Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: Screening and initial validation of cancer cell‐secreted proteins capable of significantly inducing iCAF phenotype. A) The t‐distributed stochastic neighbor embedding (t‐SNE) plot of the 92,222 cells in the single‐cell sequencing profile revealed distinct cell types observed in PDAC. B) The t‐SNE plot exhibited diverse subtypes of fibroblasts observed in PDAC. C) The top 10 up‐regulated and down‐regulated expressed marker genes of each CAF subgroup. D) The expression levels of myCAF markers (ACTA2, COL1A1, COL11A1, MMP11), iCAF markers (CXCL12, IL‐6, CCL2, CXCL2), and apCAF markers (HLA‐DRA, HLA‐DRB1) in different fibroblast subsets. E) Pathway activities scored by GSVA between different fibroblast subsets. F) The volcano plot depicts the differential expression of genes encoding secreted proteins in cancer cells derived from patients with high versus low iCAF. G–I) qRT‐PCR analysis was conducted to assess alterations in the expression levels of iCAF markers (IL‐6, CXCL12, and CCL2) in CAFs isolated from KPC mice following treatment with conditioned medium (CM) containing potential candidates for 12 (G), 24 (H), and 36 h (I). J) qRT‐PCR analysis of changes in the expression levels of myCAF markers (ACTA2 and CTGF) in CAFs isolated from KPC mice after treating them with CM containing PPY for 12, 24, and 36 h. K) Flow cytometry analysis of iCAF (Ly6C+MHC‐II‐), myCAF (Ly6C+MHC‐II‐), and apCAF (Ly6C+MHC‐II‐) populations after treating CAFs with CM containing PPY for 24 h. L,M) The changes in expression of iCAF markers (IL‐6, CXCL12, and CCL2) (L) and myCAF markers (ACTA2 and CTGF) (M) in CAFs isolated from three patients with PDAC were quantified by qRT‐PCR; following a 24‐h treatment with CM containing PPY. N) After treating CAFs derived from three PDAC patients with CM containing PPY for 24 h, ELISA was performed to assess the secretion of IL‐6, CCL2, and CXCL12. Each experiment was performed three times independently, and Student's t ‐test was used to analyze the data. The results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.001; ns, not statistically significant.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: Biomarker Discovery, Sequencing, Marker, Expressing, Quantitative Proteomics, Derivative Assay, Quantitative RT-PCR, Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: PPY significantly induces the iCAF phenotype in PDAC CAFs both in vitro and in vivo. A–C) After treating CAFs derived from human PDAC tissues for 12 h, qRT‐PCR analysis was performed to assess their alterations in the expression of iCAF markers (CXCL12 (A), IL‐6 (B), CXCL12 (C)). D–F) qRT‐PCR analysis of iCAF markers (CXCL12 (D), IL‐6 (E), CXCL12 (F)) after treating the human CAFs for 24 h. G–I) qRT‐PCR analysis of iCAF markers (CXCL12 (G), IL‐6 (H), CXCL12 (I)) after treating the human CAFs for 36 h. J) qRT‐PCR analysis of the expression levels of myCAF markers (ACTA2 and CTGF) after treating the human CAFs with PPY proteins (40ng/ml) for 24 h. K) Flow cytometry analysis was performed to evaluate the populations of iCAFs (Ly6C+MHC‐II‐), myCAFs (Ly6C+MHC‐II‐), and apCAFs (Ly6C+MHC‐II‐), after treating CAFs derived from cancer tissues of KPC mice with PPY recombinant proteins. L,M) After co‐culturing the human CAFs together with BxPC‐3 cells overexpressing PPY, the expression levels of iCAF markers (IL‐6, CCL2, and CXCL12) and myCAF markers (ACTA2 and CTGF) were quantified using qRT‐PCR (L), and the secretion levels of IL‐6, CCL2, and CXCL12 were measured using ELISA (M). N) The CAFs derived from cancer tissues of KPC mice were cocultured with Panc02 overexpressed PPY, and flow cytometry was applied to analyze iCAF, myCAF, and apCAF populations. O,P) After co‐culturing the human CAFs with PANC‐1 cells that had down‐regulated PPY expression, the expression levels of iCAF markers (IL‐6, CCL2, and CXCL12) and myCAF markers (ACTA2 and CTGF) were analyzed by qRT‐PCR (L), and secretion levels of IL‐6, CCL2, and CXCL12 were assessed by ELISA (M). Q) The murine CAFs were cocultured with Panc02 that had down‐regulated PPY expression, and flow cytometry was applied to analyze iCAF, myCAF, and apCAF populations. R) Schematic diagram of co‐injection of mouse cancer cells and CAFs (4:1) derived from KPC mice to construct the orthotopic allograft tumor model in C57BL/6J mice (n = 8), and the tumor tissues were isolated and analyzed by flow cytometry. S,T) The tumor tissues of PPY upregulated and downregulated groups and their respective control groups were dissociated into single cells, and flow cytometry was utilized to analyze the iCAF, myCAF, and apCAF populations in the tumor tissue. Student's t ‐test was used to analyze the data, and the results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.001; ns, not statistically significant. OE, overexpression.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: In Vitro, In Vivo, Derivative Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Recombinant, Enzyme-linked Immunosorbent Assay, Injection, Construct, Isolation, Control, Over Expression

Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: The inhibition of EGFR expression in CAFs impeded the induction of iCAFs by PPY. A) qRT‐PCR (A) and B) ELISA analyses of the expression levels of IL‐6, CCL2, and CXCL12 in human EGFR‐knockdown CAFs treated with PPY proteins. C) The efficiency of EGFR knockdown in KPC CAFs was examined by qRT‐PCR. D,E) qRT‐PCR (D) and ELISA (E) analyses of the expression levels of IL‐6, CCL2, and CXCL12 in murine EGFR knockdown CAFs treated with PPY proteins. F) Flow cytometry analysis was performed to evaluate the populations of iCAFs, myCAFs, and apCAFs in murine EGFR‐knockdown CAFs treated with PPY proteins. G,H) The IVIS image (G) and gross image (H) of tumors in model mice (n = 7), that was constructed by co‐injecting cancer cells with up‐regulated PPY expression and KPC CAFs with down‐regulated EGFR expression. I–N) Flow cytometric analysis was performed to evaluate the presence of iCAFs (I), M2 macrophages (J), MDSCs (K), T cells (L), CD8 + T cells (M), and exhausted CD8 + T cells (N) within the tumor microenvironment. O) Map of scientific hypotheses of this article. The statistical data is presented as mean ± SD and analyzed using the unpaired t‐ test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knockdown, Flow Cytometry, Construct

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet: ( A–C ) DUX4 and DUX4 target gene expression as determined by RT-qPCR in MB2401 control ( A ), MB073 FSHD1 ( B ) or MB200 FSHD2 ( C ) myoblasts without (-) or with (+) doxycycline (Dox) treatment for 48 hr to induce MBD3L2 transgene expression in clonal cell lines. ( D–E ) DUX4-positive nuclei upon overexpression of MBD3L2 in MB200 FSHD2 myoblasts as in ( C ) were detected by immunofluorescence ( D ) and quantified by counting three fields representing >125 nuclei ( E ). ( F–G ) DUX4 and DUX4 target gene expression as determined by RT-qPCR following control (CTRL) or MBD3L family gene shRNA knockdown in MB073 FSHD1 ( F ) or MB200 FSHD2 ( G ) myotubes. Error bars denote the standard deviation from the mean of three biological replicates. Statistical significance was calculated by comparing the specific knockdown to the control knockdown for each gene using a two-tailed, two-sample Mann-Whitney U test and p was ≤0.05 for all comparisons except in ( A ). See also . 10.7554/eLife.31023.038 Figure 6—source data 1. Source data for . This file contains the source data used to make the graphs presented in and – . GraphPad Prism was utilized to visually represent the quantitative data.

Article Snippet: To construct the doxycycline-inducible MBD3L2 plasmid, the MBD3L2 coding region was subcloned into the NheI and SalI sites of the pCW57.1 vector (a gift from David Root, Addgene plasmid #41393).

Techniques: Targeted Gene Expression, Quantitative RT-PCR, Control, Expressing, Over Expression, Immunofluorescence, shRNA, Knockdown, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet: ( A–E ) The ectopic ( A–C ) or endogenous ( D–E ) expression of MBD3L2 as determined by RT-qPCR in MB2401 control ( A ), MB073 FSHD1 ( B ) or MB200 FSHD2 ( C ) myoblasts cultured without (-) or with (+) doxycycline (Dox) for 48 hr, or in MB073 FSHD1 ( D ) or MB200 FSHD2 ( E ) myotubes expressing control (CTRL) or MBD3L gene shRNAs. Error bars denote the standard deviation from the mean of three biological replicates. See also .

Article Snippet: To construct the doxycycline-inducible MBD3L2 plasmid, the MBD3L2 coding region was subcloned into the NheI and SalI sites of the pCW57.1 vector (a gift from David Root, Addgene plasmid #41393).

Techniques: Expressing, Quantitative RT-PCR, Control, Cell Culture, Standard Deviation

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet: ( A–D ) MBD3L2, DUX4, and DUX4 target gene expression as determined by RT-qPCR in two additional independent experiments with control (CTRL) or MBD3L shRNA-expressing MB073 FSHD1 ( A–B ) or MB200 FSHD2 ( C–D ) muscle cell lines differentiated into myotubes. Error bars denote the standard deviation from the mean of three biological replicates. See also .

Article Snippet: To construct the doxycycline-inducible MBD3L2 plasmid, the MBD3L2 coding region was subcloned into the NheI and SalI sites of the pCW57.1 vector (a gift from David Root, Addgene plasmid #41393).

Techniques: Targeted Gene Expression, Quantitative RT-PCR, Control, shRNA, Expressing, Standard Deviation

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet:

Article Snippet: To construct the doxycycline-inducible MBD3L2 plasmid, the MBD3L2 coding region was subcloned into the NheI and SalI sites of the pCW57.1 vector (a gift from David Root, Addgene plasmid #41393).

Techniques: Recombinant, Plasmid Preparation, Expressing, Generated, Modification, Knock-Out, Software

Journal: Cell Death Discovery

Article Title: Germline mutations in apoptosis pathway genes in ovarian cancer; the functional role of a TP53I3 (PIG3) variant in ROS production and DNA repair

doi: 10.1038/s41420-021-00442-y

Figure Lengend Snippet: Materials for knockdown, protein immunoblotting, and plasmid constructs.

Article Snippet: PIG3 antibody- mouse (A-5) , Santa Cruz , SC-166664 , 1:1000 overnight incubation.

Techniques: Knockdown, Western Blot, Plasmid Preparation, Construct, Concentration Assay, Negative Control, Incubation

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 1 Nonselective and ADAM10-selective metalloprotease inhibitors increase the amount of full-length hGPR37 at the cell surface. A, Schematic model of hGPR37 with the metalloprotease cleavage site at Glu167↓Gln16816 and with three N-glycosylation sites16 shown as blue glycan structures. The added Myc and FLAG epitope tags are also indicated. B, HEK293i cells stably transfected with Myc-hGPR37-FLAG were induced to express the receptor for 24 hours and treated for the last 23 hours with the indicated metalloprotease inhibitors (20 µM marimastat, 2 µM GI254023X) or vehicle. Cells were fixed, permeabilized, and stained with the indicated antibodies followed by Alexa-Fluor-488- and -568- conjugated secondary antibodies. The nuclei were stained with TO-PRO-3 iodine. The cellular localization of cMyc antibody-labeled full-length receptors was analyzed by confocal microscopy. Scale bars: 10 µm. C, Induced HEK293i cells were treated with the indicated concentrations of GI254023X for 23 hours and analyzed by flow cytometry after labeling cell surface receptors with cMyc antibody and the phycoerythrin- conjugated secondary antibody. The results represent five independent experiments performed with triplicate samples. The fluorescence intensity values were normalized to the mean value obtained from cells treated with vehicle only. The results were analyzed before normalization using repeated measures one-way ANOVA followed by Dunnett's multiple comparison test. ***P < .001; **P < .01. Ab, antibody

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Glycoproteomics, FLAG-tag, Stable Transfection, Transfection, Staining, Labeling, Confocal Microscopy, Flow Cytometry, Fluorescence, Comparison

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 2 ADAM10 selective inhibitor GI254023X inhibits N-terminal cleavage of human and mouse GPR37. Stably transfected HEK293i cells were induced to express Myc-hGPR37-FLAG (A, C, G, H) or, alternatively, mGPR37-FLAG and Myc-hGPR37-FLAG were transiently expressed in either SH-SY5Y (E) or HEK293 (F) cells for 24 hours. The cells were treated with the indicated concentrations of protease inhibitors or vehicle for 20-23 hours. The shed N-terminal receptor fragment from the conditioned culture medium (A) and membrane-bound receptors from cellular lysates (C, E-H) were immunoprecipitated and analyzed by SDS-PAGE and Western blotting. The outlined area in panel E is shown with a longer exposure time. The identified hGPR37 species are depicted in panel B. The 67-kDa precursor and the 96-kDa full-length mature receptor are indicated with closed and open circles, respectively. The shed 32- 35-kDa N-terminal receptor fragment is indicated with a closed triangle and the 53- and 34-kDa cleaved C-terminal fragments with open and closed squares, respectively. The 70-kDa receptor species are indicated with an open triangle. Arrows in panel C indicate receptor oligomers and higher molecular mass aggregates. Panel D shows relative changes in the intensity of the 96-kDa full-length and 53-kDa cleaved receptors seen in panel C (left and right panels, respectively). The values were normalized to the 67-kDa precursor and are shown as means ± SD from four to nine independent experiments. The repeated-measures one-way ANOVA followed by Dunnett's multiple comparison test was used for the statistical comparison. **P < .01; *P < .05; ns, nonsignificant. Ab, antibody, decCMK, Decanoyl-RVKR-CMK; Hexa-D-Arg, hexa-D-arginine; IP, immunoprecipitation; WB, Western blotting

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Stable Transfection, Transfection, Membrane, Immunoprecipitation, SDS Page, Western Blot, Comparison

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 3 ADAM10 downregulation in HEK293 cells inhibits hGPR37 ectodomain cleavage and shedding. Inducible HEK293i cells were treated with siRNA pools targeting ADAM10 or ADAM17 or with the control siRNA pool for 72 hours and were induced to express hGPR37 for 8 hours at the end of the siRNA transfection. A, Receptors from solubilized cellular membranes were analyzed by SDS-PAGE and Western blotting. The knock-down of ADAM10 and ADAM17 was confirmed with the corresponding selective antibodies. The translocon-associated protein α-subunit (TRAPα) was used as a loading control. B, N-terminal receptor fragments in the cell culture medium were immunoprecipitated and analyzed by Western blotting with cMyc antibody. D, Cell surface cMyc antibody labeled receptors from five independent experiments were analyzed in triplicate by flow cytometry. Relative changes in the 96-kDa full-length and 53-kDa cleaved receptors seen in panel A are shown in panel C as means ± SD from 4-5 independent experiments. The values were normalized to the loading control before statistical analysis. The data were analyzed by repeated measures one-way ANOVA and Dunnett's multiple comparison test. *P < .05; ns, nonsignificant. The abbreviations and symbols describing GPR37 species are explained in the legend for Figure 2. i, immature ADAM17; m, mature ADAM10/17

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Control, Transfection, SDS Page, Western Blot, Knockdown, Cell Culture, Immunoprecipitation, Labeling, Flow Cytometry, Comparison

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 4 N-terminal cleavage of GPR37 is impaired in MEF and HEK293T cells lacking ADAM10 and is induced in vitro with rADAM10. The Myc- and FLAG-tagged hGPR37 was transiently expressed for 24 hours in WT and ADAM10KO MEF cells (A) or in WT, ADAM10KO, and ADAM17KO HEK293T cells that were treated or not with marimastat for 20 hours (B). For C-D, WT and ADAM10KO HEK293T cells were transiently transfected with Myc-hGPR37-FLAG together with the HA-tagged WT hADAM10 or its inactive E384A mutant (1:1 DNA ratio) for 48 hours. For E, mGPR37-FLAG was transiently expressed for 24 hours in WT and ADAM10KO HEK293T cells treated or not with marimastat for 20 hours, and for F, hGPR37 was expressed for 24 hours in stably transfected HEK293i cells treated with decCMK (10 µM) and GI254023X (5 µM) for 20 hours. Receptors and ADAM10 were immunoprecipitated from cellular lysates with FLAG and HA-antibodies, respectively, before analysis by Western blotting. For F, receptors were treated before elution from the FLAG antibody resin with rADAM10 as indicated. The additional GPR37 C-terminal fragment seen in HEK293T cells is indicated with an arrowhead (B, E). The other symbols and abbreviations are explained in the legends for Figures 2 and 3

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: In Vitro, Transfection, Mutagenesis, Stable Transfection, Immunoprecipitation, Western Blot

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 5 GPR37 is cleaved in its N-terminal domain by a non-metalloprotease. A, B and D, HEK293i cells were induced to express hGPR37 for 6 hours (A, B) or 16 hours (D), labeled with [35S]methionine/cysteine for 30 minutes and chased for various time periods before cell surface proteins were biotinylated (A) or not (B, D) with sulfo-NHS-biotin. GI254023X, decCMK, or vehicle were added to the medium during depletion, labeling, and chase. Receptors were subjected to two-step immunoprecipitation with FLAG antibody (A, upper panel, B, D) or were first purified with streptavidin agarose and then with FLAG antibody (A, lower panel) before analysis by SDS-PAGE and fluorography. For panel B, immunoprecipitated receptors from 120-minute chase samples were deglycosylated with Endo H (50 mU/mL) or PNGase F (20 U/mL) before SDS- PAGE. C, HEK293i cells were induced for 24 hours and treated with the indicated protease inhibitors or vehicle for 20 hours. Immunoprecipitated receptors were analyzed by SDS-PAGE and Western blotting. Note that GI254023X inhibits receptor cleavage more efficiently at 5 µM (B) than at 3 µM (A). PA, phosphoramidon. Other abbreviations and symbols are as described in the legends for Figures 2 and 3

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Labeling, Immunoprecipitation, Purification, SDS Page, Western Blot

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 6 Conservation of GPR37 N-terminal tail. WebLogo40 was used to generate a graphical representation of conservation between fourteen placental mammalian species (see Table S1). The generated sequence logo corresponding to residues 27-178 of hGPR37 (shown as a gray bar below the schematic model of the N-terminal domain) displays the conservation of each residue, indicated by the overall height of the letter. The relative height of each letter in the stack indicates the frequency of the corresponding amino acid at the site. The positively charged amino acids Arg and Lys are shown in red. The consensus sequence for N-glycosylation and putative PC cleavage sites are indicated with blue and yellow bars, respectively, below the sequence. The metalloprotease cleavage site (Glu167↓Gln168) identified for hGPR3716 is indicated with the red scissors. The corresponding sites for hGPR37 are also shown in the schematic model

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Generated, Sequencing, Residue, Glycoproteomics

Journal: The FASEB Journal

Article Title: GPR37 is processed in the N‐terminal ectodomain by ADAM10 and furin

doi: 10.1096/fj.202002385rr

Figure Lengend Snippet: FIGURE 7 GPR37 is cleaved at the conserved Arg54↓Asp55 site in the N-terminus by furin. HEK293 (A-E) and LoVo (F) cells were transiently transfected with the indicated Myc-hGPR37-FLAG constructs (A-D, F), or alternatively, with the NanoLuc- and HA-tagged hGPR37 (E) for 16-24 hours and treated or not with GI254023X (5 µM), decCMK (20 µM) or marimastat (20 µM) for 20 (A, B, F), 19 (C) or 12 (D, E) h. Immunoprecipitated receptors from cellular lysates (A, B, F) or aliquots of the concentrated conditioned culture medium without immunoprecipitation (D, E) were analyzed by SDS-PAGE and Western blotting. For panel C, cell surface cMyc antibody labeled receptors were analyzed by flow cytometry. Triplicate or quadruplicate samples from four independent experiments were analyzed by repeated-measures two- way ANOVA and Tukey's multiple comparison test before normalization to the respective controls without the inhibitor-treatment. *P < .05; ns, nonsignificant. Abbreviations and symbols are as in the legend for Figure 2

Article Snippet: The stably transfected tetracycline- inducible HEK293i cell line expressing Myc- hGPR37- FLAG was prepared using Invitrogen's T- REx System, as described.16 Briefly, the receptor construct and pOG44 plasmid were co- transfected into the Tet repressor expressing HEK293i cells under blasticidin S (4 μg/mL, InvivoGen, Toulouse, France) and hygromycin (400 μg/mL, InvivoGen) selection.

Techniques: Transfection, Construct, Immunoprecipitation, SDS Page, Western Blot, Labeling, Flow Cytometry, Comparison

Journal: Scientific reports

Article Title: Transcription factor FoxO1 regulates myoepithelial cell diversity and growth.

doi: 10.1038/s41598-024-51619-1

Figure Lengend Snippet: Figure 2. Overexpression of FoxO1 in ME cells. (A) Scatter plot of CD49f (x-axis) and EpCAM (y-axis). The cells were isolated from SMG in TP53 mutant female mice (n = 4) and analyzed by flow cytometry. EpCAMlowCD49fhigh-cells were sorted as ME cells (6.5%). (B) A schematic for integration of PiggyBac transposon vector plasmid. The Tet-On inducible gene expression system was used. FoxO1 expression was induced by doxycycline (Dox). (C) mCherry fluorescence merged with phase contrast in MEPB-FoxO1 cells treated with and without Dox (2 µg/mL) for 48 h. (D) Expression of FoxO1 mRNA in MEPB-FoxO1 cells treated with and without Dox for 24 h. *P < 0.05. n = 3. (E) Immunoblotting for FoxO1, αSMA, Krt14, Krt5, and β-actin in MEPB-FoxO1 cells treated with and without Dox for 72 h. (F) FoxO1 luciferase assay in the presence of FoxO1 inhibitor (Inh.; AS1842856) at the indicated concentrations. pGL4 luciferase reporter vector (upper) was constructed to include three FoxO1-binding elements (daf16:TTGTTTA and mdaf16:TTGCTTA). FoxO1 transcriptional activity was measured. pRL-TK was used as internal control. The Renilla luciferase normalized the firefly luciferase. #P < 0.05 vs. control (Ctrl). *P < 0.05 vs. Dox. n = 5. (G) Expression of αSMA mRNA in ME cells treated with and without FoxO1 inhibitor (Inh.; AS1842856, 1 μM) for 72 h. *P < 0.05. n = 3. (H) Expression of FoxO1 and αSMA mRNA in siRNA-mediated knockdown of FoxO1 (siFoxO1) or control (si Ctrl) in ME cells. *P < 0.05. n = 3. (I) Immunoblotting for NF-κB/p65 and phospho-NF-κB/p65 in MEPB-FoxO1 cells treated with and without Dox at the indicated time-points. The signal intensity of phospho-NF-κB/p65 was normalized to that of NF-κB/p65 (ratio). All data were representative of three independent experiments. See also Supplementary Figs. S2 and S3.

Article Snippet: The PiggyBac plasmid carried a tetracycline response element to drive doxycycline (Dox) induction (2 μg/mL; LKT Labs, St. Paul, MN).

Techniques: Over Expression, Isolation, Mutagenesis, Flow Cytometry, Plasmid Preparation, Gene Expression, Expressing, Fluorescence, Western Blot, Luciferase, Construct, Binding Assay, Activity Assay, Control, Knockdown

Journal: Cancer Research

Article Title: Receptor Activator of NF-κB Ligand Enhances Breast Cancer–Induced Osteolytic Lesions through Upregulation of Extracellular Matrix Metalloproteinase Inducer/CD147

doi: 10.1158/0008-5472.can-09-2758

Figure Lengend Snippet: Figure 1. Effect of EMMPRIN overexpression on MDA-231 cells. A to E, the human breast cancer cell line MDA-MB-231 (MDA-231) was stably transfected with a construct carrying the EMMPRIN gene (MDA-231-EMMPRIN) or with an empty vector (MDA-231-empty) as control. Cells were cultured in DMEM plus 10% FBS. A to C, cells were collected at confluence and RNA was extracted and reverse transcribed. The resulting cDNA was subjected to comparative real-time PCR using primer pairs and conditions specific for (A) EMMPRIN, (B) MMP-9, and (C) VEGF. Data are normalized versus the housekeeping gene GAPDH. Columns, mean of three independent experiments; bars, SEM; *, P < 0.05 versus MDA-231-empty. Inset, (A) EMMPRIN membrane surface expression detected by FACS analysis (gray profile), (B) MMP-9 activity by zymography in conditioned media from MDA-231-empty and MDA-231-EMMPRIN cells, and (C) VEGF protein expression by Western blot. Pictures are representative of three experiments with similar results. D, invasion assay. Cells were trypsinized and plated in Transwells onto 12-μm pore membrane precoated with Matrigel. After 8 h, the number of cells invading the Matrigel substrate was assessed as described in Materials and Methods. Columns, mean of three independent experiments; bars, SEM; *, P < 0.05 versus MDA-231-empty. E, proliferation assay. Cells were cultured in 96-well cell culture plates for 72 h, then proliferation was assessed by the XTT cell proliferation assay (black line, MDA-231-empty; gray line, MDA-231-EMMPRIN). F, migration assay. HUVECs were cultured onto filters coated with gelatin in the upper compartment of the Transwell chambers and were allowed to migrate using as chemoattractants conditioned media from MDA-231 cells (MDA-231-CM), MDA-231 cells transfected with empty vector (MDA-231-empty) and MDA-231 cells overexpressing EMMPRIN (MDA-231-EMMPRIN). Six hours after plating, cells that had migrated were evaluated as described in Materials and Methods. Columns, mean of three independent experiments; bars, SEM; *, P < 0.05 versus MDA-231-empty.

Article Snippet: Anti-VEGF antibody (#MAB293) was purchased from R&D Systems, Inc. Anti-EMMPRIN antibody (#sc-53582), goat anti-rabbit IgG horseradish peroxidase (HRP)–conjugated antibody (#sc-2004), small interfering RNA (siRNA) duplexes specific for human EMMPRIN/CD147 (#sc-35298), and siRNA transfection reagent (#sc-29528) were from Santa Cruz Biotechnology, Inc.

Techniques: Over Expression, Stable Transfection, Transfection, Construct, Plasmid Preparation, Control, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Membrane, Expressing, Activity Assay, Zymography, Western Blot, Invasion Assay, Proliferation Assay, Migration

Journal: Cancer Research

Article Title: Receptor Activator of NF-κB Ligand Enhances Breast Cancer–Induced Osteolytic Lesions through Upregulation of Extracellular Matrix Metalloproteinase Inducer/CD147

doi: 10.1158/0008-5472.can-09-2758

Figure Lengend Snippet: Figure 2. Knockdown of EMMPRIN by siRNA. MDA-231 cells were treated with EMMPRIN-specific siRNA (MDA-231- siEMMPRIN) or scramble siRNA (MDA-231- siCTRL) as control. A to C, cells were collected, the RNA was extracted, and were reverse transcribed. The resulting cDNAs was subjected to comparative real-time PCR using primer pairs and conditions specific for (A) EMMPRIN, (B) MMP-9, and (C) VEGF. Data are normalized versus the housekeeping gene GAPDH. Columns, mean of three independent experiments; bars, SEM; *, P < 0.05 versus MDA-231- siCTRL. Inset, the protein expression of (A) EMMPRIN, (B) MMP-9 activity of conditioned media from MDA-231-siCTRL and MDA-231-siEMMPRIN, and (C) VEGF protein expression. Pictures are representative of three experiments with similar results. D, invasion assay. Cells were trypsinized and plated in Transwells onto a 12-μm pore membrane precoated with Matrigel. After 8 h, the number of cells invading the Matrigel substrate was evaluated as described in Materials and Methods. Columns, mean of three independent experiments; bars, SEM; *, P < 0.05 versus MDA-231-siCTRL.

Article Snippet: Anti-VEGF antibody (#MAB293) was purchased from R&D Systems, Inc. Anti-EMMPRIN antibody (#sc-53582), goat anti-rabbit IgG horseradish peroxidase (HRP)–conjugated antibody (#sc-2004), small interfering RNA (siRNA) duplexes specific for human EMMPRIN/CD147 (#sc-35298), and siRNA transfection reagent (#sc-29528) were from Santa Cruz Biotechnology, Inc.

Techniques: Knockdown, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Invasion Assay, Membrane

Journal: Cancer Research

Article Title: Receptor Activator of NF-κB Ligand Enhances Breast Cancer–Induced Osteolytic Lesions through Upregulation of Extracellular Matrix Metalloproteinase Inducer/CD147

doi: 10.1158/0008-5472.can-09-2758

Figure Lengend Snippet: Figure 4. Effect of RANKL on the expression of EMMPRIN and its downstream genes. A, MDA-231 cells were treated with DMEM (CTRL) or with conditioned medium from primary mouse osteoblast cultures treated with vehicle (OBsCM) or 100 ng/mL OPG (OBsCM+OPG) for 48 h. The cells were collected, and the extracted RNA was reverse transcribed. The resulting cDNA was subjected to comparative real-time PCR using primer pairs and conditions specific for EMMPRIN, MMP-9, and VEGF. Data are normalized versus the housekeeping gene GAPDH. Columns, mean of three independent experiments; bar, SEM. *, P < 0.05 versus OBsCM; #, P < 0.05 versus CTRL. B-D, MDA-231 cells were treated with 30 ng/mL RANKL for 4, 8, and 16 h. Cells were collected, and the extracted RNA was reverse transcribed. The resulting cDNA was subjected to comparative real-time PCR using primer pairs and conditions specific for (B) EMMPRIN, (C) MMP-9, and (D) VEGF. Data are normalized versus the housekeeping gene GAPDH. Columns, mean of three independent experiments; bars, SEM. *, P < 0.05 versus vehicle. Inset, (B) protein expression of EMMPRIN, (C) MMP-9 activity in conditioned media, and (D) VEGF protein expression 16 h after RANKL administration, and are representative of three experiments with similar results.

Article Snippet: Anti-VEGF antibody (#MAB293) was purchased from R&D Systems, Inc. Anti-EMMPRIN antibody (#sc-53582), goat anti-rabbit IgG horseradish peroxidase (HRP)–conjugated antibody (#sc-2004), small interfering RNA (siRNA) duplexes specific for human EMMPRIN/CD147 (#sc-35298), and siRNA transfection reagent (#sc-29528) were from Santa Cruz Biotechnology, Inc.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Activity Assay

Journal: Cancer Research

Article Title: Receptor Activator of NF-κB Ligand Enhances Breast Cancer–Induced Osteolytic Lesions through Upregulation of Extracellular Matrix Metalloproteinase Inducer/CD147

doi: 10.1158/0008-5472.can-09-2758

Figure Lengend Snippet: Figure 5. Effect of EMMPRIN knockdown on RANKL modulation. A to C, MDA-231 cells were subjected to RNA silencing using EMMPRIN-specific siRNA (MDA-231-siEMMPRIN) or scramble siRNA (MDA-231-siCTRL) as control for 48 h. Cells were subsequently treated with vehicle or 30 ng/mL RANKL for 16 h. RNA was then extracted and reverse transcribed. The resulting cDNA was subjected to comparative real-time PCR using primer pairs and conditions specific for (A) EMMPRIN, (B) MMP-9, and (C) VEGF. D, HUVECs were treated with conditioned media from MDA-231 silenced for EMMPRIN or scramble siRNA, and treated with vehicle or with 30 ng/mL RANKL. Average numbers of branching points were counted from triplicate wells. Columns, mean of three independent experiments; bars, SEM. *, P < 0.05 versus MDA-231-siCTRL; §, P < 0.05 versus MDA-231-siCTRL+RANKL.

Article Snippet: Anti-VEGF antibody (#MAB293) was purchased from R&D Systems, Inc. Anti-EMMPRIN antibody (#sc-53582), goat anti-rabbit IgG horseradish peroxidase (HRP)–conjugated antibody (#sc-2004), small interfering RNA (siRNA) duplexes specific for human EMMPRIN/CD147 (#sc-35298), and siRNA transfection reagent (#sc-29528) were from Santa Cruz Biotechnology, Inc.

Techniques: Knockdown, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cancer Research

Article Title: Receptor Activator of NF-κB Ligand Enhances Breast Cancer–Induced Osteolytic Lesions through Upregulation of Extracellular Matrix Metalloproteinase Inducer/CD147

doi: 10.1158/0008-5472.can-09-2758

Figure Lengend Snippet: Figure 6. Schematic representation of the role played by EMMPRIN in the mechanism inducing osteolytic metastases. Molecular interactions among osteoblasts, osteoclasts, and tumor cells are shown. Tumor cells localized in the bone marrow produce factors (i.e., pro-osteolytic factors) that stimulate osteoclast formation by increasing the expression of RANKL on osteoblasts. RANKL binds to its receptor RANK and stimulates EMMPRIN expression in tumor cells. This in turn promotes the release of MMP-9 and VEGF. MMP-9 contributes to the digestion of organic matrix, whereas VEGF stimulates osteoclastogenesis. These two events favor tumor cells proliferation providing growth factors to tumor cells, thus contributing to the creation of the vicious cycle (Pre-OC, preosteoclast; OC, osteoclast; OB, osteoblast).

Article Snippet: Anti-VEGF antibody (#MAB293) was purchased from R&D Systems, Inc. Anti-EMMPRIN antibody (#sc-53582), goat anti-rabbit IgG horseradish peroxidase (HRP)–conjugated antibody (#sc-2004), small interfering RNA (siRNA) duplexes specific for human EMMPRIN/CD147 (#sc-35298), and siRNA transfection reagent (#sc-29528) were from Santa Cruz Biotechnology, Inc.

Techniques: Expressing

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a , b Immunofluorescence staining and quantification of phalloidin, γ-H2AX and L1-CT 48 h post IR (2 Gy, 5 Gy, or 10 Gy) in HUVECs (left panels, magnification, ×400). Scale bar = 20 µm (enlarged, 5 µm). Bar graphs quantifying the number of γ-H2AX foci, the phalloidin density, and the number of colocalized foci (right panels). For quantification of phalloidin density, error bars represent mean ± SEM (2 Gy vs. 10 Gy p < 0.0001; 5 Gy vs. 10 Gy p = 0.0015). For quantification of the number of γ-H2AX foci, error bars represent mean ± SD (**** p < 0.0001). Colocalized foci (marked by white arrow) are amplified and graphs represent quantification of L1-CT and γ-H2AX signals in selected regions of dotted lines (bottom panels). Error bars represented mean ± SEM (**** p < 0.0001). c Heat ma p of the RNA-seq analysis results showing radiation-induced EndMT and the mesenchymal phenotype. Total RNA was isolated from HUVECs before and after 10 Gy IR (5 h, 72 h). d Immunofluorescence staining of L1-CT and γ-H2AX in HUVECs at 0, 1, 24, and 48 h post IR (10 Gy; left panels). Quantification of γ-H2AX foci and nuclear L1CAM (magnification, ×400; right panels). The number of γ-H2AX foci with an intensity greater than 40 and a foci diameter of 0.1 μm was counted. Scale bar = 20 µm (enlarged, 5 µm). e Immunoblotting and quantification of full-length L1CAM and L1-CT fragments from HUVECs transfected with lentiviral shRNA targeting L1CAM 48 h after IR (10 Gy; upper panels). Error bars represent mean ± SD (full-length L1CAM: sh-Control IR – vs. + p = 0.0003; sh-Control IR + vs. sh-L1CAM IR + p < 0.0001, L1-CT fragments: sh-Control IR – vs. + p = 0.0013; sh-Control IR + vs. sh-L1CAM IR + p = 0.0006). Immunofluorescence staining of γ-H2AX and L1-CT 48 h post IR (10 Gy) in HUVECs (magnification, ×400). Scale bar = 5 µm. f Immunoblotting (upper panel) of full-length L1CAM and L1-CT fragments in the cytoplasmic (C) and nuclear (N) fractions of HUVECs 48 h post IR (10 Gy). Quantification of full-length L1CAM in the cytoplasmic fractions and L1-CT fragments in the nuclear fractions. HUVECs were treated with control IgG or Ab417 (20 µg/mL) before IR. GAPDH and lamin B were used as cytoplasmic and nuclear markers, respectively. Error bars represent mean ± SD from independent experiments (full-length L1CAM: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0001, L1-CT fragments: No IR vs. IR + IgG p = 0.0228; IR + IgG vs. IR + Ab417 p = 0.0448). g Immunofluorescence staining for L1-CT and γ-H2AX 0 and 48 h post IR (10 Gy) in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL) (upper panel). Quantification of colocalization of γ-H2AX foci with L1CAM (magnification, ×400) (lower panel). Scale bar = 20 µm (enlarged, 5 µm). h Immunofluorescence staining (upper panel) and quantification (lower panel) of phalloidin and L1-CT 72 h post IR (10 Gy) in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL; magnification, ×400). Scale bar = 20 µm. Error bars represent mean ± SD ( p = 0.0007). i Immunofluorescence staining for L1-CT and γ-H2AX at 0 and 24 h after Dox treatment in HUVECs pre-treated with control IgG or Ab417 (20 µg/mL; left panel). Colocalized foci (marked by white arrow) are amplified and graphs represent quantification of L1-CT and γ-H2AX signals in selected regions of dotted lines (middle panels). Quantification of colocalization of γ-H2AX foci with L1CAM (magnification, ×400; right panel). Scale bar = 5 µm. j Immunofluorescence staining and quantification of phalloidin and γ-H2AX 0 and 24 h after Dox treatment in HUVECs pre-treated with control IgG or Ab417 (magnification, ×400). Scale bar = 10 μm. Error bars represent mean ± SD (IgG vs. Dox + IgG p = 0.0002; Dox + IgG vs. Dox + Ab417 p = 0.0482; Ab417 vs. Dox + Ab417 p = 0.0065). For quantification of γ-H2AX foci and γ-H2AX foci colocalized with L1CAM, the foci in each sample were counted at least 70 cells per field (magnification, ×100). The average number of foci/cell was determined from >6 fields (magnification, ×100). Data are representative of three independent experiments. ( h : two-talied Student’s t -test, all other panels: one-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Amplification, RNA Sequencing, Isolation, Western Blot, Transfection, shRNA, Control

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a Immunofluorescence staining and quantification of γ-H2AX foci colocalized with L1-CT (upper left panel; magnification, ×400) and immunoblotting (lower left panel) and quantification (right panels) of full-length L1CAM, L1-CT fragments, and γ-H2AX at 48 h post-irradiation (10 Gy) in HUVECs with or without γ-secretase inhibitor L-685,458 (3 µM) treatment. For quantification of γ-H2AX foci colocalized with L1-CT, error bars represent mean ± SEM ( p = 0.0017). For quantification of full-length L1CAM and L1-CT fragments, error bars represent mean ± SD (full-length L1CAM: (-) vs. IR p = 0.0011; IR vs. IR + L-685,458 p = 0.0233, L1-CT fragments: (-) vs. IR p = 0.0017; IR vs. IR + L-685,458 p = 0.0177). b , c HUVECs were transfected with human full-length (L1-WT), NLS-mutated (L1-4A), and endocytosis-deficient (L1-dRSLE) L1CAM vectors after knockdown of endogenous L1CAM. b Scheme of L1-WT, L1-4A and L1-dRSLE constructs (top left panel). Immunoblotting of full-length L1CAM in HUVECs (top right panel) and immunofluorescence staining (middle panels) and quantification (bottom panels) of L1-CT and γ-H2AX in HUVECs 48 h post-irradiation (10 Gy; magnification, ×400). Scale bar = 5 µm. Error bars represent mean ± SEM (No. γ-H2AX foci: Control vs. L1CAM-WT p = 0.001; L1CAM-WT vs. L1CAM-4A p = 0.0001; L1CAM-WT vs. L1CAM-dRSLE p = 0.0002, No. L1-CT foci: **** p < 0.0001; L1CAM-WT vs. L1CAM-dRSLE p = 0.0004, No. colocalized foci: Control vs. L1CAM-WT p = 0.006; **** p < 0.0001). c Immunofluorescence staining and quantification of phalloidin and γ-H2AX in HUVECs 48 h post-irradiation (10 Gy; magnification, ×400). Scale bar = 20 µm. Error bars represent mean ± SD (Control vs. L1CAM-WT p = 0.0096; L1CAM-WT vs. L1CAM-4A p = 0.0014; **** p < 0.0001). d Immunofluorescence staining and pearson’s correlation coefficient of L1-CT colocalized with p-ATM, 53BP1, and DNA-PKcs in the nuclei of Ab417-pre-treated HUVECs 48 h post-irradiation (10 Gy magnification, ×400). Scale bar = 5 µm. Error bars represent mean ± SD (* * ** p < 0.0001). e Flow cytometry analysis of cells with GFP positivity resulting from DNA repair and quantification of HR (upper panel) and NHEJ (lower panel) efficiency in L1CAM-knockdown HUVECs. The HUVECs were transiently transfected with the DR-GFP or EJ5-GFP construct along with control or L1CAM siRNA and were then transfected with a SceI plasmid to induce DNA damage. Error bars represent mean ± SD (HR efficacy p = 0.0005, NHEJ efficacy p = 0.0011). f Immunofluorescence staining (upper panel) for GFP, phalloidin, γ-H2AX in HUVECs 48 h post-irradiation (10 Gy). Quantification of phalloidin is shown (magnification, ×400; right panel). Scale bar = 20 µm. Error bars represent mean ± SD (No IR vs. IR + Control p = 0.0001; IR + Control vs. IR + L1CAM-WT p = 0.0063; IR + L1CAM-WT vs. IR + L1CAM-C-term p = 0.0027, IR + Control vs. IR + L1CAM-C-term p < 0.0001). HUVECs were transfected with L1-WT or L1-CT lentiviral vectors tagged with N-terminal GFP and C-terminal His. Transfection efficiency (left panel) was tested by immunoblotting for L1-WT and L1-CT. For quantification of foci colocalized with L1CAM, the colocalized foci in each sample were counted in a minimum of 70 cells per field (magnification, ×100). The average number of colocalized foci/cell was determined from five fields (magnification, ×100). The data are presented the means ± SDs and ±SEMs from three independent experiments. ( a upper left panel: two-talied Student’s t -test; a all other panels and b , c , f : one-way ANOVA for multiple comparisons; d , e two-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Western Blot, Irradiation, Transfection, Knockdown, Construct, Control, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a RT-qPCR analysis of L1CAM in p53-knockdown HUVECs 48 h post-irradiation (10 Gy). Error bars represent mean ± SD from three independent experiments ( p < 0.0001). b Immunoblotting (upper panel) and quantification (lower panel) of full-length L1CAM and L1-CT 48 h post-irradiation (10 Gy) in p53-knockdown HUVECs pre-treated with control IgG or Ab417 (20 µg/mL). Error bars represent mean ± SD from four independent experiments (full-length L1CAM: No IR vs. si-Control IR + IgG p = 0.0468; si-Control IR + IgG vs. si-Control IR + Ab417 p = 0.0468; si-Control IR + IgG vs. si-p53 IR + IgG p = 0.0462; si-p53 IR + IgG vs. si-p53 IR + Ab417 p = 0.0218, L1-CT: No IR vs. si-Control IR + IgG p = 0.0124; si-Control IR + IgG vs. si-Control IR + Ab417 p = 0.0484; si-Control IR + IgG vs. si-p53 IR + IgG p = 0.0206; si-p53 IR + IgG vs. si-p53 IR + Ab417 p = 0.0004). c Immunofluorescence staining (upper panel) and quantification (lower panel) for γ-H2AX and L1-CT. The results of quantification of γ-H2AX foci, nuclear L1CAM, and colocalization of γ-H2AX foci with L1CAM are shown (magnification, ×400). Scale bar = 5 μm. Error bars represent mean ± SEM (No. γ-H2AX foci: si-Control vs. si-p53+IgG p = 0.0107; si-p53+IgG vs. si-p53 + Ab417 p = 0.0242, No. L1-CT foci: si-Control vs. si-p53+IgG p = 0.0425; si-p53+IgG vs. si-p53 + Ab417 p = 0.0119, No. colocalized foci: si-Control vs. si-p53 + IgG p = 0.0131; si-p53 + IgG vs. si-p53 + Ab417 p = 0.0097). For quantification of foci colocalized with L1CAM, the colocalized foci in each sample were counted in at least 70 cells per field (magnification, ×100, n = 5). d , e Wild-type or EC-p53KO mice were injected intravenously with control IgG or Ab417 (10 mg/kg) and subjected to 17.5 Gy thoracic irradiation ( n = 5 animals per group). d Immunofluorescence staining (upper panels) for γ-H2AX, L1CAM, and CD31 in heart tissues 3 weeks post-irradiation and quantification (lower panels) of γ-H2AX + cells and nuclear L1CAM + cells among CD31 + cells (magnification, ×400). Scale bar = 5 μm. Error bars represent mean ± SEM (γ-H2AX + in CD31 + nuclei: WT + IR vs. p53KO+IR p = 0.0005; p 53KO + IgG vs. p53KO + Ab417 p < 0.0001, L1CAM + in CD31 + nuclei: WT + IR vs. p53KO+IR p = 0.0001; p53KO+IgG vs. p 53KO + Ab417 p = 0.0002). e Haematoxylin and eosin staining, Masson’s trichrome staining, and immunohistochemical detection of CD31 in heart tissues 3 weeks post-irradiation (upper panels) and quantification of ventricular inflammation, perivascular fibrosis area, and microvessel density per field (magnification, ×200; lower panels). Scale bar = 100 μm. Error bars represent mean ± SEM (ventricular inflammation: No IR vs. WT IR + IgG p = 0.0003; WT IR + IgG vs. WT IR + Ab417 p = 0.0125; WT IR + IgG vs. p53KO IR + IgG p = 0.0104, perivascular fibrosis area: No IR vs. WT IR + IgG p = 0.0021; WT IR + IgG vs. WT IR + Ab417 p = 0.0328; WT IR + IgG vs. p 53KO IR + IgG p = 0.0203; WT IR + IgG vs. WT IR + Ab417 p = 0.0165, MVD: No IR vs. WT IR + IgG p < 0.0001; WT IR + IgG vs. WT IR + Ab417 p = 0.0055; WT IR + IgG vs. WT IR + Ab417 p = 0.0068). f Immunofluorescence staining (left panels) of α-SMA and CD31 in heart tissues 3 weeks post-irradiation ( n = 5 animals per group) and quantification (right panels) of the α-SMA + CD31 + area in the CD31 + area (magnification, ×400). Scale bar = 20 μm. Error bars represent mean ± SEM (No IR vs. WT IR + IgG p < 0.0001; WT IR + IgG vs. WT IR + Ab417 p = 0.0251; WT IR + IgG vs. p53KO IR + IgG p = 0.011; p 53KO IR + IgG vs. p53KO IR + Ab417 p = 0.0004). g Immunofluorescence staining (left panel) of WGA, cTnT, and CD31 ( n = 5 animals per group) and quantification (right panel) of the cTnT area per field (magnification, ×400). Scale bar = 20 μm. Error bars represent mean ± SEM (No IR vs. WT IR + IgG p = 0.0014; WT IR + IgG vs. WT IR + Ab417 p = 0.0489; WT IR + IgG vs. p53KO IR + IgG p = 0.0253; p53KO IR + IgG vs. p53KO IR + Ab417 p = 0.001). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant ( a Two-way ANOVA for multiple comparisons; all other panels: one-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Knockdown, Irradiation, Western Blot, Control, Immunofluorescence, Staining, Injection, Immunohistochemical staining

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a – e Mice were injected intravenously with control IgG or Ab417 (10 mg/kg) three times a week for 2 weeks and received 16 Gy thoracic IR (No IR n = 7; IR + IgG n = 8; IR + Ab417 n = 8). f – j Mice were injected intravenously with control IgG or Ab417 (10 mg/kg) with or without intraperitoneal Dox injection (4 mg/kg) three times a week for 2 weeks (No Dox n = 7; Dox+IgG n = 8; Dox+Ab417 n = 8). a , f Immunohistochemical detection (left panels) of L1-CT and γ-H2AX in heart tissues 1 week post IR ( a ) and 2 weeks after Dox treatment ( f ). The quantified L1-CT + cells and γ-H2AX + cells among ECs are shown (magnification, ×200; right panels). Scale bar = 50 μm. Error bars represent mean ± SEM (L1-CT: IR + IgG vs. IR + Ab417 p = 0.0004, γ-H2AX: No IR vs. IR + IgG p = 0.0076; IR + IgG vs. IR + Ab417 p = 0.0017; Dox + IgG vs. Dox+Ab417 p = 0.0003, **** p < 0.0001). b , g Haematoxylin and eosin staining, Masson’s trichrome staining, and immunohistochemical detection of CD31 in heart tissues (left panels); and quantification of arterial wall thickness, perivascular fibrosis area, and microvessel density per field in heart tissues (magnification, ×200; right panels). Scale bar = 100 μm. The arrow in b indicates inflammatory cell infiltration. Error bars represent mean ± SEM (Arterial wall thickness: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0159; No Dox vs. Dox p = 0.0009; Dox + IgG vs. Dox + Ab417 p = 0.0117, perivascular fibrosis area: No IR vs. IR + IgG p = 0.0011; IR + IgG vs. IR + Ab417 p = 0.0168; No Dox vs. Dox p = 0.0001; Dox + IgG vs. Dox + Ab417 p = 0.0492, MVD: No IR vs. IR + IgG p = 0.0012; IR + IgG vs. IR + Ab417 p = 0.0184; No Dox vs. Dox p = 0.0093; Dox + IgG vs. Dox + Ab417 p = 0.0378). c , h Serum CRP, E-selectin, and ICAM-1 levels 1 week post IR ( c ) and 1 week after Dox treatment ( h ). Error bars represent mean ± SD (CRP: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0015; No Dox vs. Dox p = 0.0007; Dox+IgG vs. Dox+Ab417 p = 0.0003, E-selectin: No IR vs. IR + IgG p = 0.0031; IR + IgG vs. IR + Ab417 p = 0.02; No Dox vs. Dox p = 0.0003; Dox+IgG vs. Dox+Ab417 p = 0.0082, ICAM-1: No IR vs. IR + IgG p = 0.0053; IR + IgG vs. IR + Ab417 p = 0.01; No Dox vs. Dox p = 0.0154). d , i Immunofluorescence detection (upper panel) of L1-CT and CD31 in mouse heart tissues 1 week post IR ( d ) and 2 weeks after Dox treatment ( i ) (magnification, ×400). Scale bar = 5 μm. Quantification of L1CAM in CD31 nuclei (lower panel). Error bars represent mean ± SEM (No IR vs. IR + IgG p = 0.0012; IR + IgG vs. IR + Ab417 p = 0.0033; No Dox vs. Dox p < 0.0001; Dox IgG vs. Dox+Ab417 p = 0.0004). e , j Immunofluorescence staining of α-SMA and CD31 (scale bar = 10 μm) and of WGA and cTnT (scale bar = 20 μm) in heart tissues 1 week post IR ( e ) and 2 weeks post Dox treatment ( j ) (magnification, ×400; upper panels). Quantification of the α-SMA + CD31 + area in the CD31 + area and quantification of the cTnT area per field (lower panels). Error bars represent mean ± SEM (SMA + CD31 + area in the CD31 + area: No IR vs. IR + IgG p < 0.0001; IR + IgG vs. IR + Ab417 p = 0.0011; No Dox vs. Dox p = 0.0013; Dox + IgG vs. Dox + Ab417 p = 0.0103, cTnT area: No IR vs. IR + IgG p = 0.0019; IR + IgG vs. IR + Ab417 p = 0.0107; No Dox vs. Dox p = 0.0126; Dox + IgG vs. Dox + Ab417 p = 0.0494, one-way ANOVA for multiple comparisons). Data are representative of three independent experiments.

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Injection, Control, Immunohistochemical staining, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a , b Co-culture of iPSC-CMs and irradiated ECs transfected with control or L1CAM siRNA. a Schematic of the procedure for co-culture of iPSC-CMs and irradiated ECs. ECs were transfected with control or L1CAM-specific siRNA and irradiated with 10 Gy. Forty-eight hours after IR, the ECs were co-cultured with iPSC-CMs (top). The beating rates of the iPSC-CMs were examined after 5 days of co-culture with irradiated ECs (bottom). Error bars represent mean ± SD (No IR vs. si-Control+IR p = 0.0005; si-Control+IR vs. si-L1CAM + IR p > 0.0001). b Immunofluorescence staining (left panel) and quantification (right panel) of cTnT expression in cardiomyocytes and the collagen I deposition area in co-cultured cells (magnification, ×200). Scale bar = 50 μm. Error bars represent mean ± SD (cTnT expression: No IR vs. si-Control+IR p = 0.0239; si-Control+IR vs. si-L1CAM + IR p = 0.0475, collagen I deposition: No IR vs. si-Control + IR p = 0.027; si-Control + IR vs. si-L1CAM + IR p = 0492). c – e Mice were injected intravenously with control IgG or Ab417 (10 mg/kg) three times a week for 2 weeks and received 16 Gy thoracic IR. f – h Mice were injected intravenously with control IgG or Ab417 (10 mg/kg) with or without intraperitoneal Dox injection (4 mg/kg) three times a week for 2 weeks. c , f Cumulative survival analysis measured in days after treatment ( n = 8 animals per group). d , g Echocardiography results (upper panels) and quantification (lower panels) of FS (%), LVEF (%), LVESV (μL), and LVEDV (μL) (No IR n = 10; IR + IgG n = 5, IR + Ab417 n = 5; No Dox n = 10; Dox+IgG n = 5, Dox + Ab417 n = 5). Scale bar = 1 mm. Error bars represent mean ± SD (FS: No IR vs. IR + IgG p = 0.0331; IR + IgG vs. IR + Ab417 p = 0.0172; Dox+IgG vs. Dox + Ab417 p = 0.0046, LVEF: No IR vs. IR + IgG p = 0.0284; IR + IgG vs. IR + Ab417 p = 0.0182; Dox + IgG vs. Dox + Ab417 p = 0.0027, LVEDV: No IR vs. IR + IgG p = 0.0099; IR + IgG vs. IR + Ab417 p = 0.0054; No Dox vs. Dox p = 0.0003; Dox + IgG vs. Dox + Ab417 p = 0.003, LVESV: No IR vs. IR + IgG p = 0.0005; IR + IgG vs. IR + Ab417 p = 0.0002, **** p < 0.0001). e , h Haematoxylin and eosin–stained ventricular myocardium (upper panels) and quantification (lower panels) of cardiomyocyte cross-sectional area (No IR n = 7; IR + IgG n = 8; IR + Ab417 n = 8; No Dox n = 7; Dox + IgG n = 8; Dox+Ab417 n = 8) (magnification, ×400). Scale bar = 20 μm. Error bars represent mean ± SEM (No IR vs. IR + IgG p = 0.0003; IR + IgG vs. IR + Ab417 p = 0.0002; No Dox vs. Dos+IgG p = 0.024; Dox + IgG vs. Dox + Ab417 p = 0.0493). ( a , d : log-rank Mantel-Cox test; all other panels: one-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Co-Culture Assay, Irradiation, Transfection, Control, Cell Culture, Immunofluorescence, Staining, Expressing, Injection

Journal: Nature Communications

Article Title: An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

doi: 10.1038/s41467-021-23478-1

Figure Lengend Snippet: a Immunofluorescence staining of L1CAM and VE-cadherin in heart tissues from patients with cardiomyopathy ( n = 12) and patients without cardiomyopathy ( n = 8) (magnification, ×400). Scale bar = 10 μm (enlarged, 2 μm). Error bars represent mean ± SEM ( p = 0.0004). b Immunofluorescence staining of γ-H2AX and CD31 in heart tissues from patients with cardiomyopathy and patients without cardiomyopathy (upper panels) and quantification (lower panel) of nuclear L1CAM + cells among CD31 + cells (magnification, ×400). Scale bar = 10 μm (enlarged, 2 μm). Error bars represent mean ± SEM (Normal vs. γ-H2AX + CD31 + p > 0.0001; γ-H2AX ‒ CD31 + vs. γ-H2AX + CD31 + p = 0.0003) c Immunofluorescence staining of L1CAM, α-SMA, and CD31 in heart tissues from patients with cardiomyopathy and from patients without cardiomyopathy (upper panels) and quantification (lower panel) of nuclear L1CAM + cells among α-SMA - CD31 + and α -SMA + CD31 + cells (magnification, ×400). Scale bar = 10 μm (enlarged, 2 μm). Error bars represent mean ± SEM ( p > 0.0001). ( a two-tailed student’s t -test; b , c one-way ANOVA for multiple comparisons).

Article Snippet: Immunoblotting, immunohistochemistry, and immunofluorescence staining were performed using primary antibodies against the N-terminal domain of L1CAM (immunohistochemistry/immunofluorescence 1:100; sc-31032; Santa Cruz Biotechnology), L1-CT (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; LS-B9803; LSBio; immunofluorescence 1:200; sc-53386; Santa Cruz Biotechnology; immunofluorescence 1:200; ab123990; Abcam), complete L1CAM (immunoblotting 1:1000; sc-53386; Santa Cruz Biotechnology), γ-H2AX (immunohistochemistry/immunofluorescence 1:500, immunoblotting 1:1000; 05-636; Millipore), CD31 (immunohistochemistry/immunofluorescence 1:200; #AF3628; R&D Systems; immunoblotting 1:1000; 28364; Abcam), GAPDH (immunoblotting 1:1000; sc-47724; Santa Cruz Biotechnology), lamin B (immunoblotting 1:1000; sc-6216; Santa Cruz Biotechnology), αSMA (1:2000; A5228; Sigma-Aldrich), VCAM-1 (immunoblotting 1:1000; sc-1504; Santa Cruz Biotechnology), p-ATM (immunofluorescence 1:200; 05-740; Millipore), 53BP1 (immunofluorescence 1:200; sc-10914; Santa Cruz Biotechnology), and DNA-PKcs (immunofluorescence 1:200; sc-135886; Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Cell Death Discovery

Article Title: MiR-150 blunts cardiac dysfunction in mice with cardiomyocyte loss of β 1 -adrenergic receptor/β-arrestin signaling and controls a unique transcriptome

doi: 10.1038/s41420-022-01295-9

Figure Lengend Snippet: A , B Genome-wide profiling and filtering strategies of array dataset based on the correlation between transcript signatures and cardiac phenotypes. Sixteen dysregulated (DE) genes, which are upregulated in the I dataset (ISO GRK − β 1 AR TG compared to vehicle GRK − β 1 AR TG controls) but are downregulated in the II dataset (ISO GRK − β 1 AR;miR-150 DTG compared to ISO GRK − β 1 AR TG) at 1-week post-treatment, were chosen for additional analyses. Twenty other DE genes, which are downregulated in the I dataset (ISO GRK − β 1 AR TG compared to vehicle GRK − β 1 AR TG controls) but are upregulated in the II dataset (ISO GRK − β 1 AR;miR-150 DTG compared to ISO GRK − β 1 AR TG) at 1-week post-treatment, were chosen for further analyses. N = 3 per group. C – K Validation strategy of array dataset. Seven potentially deleterious DE genes ( Cspg5 , Cfl1 , Gdap1l1 , Mfsd12 , Arhgef39 , Map2k7 , and Cdk14 ) were validated by QRT-PCR analyses in LVs from GRK − β 1 AR TG and GRK − β 1 AR;miR-150 DTG mice at 1-week post-treatment ( D–J ). The other potentially beneficial DE gene ( Slitrk6 ) was validated by QRT-PCR analyses in LVs from GRK − β 1 AR TG and GRK − β 1 AR;miR-150 DTG mice at 1-week post-treatment ( K ). Of note, other genes are not validated as being dysregulated as shown in ( B ) or are undetectable in LVs. Data are presented as fold induction of gene expression normalized to Gapdh . N = 3 per group. Two-way ANOVA with Tukey multiple comparison test. * P < 0.05, ** P < 0.01, or *** P < 0.001 vs. vehicle; # P < 0.05, ## P < 0.01, or ### P < 0.001 vs . ISO GRK − β 1 AR TG. All data are presented as mean ± SEM.

Article Snippet: Expression of genes or lncRNAs was detected using TaqMan expression assays for the mouse ( Anp , Mm00435329_m1 ; Tnf-α , Mm00443258_m1; Col3a1 , Mm00802300_m1 ; Bax , Mm00432051_m1 ; Bak1 , Mm00432045_m1; Egr2 , Mm00456650_m1; P2x7r , Mm00440578_m1; Casp14 , Mm00438040_g1 ; Ing-4 , Mm00460097_m1 ; p53 , Mm01731290_g1; Gdap1l1 , Mm00523187_m1 ; Cspg5 , Mm00516549_m1 ; Cfl1 , Mm03057591_g1 ; Mfsd12 , Mm01172867_m1 ; Arhgef39 , Mm01349584_g1 ; Map2k7 , Mm00488759_m1 ; Cdk14 , Mm00448111_m1 ; Slitrk6 , Mm07302106_m1 ; Gm41664 , Mm03960618_m1 ; Tspan2os , Mm01305745_m1 ; AK036033 , Mm01302617_m1, and Gapdh , Mm99999915_g1 for an endogenous control), and the human ( GDAP1L1 , Hs00225209_m1 ; BCL2 , Hs04986394_s1 ; BAK1 , Hs00832876_g1 ; BAX , Hs00180269_m1 ; p53 , Hs01034249_m1 , and GAPDH , Hs02786624_g1 for an endogenous control).

Techniques: Genome Wide, Biomarker Discovery, Quantitative RT-PCR, Gene Expression, Comparison

Journal: Cell Death Discovery

Article Title: MiR-150 blunts cardiac dysfunction in mice with cardiomyocyte loss of β 1 -adrenergic receptor/β-arrestin signaling and controls a unique transcriptome

doi: 10.1038/s41420-022-01295-9

Figure Lengend Snippet: A Mouse Gm41664 has the strongest miR-150 binding site with 12-mer complementary sequences. MiR-150 seed pairing in the target region and complementary sequences outside the seed region are presented as vertical lines. B MiR-150’s capability of directly repressing the activity of luciferase (LUC) reporter constructs that include either wild-type (WT) or mutated (MUT) binding sites for Gm41664 . Transfection with or without miR-150 mimic in H9c2 cells is shown as + or −. The LUC activity of Firefly was normalized to the LUC activity of Renilla and compared with empty vector measurements. N = 6. Unpaired two-tailed t -test. C Human GDAP1L1 has a conserved miR-150 binding site in the 3’-untranslated region (3’UTR). MiR-150 seed pairing in the target region and complementary sequences outside the seed region are presented as vertical lines. D The direct ability of miR-150 to inhibit the activity of LUC reporter constructs that include either WT or MUT binding sites for GDAP1L1 . Transfection with or without miR-150 mimic in AC16 cells is indicated as + or −. The LUC activity of Firefly was normalized to the LUC activity of Renilla and compared to empty vector measurements. N = 6. Unpaired two-tailed t -test. * P < 0.05 vs. miR mimic control.

Article Snippet: Expression of genes or lncRNAs was detected using TaqMan expression assays for the mouse ( Anp , Mm00435329_m1 ; Tnf-α , Mm00443258_m1; Col3a1 , Mm00802300_m1 ; Bax , Mm00432051_m1 ; Bak1 , Mm00432045_m1; Egr2 , Mm00456650_m1; P2x7r , Mm00440578_m1; Casp14 , Mm00438040_g1 ; Ing-4 , Mm00460097_m1 ; p53 , Mm01731290_g1; Gdap1l1 , Mm00523187_m1 ; Cspg5 , Mm00516549_m1 ; Cfl1 , Mm03057591_g1 ; Mfsd12 , Mm01172867_m1 ; Arhgef39 , Mm01349584_g1 ; Map2k7 , Mm00488759_m1 ; Cdk14 , Mm00448111_m1 ; Slitrk6 , Mm07302106_m1 ; Gm41664 , Mm03960618_m1 ; Tspan2os , Mm01305745_m1 ; AK036033 , Mm01302617_m1, and Gapdh , Mm99999915_g1 for an endogenous control), and the human ( GDAP1L1 , Hs00225209_m1 ; BCL2 , Hs04986394_s1 ; BAK1 , Hs00832876_g1 ; BAX , Hs00180269_m1 ; p53 , Hs01034249_m1 , and GAPDH , Hs02786624_g1 for an endogenous control).

Techniques: Binding Assay, Activity Assay, Luciferase, Construct, Transfection, Plasmid Preparation, Two Tailed Test, Control

Journal: Cell Death Discovery

Article Title: MiR-150 blunts cardiac dysfunction in mice with cardiomyocyte loss of β 1 -adrenergic receptor/β-arrestin signaling and controls a unique transcriptome

doi: 10.1038/s41420-022-01295-9

Figure Lengend Snippet: A , B AC16 cells were transfected with control scramble siRNA (si-control) or GDAP1L1 siRNA (si- GDAP1L1 ) ( A ) and with anti-miR control scramble or anti-miR-150 ( B ). QRT-PCR for GDAP1L1 ( A ) or miR-150 ( B ) was conducted to check the knockdown efficiency. Data were normalized to GAPDH ( A ) or U6 SNRNA ( B ) and expressed relative to controls. N = 6 per group. Unpaired two-tailed t -test. *** P < 0.001 vs. si-control or anti-miR control. C QRT-PCR expression analysis of anti-apoptotic BCL2 in human cardiomyocytes (HuCMs) transfected with 4 different groups as indicated. N = 6. BCL2 expression compared to GAPDH was calculated using 2 −ΔΔCt , and data are presented as fold induction of BCL2 expression levels normalized to control (si-control or anti-miR control). One-way ANOVA with Tukey multiple comparison test. * P < 0.05 or ** P < 0.01 vs. control. ## P < 0.01 vs. anti-miR-150. D – F RNA interference with GDAP1L1 protects HuCMs from the pro-apoptotic effects mediated by anti-miR-150. HuCMs were transfected as indicated and subjected to in vitro simulation of I/R (hypoxia/reoxygenation) [sI/R (H/R)]. TUNEL assays were then conducted in both normoxic ( D , F ) and sI/R conditions ( E , F ). Scale bar = 100 μm. The percentage of apoptotic nuclei (green) was calculated after the normalization of total nuclei (blue). N = 6. One-way ANOVA with Tukey multiple comparison test. * P < 0.05, ** P < 0.01, or *** P < 0.001 vs. control. # P < 0.05 vs. anti-miR-150. All data are shown as mean ± SEM.

Article Snippet: Expression of genes or lncRNAs was detected using TaqMan expression assays for the mouse ( Anp , Mm00435329_m1 ; Tnf-α , Mm00443258_m1; Col3a1 , Mm00802300_m1 ; Bax , Mm00432051_m1 ; Bak1 , Mm00432045_m1; Egr2 , Mm00456650_m1; P2x7r , Mm00440578_m1; Casp14 , Mm00438040_g1 ; Ing-4 , Mm00460097_m1 ; p53 , Mm01731290_g1; Gdap1l1 , Mm00523187_m1 ; Cspg5 , Mm00516549_m1 ; Cfl1 , Mm03057591_g1 ; Mfsd12 , Mm01172867_m1 ; Arhgef39 , Mm01349584_g1 ; Map2k7 , Mm00488759_m1 ; Cdk14 , Mm00448111_m1 ; Slitrk6 , Mm07302106_m1 ; Gm41664 , Mm03960618_m1 ; Tspan2os , Mm01305745_m1 ; AK036033 , Mm01302617_m1, and Gapdh , Mm99999915_g1 for an endogenous control), and the human ( GDAP1L1 , Hs00225209_m1 ; BCL2 , Hs04986394_s1 ; BAK1 , Hs00832876_g1 ; BAX , Hs00180269_m1 ; p53 , Hs01034249_m1 , and GAPDH , Hs02786624_g1 for an endogenous control).

Techniques: Transfection, Control, Quantitative RT-PCR, Knockdown, Two Tailed Test, Expressing, Comparison, In Vitro, TUNEL Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Interleukin-18 induces EMMPRIN expression in primary cardiomyocytes via JNK/Sp1 signaling and MMP-9 in part via EMMPRIN and through AP-1 and NF-?B activation

doi: 10.1152/ajpheart.00451.2010

Figure Lengend Snippet: IL-18 induces extracellular matrix metalloproteinase inhibitor (EMMPRIN) expression in adult cardiomycytes (ACMs). A: IL-18 induced EMMPRIN mRNA expression in a dose-dependent manner. ACMs were incubated with recombinant mouse IL-18 for 2 h. EMMPRIN mRNA expression was determined by quantitative RT-PCR. Actin served as an invariant control and is shown in the inset. Values are means ± SE; n = 12. *P < 0.01 (at least) vs. untreated ACMs. B: IL-18 specificity. The specificity of IL-18 was verified by incubating cells with IL-18-neutralizing antibodies or IL-18-binding protein (IL-18BP)-Fc (10 μg/ml for 1 h) before the addition of IL-18 (5 ng/ml for 2 h). EMMPRIN mRNA expression was quantified determined as in A. Actin levels are shown in the inset. Values are means ± SE; n = 12. *P < 0.001 vs. untreated ACMs; †P < 0.01 vs. IL-18 + control IgG or Fc. C: IL-18-induced EMMPRIN mRNA expression was confirmed by Northern blot analysis. ACMs were treated as in B and analyzed for EMMPRIN mRNA expression by Northern blot analysis. Both 28S rRNA and actin served as loading controls. Values are means ± SE; n = 3. D: IL-18 induced EMMPRIN protein expression. ACMs treated with IL-18 (5 ng/ml) were analyzed for EMMPRIN protein levels by immunoblot analysis. Tubulin served as a loading control. Values are means ± SE; n = 3. E: IL-18 stimulated EMMPRIN secretion. ACM were treated as in E but for 24 h, and EMMPRIN levels in culture supernatants were quantified by immunoblot analysis. Tubulin in the corresponding ACM cell lysates demonstrated that similar numbers of ACMs were plated in both groups. Values are means ± SE; n = 3. F and G: effects of IL-18 were more robust than IL-6 on EMMPRIN mRNA (F) and protein (G) expression. ACMs treated with IL-6 or IL-18 (5 ng/ml) for 2 h (F) or 12 h (G) were analyzed for EMMPRIN mRNA (F; n = 6) or protein expression (G; n = 3). F: *P < 0.05 vs. untreated ACMs; **P < 0.001 vs. untreated ACMs; §P < 0.01 vs. IL-6.

Article Snippet: Recombinant mouse IL-18 (B002-5), recombinant mouse IL-6 (406-ML-005/CF), IL-18-neutralizing antibodies (αIL-18 Ab; 10 μg/ml for 1 h), normal mouse IgG 1 (MAB002), IL-18 antibodies used in the immunoblot analysis (D043-3), IL-18BP-Fc (119-BP-100; 10 μg/ml for 1 h), Fc, and anti-EMMPRIN antibodies used in the immunoblot analysis (AF772) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Incubation, Recombinant, Quantitative RT-PCR, Control, Binding Assay, Northern Blot, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Interleukin-18 induces EMMPRIN expression in primary cardiomyocytes via JNK/Sp1 signaling and MMP-9 in part via EMMPRIN and through AP-1 and NF-?B activation

doi: 10.1152/ajpheart.00451.2010

Figure Lengend Snippet: IL-18 induces EMMPRIN transcription in NMCMs via Sp1. A: IL-18 induction of EMMPRIN promoter-reporter activation was Sp1 dependent. NMCMs were transfected with wild-type and mutant EMMPRIN promoter-reporter constructs before the addition of IL-18 and then processed as in Fig. 4B. *P < 0.05 (at lease) vs. untreated NMCMs. B: IL-18 stimulated Sp1 binding to the EMMPRIN promoter in vivo. NMCMs were treated with IL-18 for 2 h. Cross-linked chromatin was prepared and immunoprecipitated with anti-Sp1 antibody or control IgG before amplification of the EMMPRIN gene region containing Sp1 sites. The EMMPRIN open reading frame (ORF) with a similar amplicon size served as a control. Amplification of the input DNA is shown at the top. Values are means ± SE; n = 3. C: IL-18 induced EMMPRIN mRNA expression via Sp1. ACM were treated with mithramycin (100 nM in DMSO for 45 min) before the addition of IL-18 (5 ng/ml for 2 h). EMMPRIN mRNA expression was analyzed by Northern blot analysis, and results from three independent experiments are shown on the right. *P < 0.01 vs. untreated ACMs; †P < 0.05 vs. IL-18.

Article Snippet: Recombinant mouse IL-18 (B002-5), recombinant mouse IL-6 (406-ML-005/CF), IL-18-neutralizing antibodies (αIL-18 Ab; 10 μg/ml for 1 h), normal mouse IgG 1 (MAB002), IL-18 antibodies used in the immunoblot analysis (D043-3), IL-18BP-Fc (119-BP-100; 10 μg/ml for 1 h), Fc, and anti-EMMPRIN antibodies used in the immunoblot analysis (AF772) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Transfection, Mutagenesis, Construct, Binding Assay, In Vivo, Immunoprecipitation, Control, Amplification, Expressing, Northern Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Interleukin-18 induces EMMPRIN expression in primary cardiomyocytes via JNK/Sp1 signaling and MMP-9 in part via EMMPRIN and through AP-1 and NF-?B activation

doi: 10.1152/ajpheart.00451.2010

Figure Lengend Snippet: IL-18 induces Sp1 activation via JNK. A–C: IL-18 activated JNK (A), ERK (B), and p38 MAPK (C) in NMCMs. NMCMs were treated with SP-600125 (SP; 20 μM in DMSO for 30 min; A), PD-98059 (PD; 10 μM in DMSO for 1 h; B), or SB-203580 (SB; 1 μM in DMSO for 30 min; C) before the addition of IL-18 (5 ng/ml for 1 h). Total and phosphorylated (p-)MAPK levels were analyzed by immunoblot analysis using cleared whole cell lysates and activation-specific antibodies. Values are means ± SE; n = 3. D–F: knockdown of MyD88, IRAK4, and TRAF6 blunted IL-18-mediated JNK (D), ERK (E), and p38 MAPK (F) activations. NMCMs were transfected with MyD88, IRAK4, or TRAF6 siRNA before the addition of IL-18 (5 ng/ml for 1 h). Total and p-MAPK levels were analyzed by immunoblot analysis. Values are means ± SE; n = 3. G: IL-18 induced Sp1 activation via JNK but not via ERK and p38 MAPK. NMCMs were treated with SP, PD, or SB before the addition of IL-18 (5 ng/ml for 2 h). Sp1 activation in nuclear protein extracts was analyzed as in Fig. 3E. Values are means ± SE; n = 12. *P < 0.001 vs. untreated NMCMs; †P < 0.01 vs. IL-18 + DMSO. H: schema showing the signal transduction pathway involved in IL-18-mediated EMMPRIN expression.

Article Snippet: Recombinant mouse IL-18 (B002-5), recombinant mouse IL-6 (406-ML-005/CF), IL-18-neutralizing antibodies (αIL-18 Ab; 10 μg/ml for 1 h), normal mouse IgG 1 (MAB002), IL-18 antibodies used in the immunoblot analysis (D043-3), IL-18BP-Fc (119-BP-100; 10 μg/ml for 1 h), Fc, and anti-EMMPRIN antibodies used in the immunoblot analysis (AF772) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Knockdown, Transfection, Transduction, Expressing

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Interleukin-18 induces EMMPRIN expression in primary cardiomyocytes via JNK/Sp1 signaling and MMP-9 in part via EMMPRIN and through AP-1 and NF-?B activation

doi: 10.1152/ajpheart.00451.2010

Figure Lengend Snippet: IL-18 induces MMP-9 expression in part via EMMPRIN in ACMs. A: IL-18 induced MMP-9 mRNA expression. ACMs treated with IL-18-neutralizing antibodies or control IgG (10 μg/ml for 1 h) before the addition of IL-18 (5 ng/ml for 2 h) were analyzed for MMP-9 mRNA expression by quantitative RT-PCR. Actin served as an invariant control. Values are means ± SE; n = 12. *P < 0.001 vs. untreated ACMs; †P < 0.01 vs. IL-18. B: detection of IL-18-induced MMP-9 pro-enzyme and enzyme. ACMs were treated as in A but for 24 h. MMP-9 levels in culture supernatants were analyzed by gelatin zymography. Values are means ± SE; n = 3. C: IL-18 induced MMP-9 mRNA expression in part via EMMPRIN. ACMs were transduced with adenoviral mEMMPRIN (MOI: 50 for 24 h) before the addition of IL-18. Ad.GFP served as a control. MMP-9 mRNA expression was analyzed as in A. Values are means ± SE; n = 12. *P < 0.001 vs. untreated ACMs; †P < 0.01 vs. IL-18 + Ad.mEMMPRIN; §P < 0.05 vs. IL-18 + Ad.empty vector.

Article Snippet: Recombinant mouse IL-18 (B002-5), recombinant mouse IL-6 (406-ML-005/CF), IL-18-neutralizing antibodies (αIL-18 Ab; 10 μg/ml for 1 h), normal mouse IgG 1 (MAB002), IL-18 antibodies used in the immunoblot analysis (D043-3), IL-18BP-Fc (119-BP-100; 10 μg/ml for 1 h), Fc, and anti-EMMPRIN antibodies used in the immunoblot analysis (AF772) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Control, Quantitative RT-PCR, Zymography, Transduction, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: CMIP is oncogenic in human gastric cancer cells

doi: 10.3892/mmr.2017.7541

Figure Lengend Snippet: Correlation of CMIP expression with clinicopathological parameters from gastric cancer patients.

Article Snippet: Following blocking, the membranes were incubated with polyclonal primary antibodies against CMIP (cat. no. 12851-1-AP) and β-actin (cat. no. 20536-1-AP; both diluted at 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: CMIP is oncogenic in human gastric cancer cells

doi: 10.3892/mmr.2017.7541

Figure Lengend Snippet: Expression of CMIP in gastric cancer tissues. (A) Immunohistochemistry analysis of CMIP in human gastric cancer tissues and normal gastric tissues. Human testes tissue and gastric cancer tissues were also examined as the CMIP positive and isotype negative controls, respectively. Representative images are presented (magnification, ×200). (B) Association between CMIP protein expression and relapse-free survival and overall survival. CMIP, c-Maf inducing protein.

Article Snippet: Following blocking, the membranes were incubated with polyclonal primary antibodies against CMIP (cat. no. 12851-1-AP) and β-actin (cat. no. 20536-1-AP; both diluted at 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Expressing, Immunohistochemistry

Journal: Molecular Medicine Reports

Article Title: CMIP is oncogenic in human gastric cancer cells

doi: 10.3892/mmr.2017.7541

Figure Lengend Snippet: Expression of CMIP in gastric cancer and normal tissues.

Article Snippet: Following blocking, the membranes were incubated with polyclonal primary antibodies against CMIP (cat. no. 12851-1-AP) and β-actin (cat. no. 20536-1-AP; both diluted at 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: CMIP is oncogenic in human gastric cancer cells

doi: 10.3892/mmr.2017.7541

Figure Lengend Snippet: Knockdown of CMIP reduces the proliferation and enhances the apoptosis of MKN-28 cells. (A) The efficiency of CMIP knockdown by CMIP-siRNA was assessed by western blot analysis. Proliferation of MKN-28 cells transfected with CMIP-siRNA was measured using (B) cell counting (where Day 1=1×10 4 cells), (C) CCK-8 and (D) colony formation assays. (E) Apoptosis analysis of MKN-28 cells transfected with CMIP-siRNA was measured by flow cytometry using double-staining with Annexin V-fluorescein isothiocyanate and PI. CMIP, c-Maf inducing protein; si, small interfering; PI, propidium iodide; NC, negative control; OD, optical density. Data are presented as the mean ± standard error of the mean of at least 3 independent experiments. *P<0.05 and **P<0.01 vs. siNC.

Article Snippet: Following blocking, the membranes were incubated with polyclonal primary antibodies against CMIP (cat. no. 12851-1-AP) and β-actin (cat. no. 20536-1-AP; both diluted at 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Knockdown, Western Blot, Transfection, Cell Counting, CCK-8 Assay, Flow Cytometry, Double Staining, Negative Control

Journal: Molecular Medicine Reports

Article Title: CMIP is oncogenic in human gastric cancer cells

doi: 10.3892/mmr.2017.7541

Figure Lengend Snippet: Knockdown of CMIP suppresses the migration and invasion of MKN-28 cells. MKN-28 cells were transfected with the indicated siRNAs and the (A) migratory and (B) invasive abilities of the cells were evaluated (n=3; magnification, ×100). (C) A scratch was made in a confluent, adherent layer of MKN-28 cells that had undergone treatment with siNC or two different siRNAs against CMIP. Cells that had migrated into the wound were counted after 24 h (n=3). Representative images of the wounds are presented. Data are presented as the mean ± standard error of the mean of at least 3 independent experiments. *P<0.05 vs. siNC. CMIP, c-Maf inducing protein; si, small interfering; NC, negative control.

Article Snippet: Following blocking, the membranes were incubated with polyclonal primary antibodies against CMIP (cat. no. 12851-1-AP) and β-actin (cat. no. 20536-1-AP; both diluted at 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Knockdown, Migration, Transfection, Negative Control

Journal: Molecular Medicine Reports

Article Title: CMIP is oncogenic in human gastric cancer cells

doi: 10.3892/mmr.2017.7541

Figure Lengend Snippet: CMIP is a direct target of miR-101-3p and CMIP regulates the expression of MDM2 and MAPK. (A) The TargetScan-predicted binding site between miR-101-3p and the 3′-UTR of CMIP. The mutant 3′-UTR of CMIP is also presented. (B) miR-101-3p was down-regulated in gastric cancer tissues compared with normal gastric tissues. *P<0.05 vs. Normal. (C) Luciferase assay of MKN-28 cells cotransfected with miR-101-3p mimic or NC, and a luciferase reporter containing CMIP 3′-UTR wildtype or mutant constructs. *P<0.05 vs. NC. (D) MKN-28 cells were transfected with miR-101-3p mimics or NC. miR-101-3p overexpression inhibited the protein expression of CMIP. β-actin served as a loading control. (E) Cells transfected with CMIP-siRNA demonstrated a significant decrease in MDM2 and MAPK mRNA expression compared with cells transfected with siNC. All data were presented as the mean ± standard error of the mean of at least 3 independent experiments. *P<0.05 vs. siNC. CMIP, c-Maf inducing protein; MAPK, mitogen activated protein kinase; miR, microRNA; NC, negative control; si, small interfering; 3′UTR, 3-untranslated region; 3′UTR-WT, wild type 3′UTR construct; 3′UTR-MUT, mutant 3′UTR construct; MAPK, mitogen activated protein kinase.

Article Snippet: Following blocking, the membranes were incubated with polyclonal primary antibodies against CMIP (cat. no. 12851-1-AP) and β-actin (cat. no. 20536-1-AP; both diluted at 1:1,000; ProteinTech Group, Inc., Chicago, IL, USA) overnight at 4°C.

Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Construct, Transfection, Over Expression, Control, Negative Control

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Fold changes of top ranking molecular apocrine-signature genes in two studies.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: The regulation of molecular apocrine genes by the AR-ERK feedback loop . (A) Heat map of top ranking molecular apocrine-signature genes following the inhibition of AR-ERK signaling using qPCR data. Heat map shows fold changes for gene expression relative to control in MDA-MB-453 and HCC-1954 cell lines. Treatments were carried out by CI-1040 (CI) at 2 µM and 10 µM concentrations, flutamide (FLU) at 25 nM and 40 nM concentrations, and the combination of flutamide at 25 nM or 40 nM and CI-1040 at 2 µM concentrations. Red and green colors depict up-regulation and down-regulation, respectively. Bar indicates the range of fold changes in gene expression. (B) Box plots to demonstrate relative expression of PIP to control following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines using qPCR. CTL: control. (C) Western blot analysis to assess PIP protein levels following AR-ERK inhibition in MDA-MB-453 and HCC-1954 cell lines. Fold changes (RR) in band densities were measured relative to the control (CTL). AR, androgen receptor; ERK, extracellular signal-regulated kinase; qPCR, quantitative PCR; RR, relative risk.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Inhibition, Gene Expression, Control, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Fold changes of molecular apocrine-signature genes following treatment with AR and MEK inhibitors.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: PIP protein expression in primary breast tumors and in vivo models . (A) Immunohistochemistry (IHC) staining for PIP in ER negative (ER-) breast tumors. AR+ group: ≥20% of cells showing positive AR staining; AR- group: <20% of cells stained for AR. Percentage of cells with positive staining are demonstrated for each group. * P <0.01 is for AR+ versus AR-. Error Bars: ± 2SEM. (B) IHC staining for PIP in AR+ and AR- breast tumors. Magnification is at 60X. (C) IHC staining for PIP in xenograft tumors generated using MDA-MB-453 cell line. Control: a control tumor; FLU: a flutamide-treated tumor; PD: a PD0325901-treated tumor. Magnification is at 60X. (D) IHC for PIP in xenograft tumors. Percentage of cells positive for PIP was assessed using IHC and compared between each treatment group and control (CTL). * P <0.01 is for FLU or PD treatment versus CTL. Error Bars: ± 2SEM. AR, androgen receptor; SEM, standard error of the mean.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Expressing, In Vivo, Immunohistochemistry, Staining, Generated, Control

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Transcriptional regulation of PIP by AR and CREB1 . (A) Luciferase reporter assay. The transcriptional activation of PIP promoter by PRLR, AR, CREB1, PRLR + AR, and PRLR + CREB1 expression constructs was assessed using Dual-Luciferase assays in MCF-7 cells and relative response ratios are reported. Co-transfection with the PIP reporter vector and an empty pcDNA vector was used as a control (CTL). * P <0.01, is compared to the control group. (B) Induction of PIP expression following DHT treatment. PIP expression was assessed using qPCR following DHT treatment at 30 minute, 1 hour, 3 hour, 12 hour, 24 hour, and 48 hour time-points in MDA-MB-453 and HCC-1954 cell lines. Fold changes are measured relative to the respective control at each time point. * P <0.03, is compared to the control group (dashed line). Error Bars: ± 2SEM. (C) Putative transcription factor binding sites for CREB1 in 1.5 kb promoter region of PIP. P1 (primer set 1) and P2 (primer set 2) are regions of amplification for ChIP assays. (D) ChIP assay with CREB1 antibody. The results of qPCR amplification for ChIP assays are demonstrated with two sets of primers for PIP promoter. Percentage recovery of input chromatin is shown for each primer set. *, P <0.01 is for CREB1 Ab. versus control Ab. Error Bars: ± 2SEM. (E) Western blot analysis to show CREB1 and PIP protein levels following CREB1-knockdown using siRNA in MDA-MB-453 cell line. Fold changes (RR) in band densities were measured relative to non-targeting siRNA control (CTL). Ab, antibody; AR, androgen receptor; ChIP, chromatin immunoprecipitation; DHT, dihydrotestosterone; qPCR, quantitative PCR; RR, relative risk; SEM, standard error of the mean.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Luciferase, Reporter Assay, Activation Assay, Expressing, Construct, Cotransfection, Plasmid Preparation, Control, Binding Assay, Amplification, Western Blot, Knockdown, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: The effect of PIP knockdown on cell invasion and viability . (A) qPCR to demonstrate PIP-knockdown efficiencies with siRNA-duplex1 (D1) and siRNA-duplex2 (D2) in MDA-MB-453 cell line. PIP expression following knockdown was assessed relative to non-targeting siRNA control (CTL) and fold change is shown for each duplex. (B) Western blot analysis to show PIP protein level following PIP-knockdown in MDA-MB-453 cell line as described in (A). Fold changes (RR) in band densities were measured relative to the control (CTL). (C) The effect of PIP expression on cell invasion. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. *, P <0.03 is for each PIP-knockdown versus CTL. Error Bars: ± 2SEM. (D) MTT assay to measure cell viability following PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. CTL: non-targeting siRNA control. *, P <0.03 is for each PIP-knockdown versus CTL. Error Bars: ± 2SEM. MTT, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide; qPCR, quantitative PCR; RR, relative risk; SEM, standard error of the mean.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Knockdown, Expressing, Control, Western Blot, Transfection, MTT Assay, Real-time Polymerase Chain Reaction

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: The effect of PIP knockdown on ERK-Akt and integrin-β1signaling . (A) Western blot analysis to measure the levels of phosphorylated (Ph)-ERK, total (T)-ERK, ph-Akt, and T-Akt following PIP-knockdown with siRNA duplex1 (PIP-D1) and duplex2 (PIP-D2) in MDA-MB-453 cell line. Fold changes of Phospho/Total ratios (Ph/T-RR) were assessed relative to non-targeting siRNA control (CTL). (B) Western blot analysis to measure the level of ph-CREB1, T-CREB1, and ILK1 following PIP-knockdown as described in (A). Ph-ATF1 is the phosphorylated form of CREB-related protein that is known to be detected by this antibody. (C) Integrin-β1 immunoprecipitation (IP). IP assays were carried out with Integrin-β1 following PIP-knockdown with PIP-D1 and PIP-D2 in MDA-MB-453 cell line. Non-targeting siRNA was used as control (CTL). Western blot analysis was carried out on IP samples to measure the integrin-β1 binding to ILK1 and ErbB2. Immunoblotting with integrin-β1 antibody was used as a loading control. Fold changes (RR) of ILK1 and ErbB2 following PIP-knockdown were measured relative to that of control-siRNA. (D) Integrin-β1 immunoprecipitation following PIP-knockdown and the addition of fibronectin fragments (Fn-fs). PIP-knockdown with PIP-D1 was carried out as described in (C). Twenty-four hours after PIP-knockdown, cells were treated with α-chymotryptic fibronectin fragment 120K at 100 µg/ml concentration. Control cells were treated with vehicle only. Fold changes (RR) of ILK1 and ErbB2 following PIP-knockdown + Fn-fs were measured relative to the control. (E) The effect of fibronectin fragments on cell invasion following PIP-knockdown. Cell invasion assays were carried out after PIP-knockdown with PIP-D1 in MDA-MB-453 cell line. Transfection with non-targeting siRNA control (CTL) was used as a control. Treatment with fibronectin fragments was carried out as described in (D). Error Bars: ± 2SEM. Δ; is the difference between CTL and PIP-D1+Fn-fs groups. ERK, extracellular signal-regulated kinase; ILK1, integrin-linked kinase 1; RR, relative risk; SEM, standard error of the mean.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques: Knockdown, Western Blot, Control, Immunoprecipitation, Binding Assay, Concentration Assay, Transfection

Journal: Breast Cancer Research : BCR

Article Title: Prolactin-induced protein mediates cell invasion and regulates integrin signaling in estrogen receptor-negative breast cancer

doi: 10.1186/bcr3232

Figure Lengend Snippet: Schematic diagram of the PIP signaling pathway in ER-negative breast cancer . Red arrow denotes stimulatory effect. ER, estrogen receptor; Fn, fibronectin; Fn-f, Fibronectin fragment; ITG-β1, integrin-β1.

Article Snippet: Quantitative real-time PCR (qPCR) to assess the expression levels of PIP (assay ID: Hs00160082_m1), dual specificity phosphatase 6 (DUSP6, assay ID: Hs00737962_m1), S100A8 (assay ID: Hs00374264_g1), FOXA1 (assay ID: Hs00270129_m1), transcription factor AP2B (TFAP2, assay ID: Hs00231468_m1), SOX11 (assay ID: Hs00846583_s1), BANP (assay ID: Hs00215370_m1), PER2 (assay ID: Hs00256143_m1), TFF3 (assay ID: Hs00902278_m1), and AZGP1 (assay ID: Hs00426651_m1) was carried out using Taqman Gene Expression Assays (Applied Biosystems, Melbourne, VIC, Australia) as instructed by the manufacturer.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 1. VRK1 is phosphorylated in Ser342 by Plk3. (A) Sequence in the C-terminal region of VRK1 containing a conserved sequence that is a potential target for Plks. (B) In vitro kinase assay to determine the phosphorylation order between Plk3 and VRK1 and the residue phosphorylated. Plk3 and human VRK1 were expressed in E. coli and purified for the in vitro kinase assay. KE, K179E substitution in VRK1 that is kinase dead. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by Coomassie blue staining (bottom). (C) In vitro kinase assay to demonstrate that VRK1 does not phosphorylate kinase-dead Plk3. pGEX-GST-VRK1, pGEX-GST-p53 (1-82), and pGEX-GST-Plk3K52R were expressed in E. coli. The assay was carried out with a constant amount of GST-VRK1 and increasing amounts (as indicated) of GST-Plk3. GST-p53 was used as a positive control. The phosphorylated proteins were detected by autoradiography (top), and the proteins present in the assay are shown by immunoblotting (middle) or Ponceau staining (bottom). (D) Phos- phorylation of VRK1 by transfected Plk3. HEK293T cells were transfected with pCEFL-GST-Plk3 (5 g), and cell extracts were used for immunoprecipitation with monoclonal anti-Flag antibody (control) or monoclonal anti-GST antibody (Plk3). The immunoprecipitates were used for kinase assays using pGEX-GST-VRK1K179E as a substrate.

Article Snippet: Human Plk3 protein was detected with a monoclonal antibody (clone B37-2) (BD Pharmingen, San Diego, CA) or a polyclonal antibody (10977-1AP) (ProteinTech Group Inc., Chicago, IL).

Techniques: Sequencing, In Vitro, Kinase Assay, Phospho-proteomics, Residue, Autoradiography, Staining, Positive Control, Western Blot, Transfection, Immunoprecipitation, Control

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 2. Expression, interaction, and colocalization of endogenous human VRK1 and Plk3 proteins. (A) The expression of endogenous VRK1 and Plk3 proteins in different cell lines was determined by immunoblotting. Cell lysate (20 g) from each cell line was fractionated in a sodium dodecyl sulfate-(10%) polyacrylamide gel and transferred to an Immobilon-P membrane. VRK1 was detected with the VC1 polyclonal antibody, and Plk3 was detected with a monoclonal antibody. (B) Interaction of endogenous VRK1 and Plk3 proteins in HEK293T cells. One milligram of cellular extract was used for immunoprecipitation of the endogenous VRK1 protein with a monoclonal antibody (1F6) or with a control antibody (monoclonal anti-Flag). The endogenous Plk3 immunoprecipitated was detected with a specific polyclonal antibody for Plk3. IP, immunoprecipi- tation; IB, immunoblot. (C). Colocalization in HEK293T cells. The endogenous VRK1 protein was detected with a specific polyclonal antibody (VE1). HEK293T cells were transfected with 3 g of Plk3 tagged with an HA epitope and detected with a mouse monoclonal antibody specific for the HA tag. Nuclei were identified by DAPI staining. (D). Colocalization in A549 cells. The endogenous VRK1 protein was detected with a specific monoclonal antibody (1F6). A549 cells were transfected with 3 g of Plk3 tagged with a Flag epitope and detected with a polyclonal antibody for the Flag epitope. Nuclei were stained with DAPI. Bar, 50 m.

Article Snippet: Human Plk3 protein was detected with a monoclonal antibody (clone B37-2) (BD Pharmingen, San Diego, CA) or a polyclonal antibody (10977-1AP) (ProteinTech Group Inc., Chicago, IL).

Techniques: Expressing, Western Blot, Membrane, Immunoprecipitation, Control, Transfection, Staining, FLAG-tag

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 3. Stable interaction between Plk3 and VRK1. (A) Reciprocal immunoprecipitation of transfected proteins. HEK293T cells were transfected with empty vector (pCEFL-HA) or plasmid pCEFL-HA-VRK1 (5 g) in combination with pCEFL-Flag-Plk3 (5 g). The cell lysate (top) was used for immunoprecipitation of proteins that bind to either VRK1 or Plk3 with an antibody specific for the corresponding epitope. (Bottom) Detection of the reciprocal proteins was determined by immunoblotting. (B) In vivo interaction of transfected VRK1 and Plk3 proteins. HEK293T cells were transfected with plasmids expressing active (pCEFL-GST-Plk3) and kinase-dead (pCEFL-GST-Plk3K52R) Plk3 in combina- tion with VRK1, either active (pCEFL-HA-VRK1), inactive (pCEFL-HA-VRK1K179E), or nonphosphorylatable by Plk3 (pCEFL-HA- VRK1S342A). Their correct expression was checked in cell lysates (bottom), which were used for a pull-down assay of proteins associated with the GST-Plk3 constructs. (C) Detection of the interaction between endogenous VRK1 and transfected Plk3. HEK293T cells were transfected with plasmid pCEFL-GST-Plk3 (6 g), pCEFL-GST-Plk3K52R (6 g), or pCEFL-GST (2 g) as a control. The expression of the proteins was determined by Western blotting (bottom). The different lysates were used for pull-down assay with glutathione-Sepharose to bring down the GST-Plk3 proteins. The pull-down proteins were detected with anti-VRK1 (1F6) monoclonal antibody. (D) Detection of the interaction between endogenous Plk3 and transfected VRK1. HEK293T cells were transfected with plasmid pCEFL-GST-VRK1 (8 g), pCEFL-GST-VRK1K179E (8 g), or pCEFL-GST (2 g) as a control. The expression of the proteins was determined by Western blotting (bottom). The different lysates were used for a pull-down assay with glutathione-Sepharose to bring down the GST-VRK1 proteins. In the pull-down assay, the endogenous Plk3 protein was detected with a specific antibody.

Article Snippet: Human Plk3 protein was detected with a monoclonal antibody (clone B37-2) (BD Pharmingen, San Diego, CA) or a polyclonal antibody (10977-1AP) (ProteinTech Group Inc., Chicago, IL).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, In Vivo, Expressing, Pull Down Assay, Construct, Control

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 4. Mapping of the region of VRK1 interacting with Plk3. (A) HEK293T cells were transfected with plasmids expressing mammalian GST-VRK1 (FL, full-length; C, C-terminal region) and Plk3 tagged with a Flag epitope. HEK293T cells were cotransfected with constructs of VRK1-FL (8 g) and VRK1-C (6 g) fused to GST and with Flag-Plk3 (3 g). The correct expression levels of proteins were checked in the lysates. The lysates were used for a pull-down assay with glutathione-Sepharose beads, and the associated proteins were detected in Western blots (top). VRK1 constructs were detected with an anti-GST antibody. Plk3 was detected with an anti-Flag antibody. (B) HEK293T cells were transfected with plasmids expressing different regions of human VRK1 tagged with myc and a mammalian construct expressing GST-Plk3. HEK293T cells were cotransfected with constructs of VRK1 fused to myc expressing different regions of the VRK1 protein (pCDNA-myc-VRK1-FL [5 g], pCDNA- myc-VRK1-NL [5 g], and pCDNA-myc-VRK1-NS [9 g]) and with Plk3 (6 g) fused to GST. The lysates were used for a pull-down assay with glutathione-Sepharose beads, and the associated proteins were detected in Western blots (top). GST empty vector was used as a control. Plk3 was detected with an anti-GST antibody. VRK1 was detected with a specific rabbit polyclonal antibody (VE1). (C) Potential region of interaction between VRK1 and Plk3.

Article Snippet: Human Plk3 protein was detected with a monoclonal antibody (clone B37-2) (BD Pharmingen, San Diego, CA) or a polyclonal antibody (10977-1AP) (ProteinTech Group Inc., Chicago, IL).

Techniques: Transfection, Expressing, FLAG-tag, Construct, Pull Down Assay, Western Blot, Plasmid Preparation, Control

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 7. Effect of VRK1 on MEK1-Plk3 fragmentation pathway phenotype. (A) Effects on Golgi fragmentation induced by MEK1. At the bottom is shown the quantification of the effect. (B) Effects on Golgi fragmentation induced by Plk3. HeLa cells were transfected with constructs expressing the indicated proteins. The MEK1 protein is constitutively active. The transfected cells were identified by the use of green fluorescent protein (GFP) as a fluorescent tracer protein or of an antibody against the tag in the transfected protein. The Golgi apparatus was detected with a polyclonal antibody specific for giantin. Bar, 20 m. TC, transfection control with empty vector pEGFP-N1. The quantification of the effects in panels A and B are shown at the bottom of the corresponding column. At least 100 cells from three independent experiments were counted for each type of transfection, and the proportion of cells with fragmented Golgi complexes was determined. In the left column, the reference value is the fragmentation induced by MEK1 (b). In the right column, the reference value is the fragmentation induced by Plk3 (a). The mean values with their standard deviations are represented in the bar graph. *, P 0.05; **, P 0.005.

Article Snippet: Human Plk3 protein was detected with a monoclonal antibody (clone B37-2) (BD Pharmingen, San Diego, CA) or a polyclonal antibody (10977-1AP) (ProteinTech Group Inc., Chicago, IL).

Techniques: Transfection, Construct, Expressing, Control, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: Plk3 Interacts with and Specifically Phosphorylates VRK1 in Ser 342 , a Downstream Target in a Pathway That Induces Golgi Fragmentation

doi: 10.1128/mcb.01341-08

Figure Lengend Snippet: FIG. 8. Knockdown of VRK1 blocks the Golgi fragmentation phenotype induced by either MEK1 or Plk3. (A) Levels of VRK1 endogenous protein in nontransfected cells or cells transfected with siRNA control or with siRNA specific for VRK1 were determined after 5 days. NTC, nontransfected cells. (B) Photograph of HeLa cells transfected with siControl (left) or siVRK1 (right) for 5 days. (C) Immunofluorescence of endogenous VRK1 protein detected with VC polyclonal antibody. The Golgi marker GM130 was used to detect the fragmentation of the Golgi apparatus. Cells were treated with the indicated siControl or specific siVRK1 as well as with MEK1 or Plk3 as an inducer of Golgi fragmentation, as indicated in each row. Five days after siRNA treatment, cells were retransfected with 3 g of constitutively active MEK1(S218/222E, 32–51) or 3 g of Plk3. Fifteen or 13 h after retransfection, cells were fixed and stained with polyclonal VC1 antibody to VRK1 (green) and a specific monoclonal antibody to GM130 (red). Bar, 50 m. Transfected cells were identified by GFP or by an anti-tag antibody. The number of intracellular particles (objects) in transfected cells was analyzed by counting them using the ImageJ program (http://rsb.info.nih.gov/ij; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The mean values with their standard deviations are shown in the bar graph. A minimum of 20 cells were counted in each case.

Article Snippet: Human Plk3 protein was detected with a monoclonal antibody (clone B37-2) (BD Pharmingen, San Diego, CA) or a polyclonal antibody (10977-1AP) (ProteinTech Group Inc., Chicago, IL).

Techniques: Knockdown, Transfection, Control, Marker, Staining