Journal: Science Advances

Article Title: Blood-derived APLP1 + extracellular vesicles are potential biomarkers for the early diagnosis of brain diseases

doi: 10.1126/sciadv.ado6894

Figure Lengend Snippet: ( A ) mRNA profiles of APLP1 and L1CAM across different mouse organs. ( B and C ) Relative mRNA expression levels quantified via reverse transcription qPCR, normalized to glyceraldehyde phosphate dehydrogenase ( GAPDH ). Data are presented as the means ± SEM of eight independent experiments, and statistical analysis was performed on 2 Δ C t values using the analysis of variance (ANOVA; post hoc: Tukey). Symbol “***” indicates a value of < 0.001, respectively, compared with the brain. ( D and E ) Western blot representation (D) and immunofluorescence imaging (E) of L1CAM and APLP1 across varied tissues derived from five C57BL/6 mice. ( F ) Use of RNAscope and IHC to detect APLP1 colocalization in mouse cerebral cortex cells. The mRNA of APLP1 is visualized in red, while oligodendrocytes (Olig2), neurons (NeuN), astrocytes (GFAP), or microglial cells (Iba-1) are visualized in green fluorescence. Scale bars, 50 μm. The experiment was performed with three C57BL/6 mice. n.s., not significant; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The antibodies used for the ELISA were as follows: antihuman CD63 (1:1500 dilution; Ancell, catalog no. 215-820), antihuman CD9 (1:1500 dilution; BD Biosciences, catalog no. 555370; RRID: AB_395772), antihuman CD81 (1:1500 dilution; BD Biosciences, catalog no. 555675; RRID: AB_396028), antihuman L1CAM (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA, catalog no. 89861S; RRID: AB_2800145), antihuman APLP1 (1:500 dilution; Abcam, catalog no. ab94957; RRID: AB_10890629), antimouse immunoglobulin G (IgG) secondary HRP (1:500 dilution; Cell Signaling Technology, catalog no. 7076S; RRID: AB_330924), and antirabbit IgG secondary HRP (1:500 dilution; Cell Signaling Technology, catalog no. 7074S; RRID: AB_2099233).

Techniques: Expressing, Reverse Transcription, Western Blot, Immunofluorescence, Imaging, Derivative Assay, RNAscope, Fluorescence

Journal: Science Advances

Article Title: Blood-derived APLP1 + extracellular vesicles are potential biomarkers for the early diagnosis of brain diseases

doi: 10.1126/sciadv.ado6894

Figure Lengend Snippet: ( A ) Enzyme-linked immunosorbent assay (ELISA)–derived absorbance units representing the levels of EV markers (CD9, CD81, and CD63) and key antigens (EGFR, EGFRviii, L1CAM, and APLP1) within plasma EVs from the healthy group and the GBM patient group. Data represent means ± SEM of three independent experiments. ( B ) Representative immunostaining images contrasting plasma EVs from healthy individuals and patients with GBM ( n = 3). The white arrowheads indicates CD63 + APLP1 + EVs. Scale bars, 5 μm. ( C ) Quantification of CD63 + APLP1 − EVs and CD63 + APLP1 + EVs in each group ( n = 3). ( D ) Fold change ratio of CD63 + APLP1 − EVs and CD63 + APLP1 + EVs between the healthy and GBM groups ( n = 3). Data represent means ± SEM of three independent experiments. Statistical analysis was conducted using the ANOVA test (post hoc: Tukey). ** P < 0.01 and *** P < 0.001, statistical differences.

Article Snippet: The antibodies used for the ELISA were as follows: antihuman CD63 (1:1500 dilution; Ancell, catalog no. 215-820), antihuman CD9 (1:1500 dilution; BD Biosciences, catalog no. 555370; RRID: AB_395772), antihuman CD81 (1:1500 dilution; BD Biosciences, catalog no. 555675; RRID: AB_396028), antihuman L1CAM (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA, catalog no. 89861S; RRID: AB_2800145), antihuman APLP1 (1:500 dilution; Abcam, catalog no. ab94957; RRID: AB_10890629), antimouse immunoglobulin G (IgG) secondary HRP (1:500 dilution; Cell Signaling Technology, catalog no. 7076S; RRID: AB_330924), and antirabbit IgG secondary HRP (1:500 dilution; Cell Signaling Technology, catalog no. 7074S; RRID: AB_2099233).

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Clinical Proteomics, Immunostaining

Journal: bioRxiv

Article Title: Alteration of nociceptive Schwann cells in a mouse model of peripheral neuropathy in prediabetic condition

doi: 10.1101/2024.03.12.584541

Figure Lengend Snippet: (A) PGP9.5 immunofluorescence with DAPI staining in foot skin section of control (n=5) and HFD-fed mice (n=5). (B) Quantitation of IENFD is presented as the number of fibers/mm of epidermis. (C) L1CAM immunofluorescence with DAPI staining in foot skin section of control (n=5) and HFD-fed mice (n=5). Arrowhead indicate nerve fibers in the epidermis of the foot skin. Arrows indicate nociceptive Schwann cells and their cellular extensions at the border of the epidermis and the dermis. (D) Mean fluorescence intensity quantification of L1CAM immunostaining at the localization of nociceptive Schwann cells (at the border of epidermis and dermis). (E) Quantification of L1CAM-positive cells and their cellular extensions presented in number/mm of epidermis. ** P < 0.01, *** P < 0.001: control diet vs. HFD. Data are presented as means ±SEM. Scale bar: 50 μm.

Article Snippet: The same protocol was applied for L1 cell adhesion molecule (L1CAM, 1:500, #20659-1-AP, ProteinTech) antibody but the sections were incubated only overnight at 4°C.

Techniques: Immunofluorescence, Staining, Control, Quantitation Assay, Fluorescence, Immunostaining

Journal: Cell death & disease

Article Title: Scutellarin activates IDH1 to exert antitumor effects in hepatocellular carcinoma progression.

doi: 10.1038/s41419-024-06625-6

Figure Lengend Snippet: Fig. 7 Scu exerts antitumor effects by promoting IDH1 enzyme activity and activating the tumor immune microenvironment in vivo. A Schematic plan for the administration of Scu (60 and 100 mg/kg/day). C The tumor weight and D tumor volume were monitored every week for four weeks. After the mice were sacrificed, the resected tumors were B photographed and processed for pathological and immunohistochemical assays for E, F necrosis area and Ki67, L, M HIF1α, GLUT1, VEGFA, and N, O CD4, CD8, F4/80, CD56 and PD-L1 expression. G The relative mRNA levels of the indicated genes, H, I the expression levels of the indicated proteins, J the level of α-KG, and K IDH1 activity in tumor tissue were detected after treatment with Scu in vivo. The black arrows indicate positively stained cells. Scale bars, 50 μm. n ≥5. The data are mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control group.

Article Snippet: The sections were blocked in PBS containing 10% normal goat serum and 0.3% Triton X-100 for 60min; labeled with primary antibodies against Ki67 (1:2000, 27309-1-AP, Proteintech), IDH1 (1:200, 12332-1-AP, Proteintech), HIF1a (1:200, A22041, ABclonal), GLUT1 (1:200, 81463-1-RR, Proteintech), VEGFA (1:100, A12303, ABclonal), CD4 (1:500, 67786-1-Ig, Proteintech), CD8 (1:10,000, 66868-1-Ig, Proteintech), F4/80 (1:100, A23788, ABclonal), CD56 (1:2000, 14255-1-AP, Proteintech), and PDL1 (1:1000, 28076-1-AP, Proteintech) overnight at 4 °C; and incubated with the corresponding goat secondary antibodies for 1 h at room temperature.

Techniques: Activity Assay, In Vivo, Immunohistochemical staining, Expressing, Staining, Control

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: CD56 expression on naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on pre-treatment of the germ tubes with proteinase K (A) or periodate (B). Data for 3 independent donors are shown. (C) Significantly differentially expressed genes after 3-h stimulation of NK cells with A . fumigatus germ tubes pre-treated (60 min) with periodate or untreated (0 min). Abbreviations: adj. p = adjusted p-value, D = donor, fc = fold change. (D) Volcano plot summarizing transcriptional changes in NK cells stimulated with periodate-treated A . fumigatus germ tubes compared to cells stimulated with untreated germ tubes. Genes with a log2 fold change > 0.5 and an adjusted p-value < 0.05 are highlighted. Selected immune-related genes have been labelled. (E) CD56 expression on naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on the duration of periodate pre-treatment of the germ tubes. Representative histograms and data for cells from 5 independent donors are shown. (F) Chemokine and perforin release by naïve NK cells (Control) and NK cells stimulated for 3 h with A . fumigatus germ tubes, depending on the duration of periodate pre-treatment of the germ tubes. (A-B; E-F) Columns and error bars indicate means and standard deviations, respectively. Repeated measures one-way analysis of variance with Tukey’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Expressing, Control

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 binding to fungal carbohydrates and proteins, as determined by enzyme-linked immunosorbent assay. GAG = galactosaminogalactan, RodAp = surface rodlet protein/hydrophobin. N = 3 technical replicates. One-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., no coating. (B) Pull-down assay of CD56 with urea-insoluble galactosaminogalactan (PGG) or β-1,3-glucan. Lane M: Protein marker; Lane A: loading buffer extract of GAG + CD56 pellet; Lane B: supernatant of GAG + CD56; Lane C: CD56 alone; Lane D: supernatant of β-1,3-glucan + CD56; Lane E: loading buffer extract of β-1,3-glucan + CD56 pellet. (C) CD56 and CD69 expression on naïve NK cells (Control) and NK cells stimulated for 24 h with different concentrations of PGG. Representative histograms and data for cells isolated from 3 independent donors are shown. (D) Chemokine release by naïve NK cells (Control) and NK cells stimulated for 24 h with different concentrations of PGG. N = 3 independent donors. (C-D) Repeated measures (RM) one-way ANOVA with Tukey’s post-hoc test. (E) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 6 h with A . fumigatus (ATCC46645, AF) or A . nidulans (ATCC11267, AN) at different multiplicities of infection (MOIs). Representative histograms for one donor at MOI 4 are shown. (F) MOI-dependent stimulation of NK-cellular chemokine secretion after 6-h stimulation with AF or AN. (E-F) N = 3 independent donors. RM one-way ANOVA with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). In addition, AF and AN stimulation at each MOI was compared using paired t-Test (hash signs). (A-F) Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Pull Down Assay, Marker, Expressing, Isolation, Infection

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) Representative fluorescent micrographs (z-projection of 3–4 slices with 1 μm distance, representative dataset from ≥5 independent experiments) of hyphae of wild-type (WT) A . fumigatus Af293 and two galactosaminogalactan (GAG)-deficient A . fumigatus mutants (Δ uge3 and Δ agd3 ) co-cultured with soluble CD56, followed by staining with fluorescent anti-CD56 antibody. The shape of the hyphae is indicated by dotted lines The insets represent a control image (bottom; BSA stained with anti-CD56 antibody) of the respective strain. Scale: 10 μm. (B) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 6 h with WT Af293 or the GAG-deficient A . fumigatus mutants (Δ uge3 and Δ agd3 ) at different multiplicities of infection (MOIs). Representative histograms for one donor at MOI 4 are shown. (C) MOI-dependent induction of NK-cellular secretion of granzyme B, perforin, and chemokines after 6-h stimulation with WT Af293, Δ uge3 , and Δ agd3 . (B-C) N = 3 independent donors. Repeated measures (RM) one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). In addition, results for stimulation with the 3 strains at each MOI was compared using RM one-way ANOVA with Dunnett’s post-hoc test versus WT (hash signs). Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001. (D) CLSM micrographs of NK cells co-cultured with WT Af293 and Δ agd3 A . fumigatus hyphae. CD56 was stained with anti-CD56 Alexa Fluor 647 (red) to assess the CD56 localization. Germ tubes could be detected via their auto-fluorescence (cyan). Scale: 10 μm.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Cell Culture, Staining, Control, Expressing, Infection, Fluorescence

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 binding to fully acetylated (aPGG), fully deacetylated (dePGG), and native galactosaminogalactan (PGG), was determined by enzyme-linked immunosorbent assay (ELISA). Additional conditions with incomplete ELISA setup were included to preclude unspecific binding of the secondary antibody or anti-CD56 to the carbohydrates. N = 3 technical replicates. One-way analysis of variance (ANOVA) with Tukey’s post-hoc test was performed for each assay setup. (B) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 24 h with PGG, aPGG, or dePGG. Representative histograms for cells isolated from one donor are shown. (C) NK-cellular chemokine secretion after 24-h stimulation with PGG, aPGG, or dePGG. (B-C) N = 6 independent donors. Repeated measures (RM) one-way ANOVA with Tukey’s post-hoc test. (D) CD56, CD69, and CD107a expression on naïve NK cells (Control) and NK cells stimulated for 24 h with GalN oligomers, GalNAc oligomers, or chitosan. NK-cellular chemokine secretion after 24-h stimulation with GalN oligomers, GalNAc oligomers, or chitosan. N = 4 independent donors. RM one-way ANOVA with Tukey’s post-hoc test. (A-D) * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Control, Isolation

Journal: PLOS Pathogens

Article Title: CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan

doi: 10.1371/journal.ppat.1012315

Figure Lengend Snippet: (A) CD56 and CD69 expression of unstimulated NK cells (Control) and A . fumigatus -stimulated NK cells (6 h) depending on the fungal strain (wild type [WT] Af293 or isogenic mutants with defective GAG biosynthesis) and its enzymatic pre-treatment. (B) Secretion of granzyme B and perforin by unstimulated NK cells (Control) and A . fumigatus -stimulated NK cells depending (6 h) on the fungal strain and its enzymatic pre-treatment. (A-B) N = 6 independent donors. Repeated measures (RM) one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test versus Control, i.e., unstimulated NK cells (asterisks). Additionally, enzymatic pre-treatments of A . fumigatus WT and the Δ agd3 mutant, respectively, were compared using RM one-way ANOVA with Dunnett’s post-hoc test versus no enzymatic pre-treatment (hash signs). Columns and error bars indicate means and standard deviations, respectively. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001.

Article Snippet: Hyphae were incubated with soluble CD56 (5 μg/mL, R&D Systems, Minneapolis, MN, USA, Cat#2408-NC) or BSA (control, 5 μg/mL, Roth, Karlsruhe, Germany, #0163.2) in colorless RPMI for 2 h at 37°C, 5% CO 2 .

Techniques: Expressing, Control, Mutagenesis