Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Subcellular localization of Luman during osteoclastogenesis. BMMs were infected with a retroviral vector expressing FLAG-tagged Luman. After viral infection, BMMs were cultured with M-CSF for 2 days. Thereafter, infected BMMs were incubated with M-CSF and RANKL for the indicated time periods. Cells were treated with proteasome inhibitor MG132 4 h before fixation, and then were fixed with cold methanol. Immunostaining was performed with antibodies against FLAG and calnexin (CNX), an ER marker. Nuclear counter-staining was conducted with DAPI. At day 2, signals for Luman in the nucleus as well as in the ER were detected (arrows). Scale bar: 20 µm.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Infection, Retroviral, Plasmid Preparation, Expressing, Cell Culture, Incubation, Immunostaining, Marker, Staining

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman mediates the induction of DC-STAMP through CRE-like sequence in the DC-STAMP promoter. (A) Schematic representation of the 0.2-kb promoter region of the murine DC-STAMP gene. AP-1 site (△), NFAT site (□) and CRE-like sequence site (◆) are indicated. TSS, transcription start site. Each CRE-like sequence is indicated. (B) The reporter plasmid of the 0.2-kb murine DC-STAMP promoter fused with the luciferase gene was co-transfected with the pcDNA empty vector (Mock) or Luman N-terminus pcDNA expression vector (Luman-N) into RAW264 cells. Luciferase activity was measured at 24 h after transfection. Data are from three independent experiments. Values indicate mean±s.e.m. † P <0.001. (C) Luciferase reporter assay with a series of deletion mutants of the DC-STAMP promoter reporter plasmids. Reporter plasmids in which the NFAT-binding site or each CRE-like sequence had been deleted from the DC-STAMP promoter region were used. Reporter activities were measured in the same way as described in B. Data are from three independent experiments. Values indicate mean±s.e.m. ** P <0.01; † P <0.001; n.s., not significant. (D) Electrophoretic mobility shift assay. The biotin-labeled probes, including the CRE(2) site of the DC-STAMP promoter, were incubated with nuclear extract derived from FLAG–Luman-N expressing HeLa cells. Note that the binding of FLAG–Luman-N to the CRE(2) site was abolished by a competitor (lane 3), and a supershift after incubation with anti-FLAG antibodies (lane 4) was detected.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Sequencing, Plasmid Preparation, Luciferase, Transfection, Expressing, Activity Assay, Reporter Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Derivative Assay

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman interacts with DC-STAMP and is localized to the Golgi. (A) Co-immunoprecipitation analyses of HeLa cells expressing Luman and DC-STAMP. Cells were co-transfected with expression plasmids for Luman tagged with FLAG at the N-terminus (FLAG–Luman) and DC-STAMP tagged with HA at the C-terminus (DC-STAMP–HA). Cell lysates were immunoprecipitated with antibodies against HA (a-HA), and the immunoprecipitated (IP) samples were subjected to western blotting (WB) with an antibody against FLAG. (B) Subcellular localization of Luman and DC-STAMP that had been expressed in HeLa cells. FLAG–Luman and/or DC-STAMP–HA expression plasmids were transfected into HeLa cells. The transfected cells were immunostained with antibodies against FLAG or HA. Note that co-expression of Luman and DC-STAMP caused the emergence of perinuclear accumulation. Arrows indicate accumulated signals of Luman and DC-STAMP. Scale bars: 10 µm. (C) Double-staining for FLAG and Golgi markers on the cells expressing FLAG–Luman and DC-STAMP–HA. HeLa cells were co-transfected with FLAG–Luman and DC-STAMP–HA expression plasmids. The transfected cells were immunostained with antibodies against FLAG and GM130 (cis-Golgi marker) or against FLAG and TGN46 (trans-Golgi marker). Note that perinuclear accumulation of FLAG–Luman and DC-STAMP–HA overlaps with that of the trans-Golgi marker. Arrows indicate accumulated signals for Luman and DC-STAMP. Scale bars: 10 µm.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Immunoprecipitation, Expressing, Transfection, Western Blot, Double Staining, Marker

Journal: Journal of Cell Science

Article Title: Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

doi: 10.1242/jcs.176057

Figure Lengend Snippet: Luman defines the localization and stabilization of DC-STAMP by interacting with it. (A) Schematic representation of the domain structure of murine DC-STAMP and mapping of the binding region for Luman. Co-immunoprecipitation experiments with various truncated DC-STAMP mutants revealed the binding region in DC-STAMP for Luman. Solid squares with Roman numerals indicate the transmembrane domains of DC-STAMP. Putative binding regions are highlighted in red. The degrees of association between Luman and the DC-STAMP constructs shown are indicated as follows: circle, strong; triangle, weak; cross, none. Numbers represent amino acid residues. (B) Co-immunoprecipitation followed by western blot analysis. HeLa cells were co-transfected with expression plasmids for FLAG–Luman and full-length DC-STAMP [DC-STAMP(full)–HA] or HA-tagged truncated DC-STAMP [DC-STAMP(1-167)–HA]. Cell lysates were immunoprecipitated (IP) with antibodies against HA (a-HA), and the immunoprecipitated proteins were subjected to western blotting with antibodies against FLAG or HA. Note that Luman only interacts with the full-length DC-STAMP. (C) Immunostaining for FLAG–Luman and DC-STAMP–HA. HeLa cells were co-transfected with FLAG–Luman and DC-STAMP(full)–HA or DC-STAMP(1-167)–HA expression plasmids, and immunostained with antibodies against FLAG (green) and HA (red). Upon co-expression of FLAG–Luman and DC-STAMP(full)–HA, both proteins accumulated at the perinuclear region. DC-STAMP(1-167)–HA was scarcely detected in the absence of treatment with MG132. Arrows indicate accumulated signals for Luman and DC-STAMP. Scale bars: 10 µm. (D) Subcellular fractionation analyses of Luman and DC-STAMP. HeLa cells were co-transfected with expression plasmids for FLAG–Luman and DC-STAMP(full)–HA or DC-STAMP(1-167)–HA. The microsome membranes from cell lysates were ultracentrifuged and fractionated in an iodixanol gradient. Each fraction was subjected to western blotting. TGN46, GM130 and calnexin (CNX) were examined as specific intracellular markers for the trans-Golgi, cis-Golgi and ER, respectively. TGN46 is mainly distributed in fractions 2 and 3; GM130 in fractions 4–6; CNX in fractions 7–9.

Article Snippet: For supershift experiments, samples were treated with an anti-FLAG antibody (Rockland) at 4°C for 1 h before incubation with a biotin-labeled probe.

Techniques: Binding Assay, Immunoprecipitation, Construct, Western Blot, Transfection, Expressing, Immunostaining, Fractionation

Journal: PloS one

Article Title: The apoptotic function analysis of p53, Apaf1, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis.

doi: 10.1371/journal.pone.0039920

Figure Lengend Snippet: Figure 10. Immunofluorescence analysis of spontaneous apoptosis and stress induced apoptosis in newt early spermatids. The apoptotic germ cells were determined by TUNEL kit (red signal) (Beyotime, China) and the nucleus were dyed with DAPI (Blue signal). Apoptotic sperms were found in normal newt testis (A1-3), and severer mature sperm apoptosis detected under various stress including starvation (B1-3), cold exposure(4uC for 12 h) (C1-3), cadmium exposure (5 mg/Kg body weight for 36 h) (D1-3) and heat exposure (40uC for 2 h) (E1-3). The scale bar is 10 mm. doi:10.1371/journal.pone.0039920.g010

Article Snippet: The positive control was prepared by Positive TUNEL preparation kit (Beyotime, China), in which the sections were treated with DNase I for 30 min at 25uC and PBS wash before TUNEL labeling reaction.

Techniques: Immunofluorescence, TUNEL Assay

Journal: PloS one

Article Title: The apoptotic function analysis of p53, Apaf1, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis.

doi: 10.1371/journal.pone.0039920

Figure Lengend Snippet: Figure 11. Immunofluorescence analysis of spontaneous apoptosis and stress induced apoptosis in newt mature sperms. The apoptotic germ cells were determined by TUNEL kit (Beyotime, China) and the nucleus were dyed with DAPI. Apoptotic sperms were found in normal newt testis (A1-3), and severer mature sperm apoptosis detected under various stress including starvation (B1-3), cold exposure(4uC for 12 h) (C1-3), cadmium exposure (5 mg/Kg body weight for 36 h) (D1-3) and heat exposure (40uC for 2 h) (E1-3). The scale bar is 10 mm. doi:10.1371/journal.pone.0039920.g011

Article Snippet: The positive control was prepared by Positive TUNEL preparation kit (Beyotime, China), in which the sections were treated with DNase I for 30 min at 25uC and PBS wash before TUNEL labeling reaction.

Techniques: Immunofluorescence, TUNEL Assay

Journal: PloS one

Article Title: The apoptotic function analysis of p53, Apaf1, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis.

doi: 10.1371/journal.pone.0039920

Figure Lengend Snippet: Figure 12. Immunofluorescence analysis on apoptosis. No apoptosis occurred to spermatogonia in normal newt testis and stress treated newt. The apoptotic germ cells were determined by TUNEL kit (red signal) (Beyotime, China) and the nucleus were dyed with DAPI (Blue signal). No apoptotic spermatogonia were found in normal newt testis (A) and stress treated newts (B) while severe apoptotic signal was in spermatogonia in the testis treated with DNase I in the TUNEL positive preparation Kit (Beyotime, China). The scale bar is 10 mm. doi:10.1371/journal.pone.0039920.g012

Article Snippet: The positive control was prepared by Positive TUNEL preparation kit (Beyotime, China), in which the sections were treated with DNase I for 30 min at 25uC and PBS wash before TUNEL labeling reaction.

Techniques: Immunofluorescence, TUNEL Assay

Journal: Aging and disease

Article Title: Aging-Induced Brain-Derived Neurotrophic Factor in Adipocyte Progenitors Contributes to Adipose Tissue Dysfunction

doi: 10.14336/ad.2019.0810

Figure Lengend Snippet: Figure 5. proBDNF treatment-induced apoptosis/necroptosis of adipocytes required sortilin expression. (A) Immunoblot analysis of sortilin expression and apoptosis/necroptosis markers in adipocytes differentiated from C3H10T1/2 cells. (B) Immunoblot analysis of sortilin in adipocytes differentiated from C3H10T1/2 treated with siRNA or scrambled sequence controls (negative controls) (mean ± SEM; n = 4, *** p < 0.001). (C) Immunoblot analysis of cell surface protein detection in adipocytes differentiated from C3H10T1/2 cells treated with vehicle or pro-BDNF (10ng/ml) for 24 h (n = 4, means ± SEM, *** p < 0.001). Full images of Western blots are shown in supplementary Fig. 5.

Article Snippet: Fully differentiated adipocytes were exposed to DMEM supplemented with 10% FBS overnight and then treated with pro-BDNF (10ng/ml, Alomone Labs) [27, 28].

Techniques: Expressing, Western Blot, Sequencing

Journal: Nucleic Acids Research

Article Title: The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability

doi: 10.1093/nar/gky008

Figure Lengend Snippet:

Article Snippet: shCNTRL , 5′-CACAAGCTGGAGTACAACTACAACAGCCA-3′ , OriGene (TR30001).

Techniques: Sequencing