Journal: Cell reports

Article Title: In Situ Fixation Redefines Quiescence and Early Activation of Skeletal Muscle Stem Cells.

doi: 10.1016/j.celrep.2017.10.080

Figure Lengend Snippet: Figure 1. Isolation of MuSCs in a Fixed, G0-Arrested State (A) Graphical scheme of the in situ fixation protocol for MuSCs isolation and comparison to the standard protocol. A detailed description of the protocols is available in the Supplemental Information. BM, basement membrane; T0-SC, time-zero/quiescent MuSC; T3-SC, time 3 hr/activated MuSC. (B) FACS profiles of non-fixed (T3-SC) and fixed (T0-SC) GFP+ MuSCs from Tg:Pax7-nGFP muscle preparations. Sorted GFP+ cells are marked as red dots in all plots. SSC, side scatter, FSC, forward scatter. Values on the plots indicate mean percentage of sorted GFP+ cells of the total number of events, excluding small SSC/FSC and doublets; n = 5. (C) Proliferation of FACS-isolated T3-SCs and T0-SCs cultured for 48 hr and stained with EdU (24-hr chase); n = 3. EdU+ cells: 0% for T0-SCs and 74% ± 0.03% for T3-SCs. (D) Nascent RNA synthesis in FACS-isolated T3-SCs and T0-SCs cultured for 48 hr and incubated with labeled ethynyl uridine (EU) ribonucleoside for 2 hr; n = 3. Average of 80 nuclei counted per sample, 100% EU+ and EU for T3-SC and T0-SC, respectively. Dotted line delineates cell’s nucleus. (E) Morphology of T3 and T0 MuSCs immediately after the FACS. Cells were spun on Matrigel-coated slides, and membranes were stained with the lectin marker WGA (wheat germ agglutinin). Mean eccentricity of the cells was computationally calculated (Experimental Procedures). Data are reported as mean ± SD; n.d, not detected. Scale bars, 90 mm in (C), 20 mm in (D), 8 mm in (D, insets), and 10 mm in (E). See also Supplemental Information.

Article Snippet: The purity of the FACS-isolated cells was assessed with anti-PAX7 antibody (mouse monoclonal; Santa Cruz Biotechnology, #sc-81648) diluted 1:100 in BS and incubated for 16 hr at 37 C. For the membrane staining, WGA was used following manufacturer’s guidelines (Thermo Fisher Scientific, #W32464) on T0- or T3-SCs after a 15-min centrifugation (300 3 g, 4 C) on Matrigel (Corning, #354248)-coated 8-chamber slides (Sarstedt, #94.6140.802).

Techniques: Isolation, In Situ, Comparison, Membrane, Cell Culture, Staining, Incubation, Labeling, Marker

Journal: Cell reports

Article Title: In Situ Fixation Redefines Quiescence and Early Activation of Skeletal Muscle Stem Cells.

doi: 10.1016/j.celrep.2017.10.080

Figure Lengend Snippet: Figure 5. Histone H3 Modifications and DNA Methylation during Early MuSCs Activation (A) Left: H3K4me3 ChIP-seq averaged signal at promoters. Right: overlap between T0-SC and T3-SC genes with H3K4me3 peaks at promoters. TSS, tran- scription start site. (B) Left: H3K27ac ChIP-seq averaged signal at active distal enhancers (H3K27ac peaks outside gene body) from T0-SC and T3-SC samples. Right: overlap of genes that present at least one distal enhancer between T0-SCs and T3-SCs. (C) Examples of H3K4me3 and H3K27ac ChIP-seq tracks from T3 and T0 upregulated genes Fosl and Egr3. (D) Examples of H3K4me3 and H3K27ac ChIP-seq tracks from T3 and T0 downregulated genes Hes1 and Pax7. (E) H3K27me3 ChIP-seq averaged signal at promoters between T0-SCs, T3-SCs, and activated MuSCs from a published dataset (Liu et al., 2013). (F) Volcano plot representing genomic DNA methylation changes between T0-SCs and T3-SCs. Each dot represents one of 27,878 CpG clusters interrogated. Among all clusters (blue), those with a methylation change R 10% and an FDR < 0.01 were considered significantly different (red). FDR, false discovery rate (logarithmic scale).

Article Snippet: The purity of the FACS-isolated cells was assessed with anti-PAX7 antibody (mouse monoclonal; Santa Cruz Biotechnology, #sc-81648) diluted 1:100 in BS and incubated for 16 hr at 37 C. For the membrane staining, WGA was used following manufacturer’s guidelines (Thermo Fisher Scientific, #W32464) on T0- or T3-SCs after a 15-min centrifugation (300 3 g, 4 C) on Matrigel (Corning, #354248)-coated 8-chamber slides (Sarstedt, #94.6140.802).

Techniques: DNA Methylation Assay, Activation Assay, ChIP-sequencing, Methylation

Journal: PLoS Neglected Tropical Diseases

Article Title: Diagnostic accuracy of serological tests for the diagnosis of Chikungunya virus infection: A systematic review and meta-analysis

doi: 10.1371/journal.pntd.0010152

Figure Lengend Snippet: Characteristics of studies on IgM detection tests included in the meta-analysis.

Article Snippet: On-site CHIK IgM Combo Rapid test , CTK Biotech Inc., San Diego, CA, USA , 3 , 145 , 27.9 [10.8; 55.2] , 81.0% [40.5; 93.9] , 90.4 , 98.7 [84.9; 99.9] , 0.0% [0.0; 0.0] , 98.

Techniques: Enzyme-linked Immunosorbent Assay, Indirect ELISA, HI Assay, Plaque Reduction Neutralization Test, Neutralization

Journal: PLoS Neglected Tropical Diseases

Article Title: Diagnostic accuracy of serological tests for the diagnosis of Chikungunya virus infection: A systematic review and meta-analysis

doi: 10.1371/journal.pntd.0010152

Figure Lengend Snippet: Subgroup analysis for commercial tests.

Article Snippet: On-site CHIK IgM Combo Rapid test , CTK Biotech Inc., San Diego, CA, USA , 3 , 145 , 27.9 [10.8; 55.2] , 81.0% [40.5; 93.9] , 90.4 , 98.7 [84.9; 99.9] , 0.0% [0.0; 0.0] , 98.

Techniques: Enzyme-linked Immunosorbent Assay, Virus, Immunofluorescence

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: Tug1 in tumor cells regulates the antitumor immune response of CD8 + T cells and phagocytosis of macrophages through PD‐L1 and CD47, respectively. A) The correlation of TUG1 with PD‐L1 and CD47 in HCC patients ( n = 371). B) The correlation of METTL3 with PD‐L1 and CD47 in HCC patients ( n = 371). C) The expressions of PD‐L1 and CD47 at the protein and mRNA levels in the control and sh‐METTL3 HepG2 cells. D) The expressions of Pdl1 and Cd47 at the protein and mRNA levels in the control and sh‐Mettl3 Hepa1‐6 cells. E) Representative images of IHC staining and mean intensities of PD‐L1 and CD47 ( n = 5. Magnification: 40 ×). F–H) The frequencies of IFN‐γ, TNF‐α, and GzmB in CD8 + T cells co‐cultured with sh‐NC or sh‐Tug1 Hepa1‐6 cells ( n = 3). I) Representative images from immunofluorescence (IF) staining of peritoneal cavity‐derived macrophages and BMDMs engulfing cancer cells. The white arrows indicate macrophages that phagocytose cancer cells. Macrophages are shown in red (F4/80 + ), cancer cells are shown in green (GFP + ) and nuclei are shown in blue (DAPI). Magnification: 100 ×. J, K) Statistical analysis of phagocytosis by macrophages as detected via IF staining ( n = 3). L, M) Representative plots and statistical analysis of phagocytosis by macrophages derived from the peritoneal cavity and bone marrow as detected using a flow cytometer ( n = 3). Results are represented as the mean ± SEM. Data were statistically analyzed using unpaired two‐tailed Student's t‐test (C‐H, J‐M) or Spearman's correlation analysis (A‐B).*, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Control, Immunohistochemistry, Cell Culture, Immunofluorescence, Staining, Derivative Assay, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: Tug1 acts as a microRNA sponge to promote PD‐L1 and CD47 expressions, thereby regulating the antitumor immune response of CD8 + T cells and phagocytosis of macrophages. A) The luciferase reporter assays verify the interaction sites of miR‐141 with Tug1 and Pdl1 ( n = 3). B) The expressions of Tug1, miR‐141, and Pdl1 in the control and si‐Tug1‐transfected Hepa1‐6 cells. C) The expression of miR‐141 and Pdl1 in the control and miR‐141 mimic‐transfected Hepa1‐6 cells. D) The production frequency of IFN‐γ in CD8 + T cells co‐cultured with miR‐NC or miR‐141‐overexpressed Hepa1‐6 cells ( n = 3). E) Representative images of tumors, tumor growth curves, and tumor weights from mice bearing miR‐NC and miR‐141‐overexpressed Hepa1‐6 cells bearing mice ( n = 5). F) Immunohistochemistry (IHC) staining and mean intensity of Pdl1 ( n = 5. Magnification: 40 ×). G) The secretion of IFN‐γ in CD8 + T cells from the tumors of mice bearing miR‐NC and miR‐141‐overexpressed Hepa1‐6 cells ( n = 5). H) The luciferase reporter assays verified the interaction sites of miR‐340 with Tug1 and Cd47 ( n = 3). I) The expressions of miR‐340 and Cd47 in the control and si‐Tug1‐transfected Hepa1‐6 cells. J) The expressions of miR‐340 and Cd47 in the control and miR‐340 mimic‐transfected Hepa1‐6 cells. K) Representative plots of flow cytometry and statistical analysis of F4/80 + macrophages phagocytosing miR‐NC or miR‐340‐overexpressed Hepa1‐6 cells ( n = 3). L) Representative images of tumors, tumor growth curves, and tumor weights from mice bearing miR‐NC and miR‐340‐overexpressed Hepa1‐6 cells ( n = 5). M) Immunohistochemistry (IHC) staining of Cd47 ( n = 5. Magnification: 40 ×). N) The frequency of M1‐like macrophages in the tumors obtained from mice bearing miR‐NC and miR‐340‐overexpressed Hepa1‐6 cells ( n = 4). O) The expression of miR‐340 and miR‐141 were upregulated in sh‐Tug1 Hepa1c1c7 cells. P) Pdl1 and Cd47 expression at both mRNAs and protein levels in sh‐Tug1 Hepa1c1c7 cells. Results are represented as the mean ± SEM. Data were statistically analyzed using unpaired two‐tailed Student's t‐test or two‐way ANOVA (E, L). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Luciferase, Control, Transfection, Expressing, Cell Culture, Immunohistochemistry, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: Tug1 interacts with Ybx1 to transcriptionally regulate Pdl1 and Cd47, thereby regulating the antitumor immune response of CD8 + T cells and phagocytosis of macrophages. A) Hepa1‐6 cells were used for CHIRP assay. The enrichment efficiency of Tug1 probes is confirmed via qPCR and electrophoresis. B) Silver staining of Tug1‐associated proteins from CHIRP. C) Enrichment of Ybx1 protein in the pull‐downs of both “odd” and “even” probes targeting Tug1 relative to LacZ probes. D) RIP‐qPCR shows enrichment of Tug1 after immunoprecipitation of Ybx1. E) The tertiary structure of TUG1 was docked with the YBX1 protein, the intermolecular interaction details were presented. F, G) The correlation of YBX1 with PD‐L1 and CD47 in HCC patients ( n = 371). H) Pdl1 and Cd47 expressions in the control and Ybx1 siRNAs‐transfected Hepa1‐6 cells at protein and mRNA levels. I) Pdl1 and Cd47 expression in sh‐NC and sh‐Ybx1 Hepa1c1c7 cells at protein and mRNA levels. J) CHIP‐qPCR verifies the enrichment of YBX1 in the promoter regions of PD‐L1 and CD47. K) The CHIP‐qPCR results showed a reduction in the recruitment of YBX1 in the promoter regions of PD‐L1 and CD47 when TUG1 is downregulated. L) Representative plots and statistical analysis of macrophages phagocytosing sh‐NC or sh‐Ybx1 Hepa1‐6 cells ( n = 3). M, N) Representative images of tumors, tumor growth curves, and tumor weights from sh‐NC and sh‐Ybx1 Hepa1‐6 cells tumor‐bearing mice ( n = 5). O) The secretion of cytokines in CD8 + T cells from the tumors of sh‐NC and sh‐Ybx1 Hepa1‐6 cell‐bearing mice ( n = 5). P, Q) The immunohistochemistry (IHC) staining and mean intensities of Ybx1, Pdl1 and Cd47. ( n = 5. Magnification: 40 ×). Results are represented as the mean ± SEM. Statistical analysis was performed using the unpaired two‐tailed Student's t‐test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Electrophoresis, Silver Staining, Immunoprecipitation, Control, Transfection, Expressing, ChIP-qPCR, Immunohistochemistry, Two Tailed Test

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: TUG1, but not TUG1‐ORF, regulates the expressions of PD‐L1 and CD47 in liver cancer cells. A) The expressions of miR‐141 and miR‐340 are upregulated in TUG1‐downregulated HepG2 cells. B) PD‐L1 and CD47 expressions at both the mRNAs and protein levels in TUG1‐downregulated HepG2 cells. C) The expression of miR‐141 and PD‐L1 in the control and miR‐141 over‐expressed HepG2 cells. D) The expressions of miR‐340 and CD47 in the control and miR‐340 over‐expressed HepG2 cells. E) The expressions of miR‐141 and miR‐340 are upregulated in TUG1‐downregulated LM3 cells. F) PD‐L1 and CD47 expressions at both the mRNAs and protein levels in TUG1‐downregulated LM3 cells. G) The expression of miR‐141 and PD‐L1 in the control and miR‐141 over expressed LM3 cells. H) The expressions of miR‐340 and CD47 in the control and miR‐340 over expressed LM3 cells. I) PD‐L1 and CD47 expressions at mRNA levels in YBX1‐downregulated HepG2 cells. J) PD‐L1 and CD47 expressions at mRNA levels in YBX1‐downregulated LM3 cells. K) PD‐L1 and CD47 expressions at protein levels in YBX1‐downregulated HepG2 cells and LM3 cells. L) Pdl1 and Cd47 expressions at both the mRNAs and protein levels in Tug1 full length (Tug1‐FL)‐overexpressed Hepa1‐6 cells. M) The frequencies of IFN‐γ in CD8 + T cells co‐cultured with NC and Tug1‐FL Hepa1‐6 cells ( n = 3). N) Representative plots and statistical analysis of macrophages phagocytosing NC or Tug1‐FL Hepa1‐6 cells ( n = 3). O) Pdl1 and Cd47 expressions at both the mRNAs and protein levels in Tug1‐ORF‐overexpressed Hepa1‐6 cells. P) Representative images of tumors, tumor growth curves, and tumor weights from mice bearing NC and Tug1‐ORF‐overexpressed Hepa1‐6 cells tumor‐bearing mice ( n = 5). Results are represented as the mean ± SEM. Statistical analysis was performed using the unpaired two‐tailed Student's t‐test.*, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Expressing, Control, Cell Culture, Two Tailed Test

Journal: Advanced Science

Article Title: M 6 A‐mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8 + T cells and phagocytosis of macrophages

doi: 10.1002/advs.202400695

Figure Lengend Snippet: TUG1 is a potential biomarker and immunotherapeutic target for liver cancer. A) Expression of TUG1 in 40 pairs of clinical specimens of both normal and tumor tissues was determined by qRT‐PCR. B) Correlative analysis of the relative expression of TUG1 with PD‐L1 in human HCC tumors ( n = 40). C) Correlative analysis of the relative expression of TUG1 with CD47 in human HCC tumors ( n = 40). D) Correlative analysis of the relative expression of TUG1 with YBX1 in human HCC tumors ( n = 40). E) Correlative analysis of the relative expression of YBX1 with PD‐L1 in human HCC tumors ( n = 40). F) Correlative analysis of the relative expression of YBX1 with CD47 in human HCC tumors ( n = 40). G) Schematic of Tug1 siRNAs therapy in combination with an anti‐Pdl1 antibody. H–J) Representative images of tumors, tumor volumes, and tumor weights ( n = 3). K) Schematic of the molecular mechanism of TUG1 in regulating antitumor immune response. Results are represented as the mean ± SEM. Statistical analysis was performed using the unpaired two‐tailed Student's t‐test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti‐ Pdl1 antibodies (Clone No.10F.9G2, BioXcell) or IgG isotype control were given intraperitoneally at 200 μg/day every 4 days.

Techniques: Biomarker Discovery, Expressing, Quantitative RT-PCR, Two Tailed Test