Journal: Cancer Immunology, Immunotherapy : CII

Article Title: LincRNA-p21 knockdown reversed tumor-associated macrophages function by promoting MDM2 to antagonize* p53 activation and alleviate breast cancer development

doi: 10.1007/s00262-020-02511-0

Figure Lengend Snippet: LincRNA-p21 down-regulated TAMs promoted tumor cells apoptosis, inhibited their migration and invasion. a, e LincRNA-p21 down-regulated TAMs promoted tumor cell apoptosis. 4T1 and LLC were co-cultured with lincRNA-p21 down-regulated macrophages for 48 h, respectively. The tumor cells were collected and stained with Annexin-V and 7AAD to detect the apoptosis using flow cytometry, respectively. b LincRNA-p21 down-regulated TAMs inhibited 4T1 cell proliferation. The proliferation of 4T1 cells co-cultured with lincRNA-p21 down-regulated macrophages was measured using Cell Counting Kit-8 (CCK-8) at indicated point. c, f LincRNA-p21 down-regulated TAMs inhibited tumor cells migration. Transwell assays were performed to detect migration ability of tumor cells (4T1 and LLC) co-cultured with lincRNA-p21 down-regulated TAMs for 24 h. The left panels show representative images and right panels show statistical analysis. d LincRNA-p21 down-regulated TAMs inhibited 4T1 cells invasion. Wound-healing assay was used to detect the invasive ability of 4T1 cells co-cultured with lincRNA-p21 down-regulated TAMs. g LincRNA-p21 knockdown could not increase ROS production in TAMs. The left panels show representative images and right panels show statistical analysis. h The expression of Fas on 4T1 cell and FasL on TAMs was assessed by flow cytometry. i R7050 (10 nM) was used to pretreat 4T1 for 6 h and after that 4T1 was co-cultured with lincRNA-p21 down-regulated macrophages for 48 h; 4T1 cells were collected and stained with Annexin V and PI to detect apoptosis. All the data were obtained from three independent experiments and the representative images are shown. *P<0.05 relative to control. N.S. means no significance. LLC and SLP1 mean Lewis lung carcinoma cells and lincRNA-p21 downregulated macrophages

Article Snippet: For cellular apoptosis assay, cells were stained with Annexin-V-APC and PI using the Cell Apoptosis Analysis Kit (MultiSciences (Lianke) Biotech Co., Ltd, Hangzhou, China) according to the manufacturer’s instructions. (AnnexinV-APC) − /PI + cells were considered as necrosis, (AnnexinV-FITC) + /PI- and (AnnexinV-FITC) + /PI + cells were considered as apoptosis.

Techniques: Migration, Cell Culture, Staining, Flow Cytometry, Cell Counting, CCK-8 Assay, Wound Healing Assay, Knockdown, Expressing, Control

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: STAR Protocols

Article Title: Rapid and efficient enhancer cloning and in vivo screening using the developing chick embryo

doi: 10.1016/j.xpro.2021.100507

Figure Lengend Snippet: Nanostring screening of putative enhancers (A) Nanotag transcripts are detected by a customized Nanostring codeset , counts above 100 (in green, above dashed line) were determined to depict in vivo enhancer activity. (B) Negative control plasmids achieved Nanostring counts less than 100. In vivo background expression is observed in the posterior extra-embryonic region. (C) Enhancers with Nanostring count >100 are re-electroporated into early chick embryos at higher concentration such that fluorescence can be observed. Multiple enhancers carrying different fluorophores can be imaged in parallel, as shown for neural crest enhancers; NC1, NC2 and Sox10E2 ( Betancur et al., 2010 ; Simoes-Costa et al., 2012 ). Figure adapted and reprinted with permission from ( Williams et al., 2019 ).

Article Snippet: We provide a number of Nanotag reporter plasmids containing the FoxD3 neural crest enhancer (NC1) ( ) as positive control (Addgene #130570 - #130573).

Techniques: In Vivo, Activity Assay, Negative Control, Expressing, Concentration Assay, Fluorescence

Journal: PLoS Genetics

Article Title: Initial Genomics of the Human Nucleolus

doi: 10.1371/journal.pgen.1000889

Figure Lengend Snippet: (A) Histograms show the frequency of the nucleolar localisation of NADs and control chromosomal regions detected by 3D FISH in HeLa cervix carcinoma and IMR90 diploid fibroblast cells. Percentage of nucleolus-associated alleles is shown on the left. Red diamond indicates target, green ones negative controls, whereas yellow diamond indicates the chromosome X pericentromeric and blue diamond the 5S cluster positive controls, respectively (see for further BAC details). Single light optical sections of HeLa nuclei are shown on the right. BAC hybridization signals of RP11-90G23 target, RP5-915N17 positive control and RP11-81M8 negative control BACs are shown in green, nucleolar staining in red and DAPI counterstain in blue (scale bars: 5 µm). (B) α-H3K27Me3, α-centromere, α-active Pol II and α-B23/nucleophosmin immunostaining of HeLa and IMR90 cells. α-H3K27Me3, α-centromere and α-active Pol II signals are shown in green, nucleolar staining in red and DAPI counterstain in blue (scale bars: 5 µm).

Article Snippet: In localisation experiments α-B23/nucleophosmin (Sigma, B0556), α-H3K27Me3 (Upstate, 07-449), α-active Pol II (Covance, MMS-129R), α-centromere (Antibodies Inc., 15–134) and different fluorescence dye-conjugated secondary antibodies, furthermore BAC clones RP11-90G23, RP11-173M10, RP11-44B13, RP11-89H10, RP11-413F20, RP11-81M8, RP5-915N17, RP11-1137G4, RP11-89O2, RP11-123G19 and RP11-434B14 were used on HeLa cervix carcinoma cells and IMR90 lung embryonic fibroblasts.

Techniques: Control, Hybridization, Positive Control, Negative Control, Staining, Immunostaining

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: bioRxiv

Article Title: Integrated transcriptomics and proteomics define the TRP channel hierarchy in mouse cortex

doi: 10.64898/2026.04.07.716663

Figure Lengend Snippet: ( A ) Proteomics workflow. Tissues from adult mice were homogenised and fractionated into cytosolic, membrane-enriched, and insoluble fractions. Proteins were solubilised using detergent- or urea-based chemistries, enzymatically digested, cleaned up using FASP, SP3, or StageTip workflows, and analysed by LC–MS/MS. Sample labels used throughout (SN, Control, Pellet and Pelletpellet) correspond to cytosolic, crude membrane, urea-soluble membrane, and urea-insoluble membrane fractions, respectively (see Supplementary Fig. 7; terminology in Supplementary Table 5). ( B ) Missing-value map with unsupervised hierarchical clustering (black = observed; white = missing). Samples cluster by protocol, indicating high within-protocol consistency and distinct missingness profiles across procedures. ( C ) Proteins identified per sample; membrane-enriching inputs (FASP-Pellet, Pelletpellet) yield more identifications than supernatant (SN) or gel-only preparations; replicate variability is low ( n = 3 per condition). ( D ) PCA of the top 500 variable proteins. Replicates cluster tightly by protocol and separate along PC1/PC2, indicating reproducible and protocol-specific proteome signatures. ( E ) TRP-focused log₁₀-transformed protein intensities [log₁₀(intensity + 1)] heatmap showing evidence for TRP channels (reproducible detection criteria were met for TRPV2, TRPC4, TRPM3, TRPM7 and TRPP2) across protocols. FASP-based and pellet-derived inputs gave the strongest TRP signals. QC: 1% precursor- and protein-group–level detection FDR (DIA-NN) with downstream DEP filtering and imputation.

Article Snippet: Purified Xenoblot-positive control proteins for TRPA1 and TRPV1 were obtained from Alomone Labs (TRPA1 Xenoblot-positive control, Cat. No. XB-010; TRPV1 Xenoblot-positive control, Cat. No. XB-008).

Techniques: Membrane, Liquid Chromatography with Mass Spectroscopy, Control, Transformation Assay, Derivative Assay, Protein Extraction, Tandem Mass Spectroscopy

Journal: bioRxiv

Article Title: Integrated transcriptomics and proteomics define the TRP channel hierarchy in mouse cortex

doi: 10.64898/2026.04.07.716663

Figure Lengend Snippet: ( A ) Bulk cortex TaqMan qPCR showing mean Ct values for Trpa1 and Trpv1 in adult mouse cortex. Both transcripts are detected only at high Ct values. Red dashed line indicates Gapdh Ct. ( B ) TaqMan qPCR of FACS-sorted cortical cell populations showing mean Ct values by fraction: neurons (blue), astrocytes (orange), and NeuN⁺/ACSA2⁺ double-positive cells (grey). Dashed lines indicate fraction-specific Gapdh Ct. “‡” indicate no detectable amplification reactions. Trpv1 is neuron-only at high Ct; Trpa1 is rare/borderline. ( C, D ) Subcellular fractionation and immunoblotting for TRPA1 and TRPV1, respectively. Cortex lysates (n = 3) were separated into supernatant (S) and membrane pellet (P) fractions; Ladder (L). Faint immunoreactive bands near the expected molecular weight (∼130–140 and ∼95 kDa, respectively) are preferentially enriched in pellet fractions. GAPDH (∼37 kDa) was used as a loading control. Kidney, DRG, and testes processed in parallel serve as positive control tissues. Note: Lysates and controls were processed in the same experiment and run on the same gels/blots. ( E, F ) Immunoprecipitation (IP) from cortex (C) and DRG (D) lysates followed by SDS–PAGE/Western blotting and LC–MS/MS. As shown in Supplementary Fig. 12f, no TRPA1 peptides were detected, and only extremely low-intensity TRPV1 protein-group signals were observed without replicate consistency.

Article Snippet: Purified Xenoblot-positive control proteins for TRPA1 and TRPV1 were obtained from Alomone Labs (TRPA1 Xenoblot-positive control, Cat. No. XB-010; TRPV1 Xenoblot-positive control, Cat. No. XB-008).

Techniques: Amplification, Cell Fractionation, Western Blot, Membrane, Molecular Weight, Control, Positive Control, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Real-time Polymerase Chain Reaction, Co-Immunoprecipitation Assay