Journal: STAR Protocols

Article Title: Fluorescence-activated cell sorting and phenotypic characterization of human fibro-adipogenic progenitors

doi: 10.1016/j.xpro.2022.102008

Figure Lengend Snippet:

Article Snippet: Recombinant human PDGF-AA , Miltenyi Biotec , Cat#130-108-983.

Techniques: Recombinant, Blocking Assay, Saline, Imaging, Software, Fluorescence, FACS, Flow Cytometry, Dissection, Transferring, Cell Culture, Sterility

Journal: bioRxiv

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells

doi: 10.1101/2024.09.25.614853

Figure Lengend Snippet: (A) Each EGFRL-ScNeo included the mScarlet red fluorescent protein and the mNeonGreen green fluorescent protein in the extracellular and cytoplasmic domains, respectively. ADAM-family proteases shed mScarlet-fused EGFRL. (B) mScarlet was inserted in front of the EGF domain. mNeonGreen was fused to the C terminus except in the case of TGFα, in which mNeonGreen was inserted before the C-terminal tandem valine residues. Note that the pro-NRG1 lacks the signal peptide and that Necl-5 lacks the ADAM cleavage site. SP, signal peptide; Pro, propeptide; EGF, EGF domain; TM, transmembrane domain; Ig, immunoglobulin-like domain. (C) XY confocal images of EGFRL-ScNeos expressed in MDCK cells. (D) mScarlet/mNeonGreen fluorescence ratio of the cell membrane in (C). The bar graphs show the mean values. Each dot represents the average value for one experiment. n > 100 cells for each condition. (E) Western blotting of total cell lysates of EGFRL-ScNeo-expressing cells. Membranes were probed with an anti-mScarlet or an anti-mNeonGreen antibody. *Full-length EGFRL-ScNeo; **Cytoplasmic domain fused with mNeonGreen. (F) The proportion of cleaved EGFRL-ScNeo in (E). The intensity of cytoplasmic domain fused with mNeonGreen (**) was divided by the intensity of the total detected bands for each ligand. The bar graphs show the mean values. Each dot indicates an independent experiment. (G) Western blotting of supernatants of EGFRL-ScNeo-expressing cells. Membranes were probed with an anti-mScarlet antibody. (H) The production rates of EGFRL from a single EGFRL-ScNeo-expressing cell were calculated from the image shown in (G). The bar graphs show the mean values. Each dot indicates an independent experiment. (I) Representative mScarlet/mNeonGreen ratio images of EREG-ScNeo-expressing MDCK cells upon treatment with 10 nM TPA (Video S1) or 10 μM Marimastat (Video S2). (J) Representative images of ERK activity (FRET/CFP) in MDCK-4KO cells expressing EKARrEV-NLS stimulated with the supernatant of MDCK-4KO cells expressing HBEGF-ScNeo. (K) MDCK-4KO-EKARrEV-NLS cells observed under a fluorescent microscope were stimulated with the supernatant of MDCK-4KO cells expressing EGFRL-ScNeo. The FRET/CFP ratio was normalized to the average values over the 60 min-observation period before stimulation. ERK activity (normalized FRET/CFP) was quantified and plotted as a function of time. Data were pooled from two independent experiments. n > 1000 cells for each condition. (L) Maximum ERK activity (normalized FRET/CFP) in the time course of average ERK activity in (K). The bar graphs show the mean values. Each dot represents the average value for one experiment. n > 1000 cells from three independent experiments were used for each condition.

Article Snippet: The following reagents were used: dimethyl sulfoxide (no. 13445-74; Nacalai Tesque, Kyoto, Japan), 12-O-tetradecanoylphorbol 13-acetate (TPA) (no. P-1680; LC Laboratories, Woburn, MA), Marimastat (no. SC-202223; Santa Cruz Biotechnology, Dallas, TX), EGF (no. E9644; Sigma-Aldrich, St. Louis, MO), HBEGF (no. 100-47; PeproTech, Cranbury, NJ), EREG (no. 100-04; PeproTech), TGFα (no. 239-A-100; R&D Systems, Minneapolis, MN), trametinib (no. T-8123; LC Laboratories), Cytochalasin D (no. 250255; Calbiochem, La Jolla, CA), and bovine serum albumin (no. A2153; Sigma-Aldrich). m D cTMP was synthesized as described previously ( ).

Techniques: Fluorescence, Membrane, Western Blot, Expressing, Activity Assay, Microscopy

Journal: bioRxiv

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells

doi: 10.1101/2024.09.25.614853

Figure Lengend Snippet: (A) Identical to . Low-magnification XY images and XZ images are shown. (B) Confocal XZ projection mNeonGreen images of HBEGF-ScNeo, TGFα-ScNeo, and EREG-ScNeo-expressing MDCK cells plated either on the cover glass or permeable filter. (C) EGFRL-ScNeo-expressing cells were stimulated with 10 nM TPA for 30 min. The mScarlet/mNeonGreen ratio of the plasma membrane was quantified. Data were pooled from three independent experiments. From the images of three independent experiments, 84 cells under each condition were randomly selected for analysis. Data are shown in the box plot. (D) EGFRL-ScNeo-expressing cells were cultured in the presence of 0.1% DMSO or 10 μM Marimastat for one day before imaging. The mScarlet/mNeonGreen ratio of the plasma membrane was quantified. Data were pooled from three independent experiments. From the images of three independent experiments, 90 cells under each condition were randomly selected for analysis. Data are shown in the box plot. (E) MDCK-4KO-EKARrEV-NLS cells observed under a fluorescent microscope were stimulated with the 0.1 nM recombinant EGFRLs. The FRET/CFP ratio was normalized to the average values over the 60 min-observation period before stimulation. ERK activity (normalized FRET/CFP) was quantified and plotted as a function of time. Data were pooled from three independent experiments. n > 1000 cells for each condition. (F) Western blotting analysis of cell lysates of MDCK-4KO-EKARrEV-NLS cells stimulated with the supernatant of MDCK-4KO-EGFRL-ScNeo cells or 10 ng/mL EGF or 200 nM Trametinib. pY1068 was normalized with EGF condition. Each dot indicates an independent experiment. The bar graphs represents the means. Statistical significance was determined by unpaired two-tailed Welch’s t-test.

Article Snippet: The following reagents were used: dimethyl sulfoxide (no. 13445-74; Nacalai Tesque, Kyoto, Japan), 12-O-tetradecanoylphorbol 13-acetate (TPA) (no. P-1680; LC Laboratories, Woburn, MA), Marimastat (no. SC-202223; Santa Cruz Biotechnology, Dallas, TX), EGF (no. E9644; Sigma-Aldrich, St. Louis, MO), HBEGF (no. 100-47; PeproTech, Cranbury, NJ), EREG (no. 100-04; PeproTech), TGFα (no. 239-A-100; R&D Systems, Minneapolis, MN), trametinib (no. T-8123; LC Laboratories), Cytochalasin D (no. 250255; Calbiochem, La Jolla, CA), and bovine serum albumin (no. A2153; Sigma-Aldrich). m D cTMP was synthesized as described previously ( ).

Techniques: Expressing, Clinical Proteomics, Membrane, Cell Culture, Imaging, Microscopy, Recombinant, Activity Assay, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells

doi: 10.1101/2024.09.25.614853

Figure Lengend Snippet: (A) Schematic of the SLL-induced protein translocation (SLIPT) system. Bath application of m D cTMP, which localizes at the inner leaflet of the plasma membrane and binds to Escherichia coli dihydrofolate reductase (eDHFR), recruits the miRFP703-eDHFR-cRaf fusion protein to the plasma membrane. cRaf at the plasma membrane stimulates ERK, which in turn activates ADAM17. The activated ADAM17 at the plasma membrane induces ectodomain shedding of EGFRL-ScNeo. The miRFP703-eDHFR-cRaf fusion protein was expressed in MDCK-4KO cells ( ΔEGF, ΔHBEGF, ΔTGFA, ΔEREG ) to generate eDHFR-cRaf cells. (B) TSen, a FRET biosensor for ADAM17, was introduced into the eDHFR-cRaf cells. Cells observed under a fluorescent microscope were stimulated with various concentrations of m D cTMP. The FRET/CFP ratio was normalized to the average values over the 20 min-observation period before stimulation. The average ERK activity (normalized FRET/CFP) was quantified and plotted as a function of time with the s.d. Data were pooled from three independent experiments. n > 100 cells for each condition. (C) The AREG-ScNeo probe was introduced into the eDHFR-cRaf cells. Cells were treated with 10 μM m D cTMP. Representative images of miRFP703 and mScarlet/mNeonGreen ratio are shown (Video S3). (D) EGFRL-ScNeo probes were introduced into the MDCK-4KO cells expressing eDHFR-cRaf to generate the producer cells, which were co-cultured with an excess of MDCK-4KO cells expressing a FRET biosensor for ERK, EKARrEV-NLS. (E) Representative time-lapse ERK activity (FRET/CFP) images. The EREG-producer cells are located at the center of the image. Surrounding cells are MDCK-4KO cells expressing EKARrEV-NLS. (F) Each ligand producer is located at the center of the image. Images are snapshots of Video S4 acquired 20 min after m D cTMP addition. (G) In each cell shown in (F), the time of the highest ERK activity (FRET/CFP) is plotted against the distance to the producer cells. (H) From the scatter plot in (G), velocities of ERK waves were calculated. Each dot indicates a single producer cell population. The red bars represent the means. n = 28 (EREG), 36 (AREG), 50 (TGFα), 30 (HBEGF), and 23 (NRG1) producer cell populations from three independent experiments, depicted by the three colors. (I) Maximum radius of the ERK-activated area upon EGFRL shedding. Data in (H) are used for the analysis. The red bars represent the means. Statistical significance (J) Analysis of the m D cTMP-dependent EGFRL shedding. EGFRL-ScNeo-expressing 4KO-eDHFR-cRaf cells were incubated with or without 10 μM m D cTMP for 1 hour. The supernatant of the cells was corrected and calculated the amount of EGFRL with an anti-mScarlet antibody. (K) The production rates of EGFRL. mScarlet intensities of HBEGF sup with m D cTMP were set as one. The bar graphs show the mean values. Each dot indicates an independent experiment.

Article Snippet: The following reagents were used: dimethyl sulfoxide (no. 13445-74; Nacalai Tesque, Kyoto, Japan), 12-O-tetradecanoylphorbol 13-acetate (TPA) (no. P-1680; LC Laboratories, Woburn, MA), Marimastat (no. SC-202223; Santa Cruz Biotechnology, Dallas, TX), EGF (no. E9644; Sigma-Aldrich, St. Louis, MO), HBEGF (no. 100-47; PeproTech, Cranbury, NJ), EREG (no. 100-04; PeproTech), TGFα (no. 239-A-100; R&D Systems, Minneapolis, MN), trametinib (no. T-8123; LC Laboratories), Cytochalasin D (no. 250255; Calbiochem, La Jolla, CA), and bovine serum albumin (no. A2153; Sigma-Aldrich). m D cTMP was synthesized as described previously ( ).

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Microscopy, Activity Assay, Expressing, Cell Culture, Incubation

Journal: bioRxiv

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells

doi: 10.1101/2024.09.25.614853

Figure Lengend Snippet: (A) Representative images of ERK activity (FRET/CFP) in MDCK-dErbB1-EKARrEV-NLS receiver cells. The producer cells were located in the center of the image. Images were acquired 20 min after m D cTMP addition. (B) Representative images of ERK activity (FRET/CFP) in MDCK-dErbB134-EKARrEV-NLS receiver cells. The producer cells were located in the center of the image. Images were acquired 20 min after m D cTMP addition. (C) Representative images of ERK activity (FRET/CFP) in MDCK-4KO-EKARrEV-NLS receiver cells. ADAM17 of producer cells was deleted. The producer cells were located in the center of the image. Images were acquired 20 min after m D cTMP addition. (D) A schematic of EGFRL-mNeonGreen with mNeonGreen located in the cytoplasm. (E) The velocity of ERK wave propagated from producers expressing each ligand fused with C-terminal mNeonGreen. Each dot indicates a single producer cell population. The red bars represent the means. n = 5 (EREG), 3 (TGFα), and 3 (HBEGF) producer cell populations from a single experiment. (F) Maximum radius of ERK wave propagated from EREG- and HBEGF-producer cells to MDCK-4KO-EKARrEV-NLS receiver cells supplemented with 0.5% DMSO or 10 μM Cytochalasin D. Each dot indicates a single producer cell population. The red bars represent the means. n = 9 (EREG, DMSO), 12 (EREG, Cytochalasin D), 3 (HBEGF, DMSO), 6 (HBEGF, Cytochalasin D) producer cell populations from a single experiment. Statistical significance was determined by unpaired two-tailed Welch’s t-test.

Article Snippet: The following reagents were used: dimethyl sulfoxide (no. 13445-74; Nacalai Tesque, Kyoto, Japan), 12-O-tetradecanoylphorbol 13-acetate (TPA) (no. P-1680; LC Laboratories, Woburn, MA), Marimastat (no. SC-202223; Santa Cruz Biotechnology, Dallas, TX), EGF (no. E9644; Sigma-Aldrich, St. Louis, MO), HBEGF (no. 100-47; PeproTech, Cranbury, NJ), EREG (no. 100-04; PeproTech), TGFα (no. 239-A-100; R&D Systems, Minneapolis, MN), trametinib (no. T-8123; LC Laboratories), Cytochalasin D (no. 250255; Calbiochem, La Jolla, CA), and bovine serum albumin (no. A2153; Sigma-Aldrich). m D cTMP was synthesized as described previously ( ).

Techniques: Activity Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Low-affinity ligands of the epidermal growth factor receptor are long-range signal transmitters during collective cell migration of epithelial cells

doi: 10.1101/2024.09.25.614853

Figure Lengend Snippet: (A) (Upper) A schematic of the TGFα-EREG chimera of extracellular TGFα and cytoplasmic EREG. (Bottom) mNeonGreen XZ images of EREG, TGFα, and a TGFα-EREG chimera. (B) The velocity of ERK wave propagated from EREG-, TGFα-, or a TGFα-EREG chimera-producer cells to MDCK-4KO-EKARrEV-NLS receiver cells. Each dot indicates a single producer cell population. The red bars represent the means. N = 30 (EREG), 26 (TGFα), and 24 (TGFα-EREG chimera) producer cell populations from two independent experiments, depicted by the two colors. (C) The velocity of ERK wave propagated from EREG- or HBEGF-producer cells to WT or EGFR-overexpressing receiver cells. Each dot indicates a single producer cell population. The red bars represent the means. n = 35 (EREG, WT), 41 (EREG, EGFR O/E), 32 (HBEGF, WT), and 12 (HBEGF, EGFR O/E) producer cell populations from two independent experiments. (D) Representative images of ERK activity (FRET/CFP) in MDCK-α-1-catenin KO receiver cells. Each ligand producer cell is located in the center of the image. Images were acquired 30 min after m D cTMP addition (Video S5). (E) Maximum radius of ERK wave propagated from EREG-producer cells to MDCK-II-WT or MDCK-II-quinKO receiver cells. Each dot indicates a single producer cell population. The red bars represent the means. n = 38 (WT) and 42 (quinKO) producer cell populations from three independent experiments, depicted by the three colors. (F) The velocity of ERK wave propagated from EREG- or HBEGF-producer cells to WT, E-cadherin KO, and p120-catenin KO receiver cells. Each dot indicates a single producer cell population. The red bars represent the means. n = 21 (EREG, WT), 21 (EREG, E-cadherin KO), 24 (EREG, p120-catenin KO), 18 (HBEGF, WT), 11 (HBEGF, E-cadherin KO), and 21 (HBEGF, p120-catenin KO) producer cell populations from two independent experiments. (G) Representative images of ERK activity (FRET/CFP) in MDCK-4KO-EKARrEV-NLS receiver cells. Each ligand producer cell is located in the white area of the image. The producer cells expressing eDHFR-cRaf are derived from the cell lines listed at the top of each image. Images were acquired 20 min after m D cTMP addition. (H) Maximum radius of ERK wave propagation in (G). Each dot indicates a single producer cell population. The red bars represent the means. n = 23 (WT), 25 (TKO) producer cell populations from three independent experiments. n = 6 (4KO) producer cell populations from two independent experiments. n = 11 (dEREG) producer cell populations from a single experiment. Statistical significance was determined by unpaired two-tailed Welch’s t-test.

Article Snippet: The following reagents were used: dimethyl sulfoxide (no. 13445-74; Nacalai Tesque, Kyoto, Japan), 12-O-tetradecanoylphorbol 13-acetate (TPA) (no. P-1680; LC Laboratories, Woburn, MA), Marimastat (no. SC-202223; Santa Cruz Biotechnology, Dallas, TX), EGF (no. E9644; Sigma-Aldrich, St. Louis, MO), HBEGF (no. 100-47; PeproTech, Cranbury, NJ), EREG (no. 100-04; PeproTech), TGFα (no. 239-A-100; R&D Systems, Minneapolis, MN), trametinib (no. T-8123; LC Laboratories), Cytochalasin D (no. 250255; Calbiochem, La Jolla, CA), and bovine serum albumin (no. A2153; Sigma-Aldrich). m D cTMP was synthesized as described previously ( ).

Techniques: Activity Assay, Expressing, Derivative Assay, Two Tailed Test

Journal: Bioengineering (Basel, Switzerland)

Article Title: Artificial Vision: The High-Frequency Electrical Stimulation of the Blind Mouse Retina Decay Spike Generation and Electrogenically Clamped Intracellular Ca 2+ at Elevated Levels.

doi: 10.3390/bioengineering10101208

Figure Lengend Snippet: Figure 3. Stimulation frequency-dependent modulation of retinal ganglion cell responses: (A) Ca2+ imaging recordings of ganglion cells (GCs, mean of n = 6 retinas, m = 6 cells) elicited via sustained electrical stimulation (stim; 90 s) at various frequencies (0.5, 1.5, 3, 5, 10, 20, and 50 Hz; biphasic stimulus: −1.6/+1.5 V). Temporal magnification of the response’s initial, middle, and recovery phases is indicated in the inset boxes (15 s resolution). Experimental recording conditions: control (ctr) and in the presence of pharmaceuticals (cf. Figure 1 for site of action): mGluR6 agonist L-AP4, iGluR antagonist CNQX, VGNaC antagonist TTX, and VGCC blocker verapamil. (B) Representative Ca2+-induced Ca2+-release (CICR) events (black) were rarely recorded in a GC during subretinal stim.

Article Snippet: We used (in μM) 100 L-AP4 (mGluR6 agonist; L-2-amino-4-phosphonobutyric acid), 20 CNQX (AMPA/kainite-type GluR antagonist; 6-Cyano-7-nitroquinoxaline-2,3-dione) obtained from TocrisBioScience (Bristol, UK); 100 verapamil (L-type voltage-gated Ca2+ channel (VGCC) blocker) obtained from Sigma-Aldrich Bioengineering 2023, 10, 1208 5 of 19 (Darmstadt, Germany); and 30 TTX (sodium channel blocker; tetrodotoxin) obtained from Carl Roth (Karlsruhe, Germany).

Techniques: Imaging, Control

Journal: Bioengineering (Basel, Switzerland)

Article Title: Artificial Vision: The High-Frequency Electrical Stimulation of the Blind Mouse Retina Decay Spike Generation and Electrogenically Clamped Intracellular Ca 2+ at Elevated Levels.

doi: 10.3390/bioengineering10101208

Figure Lengend Snippet: Figure 4. Quantification of the ganglion cell Ca2+ responses modulated by various frequencies and pharmaceuticals. Stimulation (stim) frequency (x-axis: 0.5, 1.5, 3, 5, 10, 20, and 50 Hz; biphasic stimulus: −1.6/+1.5 V)-dependent modulation of ganglion cell (GC responses under control (ctr, black) and in the presence of pharmaceuticals (colored) (cf. Figure 1 for site of action): mGluR6 agonist L-AP4, iGluR antagonist CNQX, VGNaC antagonist TTX, and VGCC blocker verapamil. Quantification of (A) δ-amplitude (y-axis normalized, first response peak within early response phase; see Figure 3), and (B) estimation of elevated Ca2+ levels (y-axis normalized, average Ca2+ level of middle recording phase; see Figure 3). For each condition, n = 6 retina, m = 6 cells. Error bars indicate ± SEM. Statistical analysis is provided in Table S1.

Article Snippet: We used (in μM) 100 L-AP4 (mGluR6 agonist; L-2-amino-4-phosphonobutyric acid), 20 CNQX (AMPA/kainite-type GluR antagonist; 6-Cyano-7-nitroquinoxaline-2,3-dione) obtained from TocrisBioScience (Bristol, UK); 100 verapamil (L-type voltage-gated Ca2+ channel (VGCC) blocker) obtained from Sigma-Aldrich Bioengineering 2023, 10, 1208 5 of 19 (Darmstadt, Germany); and 30 TTX (sodium channel blocker; tetrodotoxin) obtained from Carl Roth (Karlsruhe, Germany).

Techniques: Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Targeting PDXK could promote the formation of TLSs and enhance the efficacy of immunotherapy in gastric cancer. ( A ) Heatmap displaying the metabolic feature for T-cell clusters using scMetabolism package. ( B ) Dot plot showing the expression of the genes encoding rate-limiting enzymes of vitamin B 6 metabolism in different types of T cells. Dot size encodes the percentage of cells expressing the gene, color encodes the average per cell gene expression level. ( C ) Quantification of CXCL13 in PDTFs in the presence of different enzyme inhibitors measured by ELISA. ( D ) Representative H&E staining and PDXK immunohistochemistry of gastric cancer tissues with different responses following immunotherapy. Scale bar, 500 µm. ( E ) The schematic diagram of the animal experiments. ( F–G ) Images of tumors and tumor volume curves of 615 mice treated with various agents (n=6, each group). ( H ) Paraffin sections of mouse subcutaneous graft tumor tissue stained with H&E and IHC detection for CD8, CD20 and CXCL13. Scale bar, 100 µm. ( I ) The number (left panel) and area (right panel) of TLS per tumor area were compared between groups (n=6, each group). Data are presented as the mean±SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. AOX1, aldehyde oxidase 1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CR, complete response; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PDXK, pyridoxal kinase; PDXP, pyridoxal phosphatase; PDTFs, patient-derived tumor fragments; PHOSPHO2, phosphatase orphan 2; PNPO, pyridoxamine 5'-phosphate oxidase; PR, partial response; PSAT1, phosphoserine aminotransferase 1; s.c, subcutaneous injections; SD, stable disease; TLS, tertiary lymphoid structures.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Two Tailed Test, Saline, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Vitamin B 6 could promote the expression and secretion of CXCL13 in CD160 + CD8 + T cells. ( A ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in PDTFs in the presence of different drugs measured by ELISA. ( B ) The schematic diagram of orthotopic transplanted tumor model with diets containing various amounts of vitamin B 6 (n=6, each group). ( C ) Mouse orthotopic stomach xenograft tumor tissues stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( D ) The schematic diagram of orthotopic transplanted tumor model fed with different drugs or diets (n=6, each group). ( E ) The representative images of mouse bioluminescence imaging at week 3 (left panel) and the corresponding quantification analysis (right panel). ( F ) Representative micrographs of xenografts stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( G–H ) Density of TLSs (left panel) and ratio of tumor area occupied by TLSs (right panel). ( I ) Gating strategy for CD160 + CD8 + T cells. ( J ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in supernatants from CD160 + CD8 + T-cell cultures in the presence or absence of PL measured by ELISA. ( K ) Flow cytometric analysis and corresponding quantification of CXCL13 + CD160 + CD8 + T cells with or without PL treatment. Data are presented as the mean±SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-tailed Student’s t-test. ICIs, immune checkpoint inhibitors; IFN, interferon; IHC, immunohistochemistry; IL, interleukin; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PL, pyridoxol; PD-1, programmed cell death protein 1; s.c, subcutaneous injections; TLS, tertiary lymphoid structures; TNF, tumor necrosis factor.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Imaging, Two Tailed Test, Immunohistochemistry, Saline

Journal: eLife

Article Title: NHE6 depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load

doi: 10.7554/eLife.72034

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , γ-Secretase inhibitor L-685458 , Tocris Bioscience , 2627 , .

Techniques: Staining, Plasmid Preparation, Transfection, Construct, Expressing, shRNA, Recombinant, Sequencing, Software, Imaging

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: TNFα is correlated with human oral cancer pain scores. ( a ) TNFα protein concentration is higher in cancer tissues compared to anatomically matched contralateral healthy tissues from the same patient (n = 10, * P < 0.05, paired t-test). ( b ) Patients were asked to answer the Oral Cancer Pain Questionnaire before surgery. The mean pain score from patients correlated positively with percentage change in TNFα concentration between cancer and matched contralateral normal tissues ( r = 0.7, P < 0.05).

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Protein Concentration, Concentration Assay

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: Blocking TNFα or JNK inhibits nociception in mice with cancer. ( a ) After 16 weeks of 4NQO treatment, mice exhibited significant increase in gnaw-time from its respective baseline (pre-injection). Propylene glycol (PG) treatment did not affect gnaw-time. In 4NQO tongue cancer mice, C-87 (12.5 mg/kg) IP injection significantly reduced percentage of gnaw-time change from baseline (n = 8) 1 h post-injection than the vehicle (10% DMSO) treated cancer mice (n = 5). C-87 (n = 6) or vehicle (n = 4) had no effect in non-cancer mice treated with PG alone. ( b ) Mice with paw SCC developed cancer pain at PID7. C-87 and the JNK inhibitor SP600125 treatment significantly reduced mechanical nociception compared to vehicle at 1, 3, and 6 h after treatment compared to the control group. 24 h after the treatment the analgesic effect of C-87 was gone (n = 5 per group, Two-way ANOVA). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Blocking Assay, Injection, Control

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: Blocking TNFα inhibits cancer cell growth, migration, and cytokine release. ( a ) Growth rate, measured with the RTCA, following different doses of C-87 treatment in HSC-3 cell culture. C-87 inhibited oral cancer cell growth in a dose dependent manner. One-way ANOVA with Tukey's post hoc analysis. ( b ) Mice with C-87 treatment (n = 7) exhibited a significant decrease in the paw volume compared to the vehicle control mice (n = 6) at PID14, 18, and 21 (two-way ANOVA). Arrow indicates C-87 injection. ( c ) C-87 treated paw cancer mice (n = 6) had smaller tumor area relative to the total paw area compared to vehicle treated paw cancer mice (n = 4). Tumor areas and total paw areas were quantified using H&E stained paw sections. Mann–Whitney U-test. ( d ) Representative H&E stained pictures showing a normal mouse paw, a cancer mouse paw, and a cancer paw treated with C-87 (10 × inset). Scale bar: 100 μm. Images were taken and quantified using Nikon imaging software NIS-Elements F Ver4.60.00. ( e ) C-87 treatment reduced the concentration of TNFα, NGF, IL1β, IL4, MIP3α, IL28β, and IL33 in the paw tumor. Data were presented as fold change of cytokines/chemokines measured from tumor paws over normal paws. n = 6 per group. Mann–Whitney U test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Blocking Assay, Migration, Cell Culture, Control, Injection, Staining, MANN-WHITNEY, Imaging, Software, Concentration Assay

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: TNFα mediates Schwann cell proliferation and migration in vitro. ( a ) The presence of either DOK or HSC-3 cells in cell inserts increased Schwann cell proliferation 48 h following co-culture in the MTS assay. Increased Schwann cell proliferation induced by the presence of HSC-3 cells was inhibited by adding C-87 into inserts. Representative images of cells with Hoechst stain were shown under each culture condition. OD: optical density. ( b ) Schwann cells are more migratory in the presence of HSC-3 cells compared to the DMEM control while DOK reduced Schwann cell migration. Adding C-87 into the HSC-3 culture reduced Schwann cell migration. ( c ) Adding TNFα to the media at the bottom chamber increased Schwann cells migration compared to the DMEM control. Neutralizing TNFα with C-87 decreased Schwann cell migration. ( d ) Schwann cells induced increased HSC-3 cell migration compared to the DMEM control; adding C-87 (20 µM) into the Schwann cell culture in the bottom chamber blocked this increase. ( b – d ), images shown are representative diff-quick stained migrated cells. a-d, one-way ANOVA with Tukey's post hoc analysis. SCs: Schwann cells. Scale bar: 100 μm. * P < 0.05; ** P < 0.01; *** P < 0.001. Images were taken using Nikon imaging software NIS-Elements F Ver4.60.00.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Migration, In Vitro, Co-Culture Assay, MTS Assay, Staining, Control, Cell Culture, Diff-Quik, Imaging, Software

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: The effect TNFα on the expression of Schwann cell activation markers in vitro. TNFα treatment increased c-Jun ( a , b ), GFAP ( c , d ), and p75 ( e – f ) immunofluorescence intensity and protein expression in cultured Schwann cells compared to the DMEM control. TNFα treatment decreased MBP immunofluorescence intensity and protein expression in cultured Schwann cells compared to the DMEM control ( g – h ). Full-length gel blots were provided in the Supplemental Fig. online. Scale bar: 100 μm. Student’s t-test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Expressing, Activation Assay, In Vitro, Immunofluorescence, Cell Culture, Control

Journal: Scientific Reports

Article Title: TNFα promotes oral cancer growth, pain, and Schwann cell activation

doi: 10.1038/s41598-021-81500-4

Figure Lengend Snippet: Activated Schwann cells release increased NGF and TNFα. ( a ) Schwann cells co-cultured with HSC-3 cells overexpressed c-Jun, GFAP, p75 but downregulated MBP compared to control Schwann cells (media alone). Full-length gel blots were provided in Supplemental Fig. online. ( b ) Both DOK and HSC-3 co-cultures increased TNFα mRNA expression in Schwann cells compared to control Schwann cells. ( c ) TNFα protein concentration in Schwann cells co-cultured with either DOK or HSC-3 cells compared to control Schwann cells. ( d ) HSC-3 cell or DRK co-culture increased NGF release in Schwann cells compared with control Schwann cells. ( e ) Adding TNFα in cell culture media stimulated increased NGF release compared with control Schwann cells. One-way ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Human NGF and TNFα Quantikine ELISA kits were purchased from R&D systems.

Techniques: Cell Culture, Control, Expressing, Protein Concentration, Co-Culture Assay

Journal: eLife

Article Title: Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

doi: 10.7554/eLife.69708

Figure Lengend Snippet: ( a ) Upper panels, total flow cytometry plots of HUVEC and TIME cells stained for endothelial cell markers PECAM and von Willebrand factor (vWF). Medium and lower panels, surface flow cytometry plots of HUVEC and TIME cells stained for PCDH1, β3 integrin, DAF, β1 integrin. ( b ) Surface flow cytometry of wild-type (WT) and knockout (KO) TIME cells stained as above. Histograms of WT cells are shown in gray; single- and double-KO cells are shown in color. ( c ) Western blot analysis of WT TIME cells and KO cells ± cDNA. β-Actin was used as a loading control. Figure 1—source data 1. Original blot of WT TIME cells and KO cells ± cDNA.

Article Snippet: Antibody , α–Human β3 Integrin (Rabbit polyclonal) , Cell Signaling , Cat. # 4702 , WB 1:300.

Techniques: Flow Cytometry, Staining, Knock-Out, Western Blot, Control

Journal: eLife

Article Title: Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells

doi: 10.7554/eLife.69708

Figure Lengend Snippet:

Article Snippet: Antibody , α–Human β3 Integrin (Rabbit polyclonal) , Cell Signaling , Cat. # 4702 , WB 1:300.

Techniques: Virus, Plasmid Preparation, Expressing, Transduction, Retroviral, Recombinant, Sequencing, Staining, Software, Imaging