control) in control, FLAG-tag-CPE with PC1/3 5′ UTR, or FLAG tag-CPE with PC1/3 5′ UTR in IR KD cells.

Article Snippet: Human CPE was subcloned from pCMV6XL5 (Origene) into pCDH-CMV-MSC-EF1-GreenPuro lentiviral vector (System Biosciences).

Techniques: Western Blot, Binding Assay, Control, shRNA, Infection, Isolation, Enzyme-linked Immunosorbent Assay, FLAG-tag

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Insulin regulates carboxypeptidase E by modulating translation initiation scaffolding protein eIF4G1 in pancreatic β cells.

doi: 10.1073/pnas.1323066111

Figure Lengend Snippet: Fig. 4. Insulin signaling regulates eIF4G1 via Pdx1. (A) Real-time qPCR for eIF4G1 in control or βIRKO cell lines and in islets isolated from control or βIRKO mice (n = 4). Data are mean ± SEM. *P < 0.05; **P < 0.01. (B) qPCR for eIF4G1 in islets isolated from control or HFD-fed C57BL/6J mice (n = 6). Data are mean ± SEM. *P < 0.05. (C) qPCR for eIF4G1 in islets isolated from controls or patients with T2D (n = 5). Data are mean ± SEM. *P < 0.05. (D) qPCR (Left) and Western blotting (Right) for Pdx1 in control or βIRKO cell lines and in islets isolated from control or βIRKO mice (n = 4). Data are mean ± SEM. *P < 0.05. (E) qPCR (Upper) and representative Western blotting (Lower) for Pdx1 in islets (pooled) isolated from control or HFD-fed C57BL/6J mice (n = 6). Data are mean ± SEM. *P < 0.05. (F) qPCR (Left) and repre- sentative Western blotting (Right) for Pdx1 in islets isolated from controls or patients with T2D (n = 5). Data are mean ± SEM. *P < 0.05. (G) ChIP assay for Pdx1 on eIF4G1 5′ and 3′ regions in primary mouse islets. Data are mean ± SEM. *P < 0.05. (H) qPCR (Left) for eIF4G1 and Western blotting (Right) for eIF4G1, Pdx1, and tubulin (loading control) in control, βIRKO, or βIRKO Pdx1- reexpressing cell lines. (I) Western blotting for CPE, eIF4G1, Pdx1, and tubulin (loading control) (Left) and proinsulin ELISA (Right) in islets isolated from lean control or obese ob/ob mice with or without Pdx1 reexpression. (J) Western blotting for Pdx1 or tubulin (loading control) in control or IR KD β cells harvested 24, 32, or 44 h after IR shRNA containing lentiviral infection.

Article Snippet: Human CPE was subcloned from pCMV6XL5 (Origene) into pCDH-CMV-MSC-EF1-GreenPuro lentiviral vector (System Biosciences).

Techniques: Control, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, shRNA, Infection