Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: In vitro assessment of the CD70-directed CAR-T cell anti-GB cytotoxicity (A) Measurement of secreted TNF-α and IFN-γ in the SN of GB/CAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. For comparison between MCS and CD70 (upper bar plots), an unpaired two-tailed t test was used. For comparisons among constructs (bottom), a one-way ANOVA followed by a Holm-Šídák multiple comparisons test was used. (B) Measurement of tumor cell signal during co-culture with CAR-T cells on the Incucyte platform. N = 2 biological replicates per group. Every biological replicate is the mean of N = 5 technical replicates. Time point intervals = 45 min. A two-tailed Student’s t test was performed using the values of the last measured time point to determine statistical significance. For (A) and (B), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: The following kits were used to quantify cytokine secretion: Human Granzyme-B DuoSet enzyme-linked immunosorbent assay kit (ELISA; #DY2906–05, R&D Systems), Human IFN-gamma DuoSet ELISA kit (#DY285B, R&D Systems), Human TNF-alpha DuoSet ELISA kit (#DY210, R&D Systems), and Mouse TNF-alpha DuoSet ELISA kit (#DY410, R&D Systems).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test, Construct, Co-Culture Assay

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: Evaluation of CD70-directed CAR-T cell effector function in cerebral organoids (A) Confocal microscopy of cerebral organoids, infiltrated by generated GB models. (B) Quantification of CD70 signal in organoids from (A). Each dot represents an organoid. A Welch’s t test was used to assess significance. (C) Immunofluorescence analysis of endogenous CD70 expression in cerebral organoids. (D) Confocal microscopy of cerebral organoids previously invaded by GB cells and subsequently treated with CAR-T cells for 3 d. (E) Quantification of Granzyme-B signal from (D). A two-tailed t test was used to determine significance. (F) Measurement of secreted Granzyme-B and IFN-γ levels in the SN of co-cultures from (D) by ELISA. N = 3 biological replicates per group. (G) CAR construct direct comparisons from (F). A one-way ANOVA followed by a Tukey’s post hoc test for multiple comparisons was used. For (A), (C), and (D), scale bars, 200 μm. For (D) and (E), N ≥ 3 organoids per group. For (E) and (F), a two-tailed t test was used to assess significance. For (B), (E), and (F), data presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant.

Article Snippet: The following kits were used to quantify cytokine secretion: Human Granzyme-B DuoSet enzyme-linked immunosorbent assay kit (ELISA; #DY2906–05, R&D Systems), Human IFN-gamma DuoSet ELISA kit (#DY285B, R&D Systems), Human TNF-alpha DuoSet ELISA kit (#DY210, R&D Systems), and Mouse TNF-alpha DuoSet ELISA kit (#DY410, R&D Systems).

Techniques: Confocal Microscopy, Generated, Immunofluorescence, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Construct

Journal: Molecular Therapy Oncology

Article Title: A comparative analysis of CD70-directed CAR-T cells for glioblastoma treatment demonstrates a superior efficacy of the ligand-based construct

doi: 10.1016/j.omton.2026.201134

Figure Lengend Snippet: mCD27-based anti-murine CD70 CAR-T cells are potent against murine GB in vitro (A) Murine CD27-based construct design. (B) Transduction efficiency of primary murine T cells by flow cytometry. A non-transduced (NT) sample from each donor mouse was used to determine gating. Data gated on single live mCD3 + cells. (C) Measurement of mCD70 gene expression levels in generated OE models by RT-qPCR. N = 3 technical replicates per cell line. (D) Measurement of mCD70 on the surface of generated murine OE GB models by flow cytometry (blue histograms). Signal was compared to that of an isotype control (red histograms). (E) Quantification of secreted TNF-α in the SN of mGB/mCAR-T cell co-cultures by ELISA. N = 3 biological replicates per group. (F) Pairwise comparisons of secreted TNF-α levels from (E) among targeting constructs. A one-way ANOVA with a post hoc Holm-Šídák test was used for significance. (G) Schematic representation of the live-cell imaging pipeline. (H) Quantification of tumor cell signal from (G) over time. A one-way ANOVA with a Dunnett’s multiple comparisons test was used with data from the t = 660 min mark. For (C) and (E), an unpaired two-tailed t test was used for significance. Data are presented as mean (SD). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n.s., not significant; n.d., not detected.

Article Snippet: The following kits were used to quantify cytokine secretion: Human Granzyme-B DuoSet enzyme-linked immunosorbent assay kit (ELISA; #DY2906–05, R&D Systems), Human IFN-gamma DuoSet ELISA kit (#DY285B, R&D Systems), Human TNF-alpha DuoSet ELISA kit (#DY210, R&D Systems), and Mouse TNF-alpha DuoSet ELISA kit (#DY410, R&D Systems).

Techniques: In Vitro, Construct, Transduction, Flow Cytometry, Gene Expression, Generated, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Live Cell Imaging, Two Tailed Test

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: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: Establishment of an EMT imaging system. (A) Schematic overview of EMTimage, expressing RFP under the control of the E‐cadherin promoter. Differential interference contrast (DIC)/RFP fluorescence overlay images of living E‐cadherin ‐RFP/Py2T cells during EMT/MET. EMT was induced by 5 ng·mL −1 TGFβ1 stimulation for 7 days and MET was induced by withdrawal of TGFβ1 for 7 days. Scale bar, 50 μm. (B) Relative expression of epithelial and mesenchymal genes normalized to Gapdh in E‐cadherin ‐RFP/Py2T cells responding to TGFβ for the indicated time periods. Average values and SD from n = 3 biological replicates, each with technical triplicates, and P ‐values (** P < 0.01, *** P < 0.001) after two‐tailed paired Student’s t ‐test. (C) Immunoblot of parental Py2T and E‐cadherin ‐RFP/Py2T cells for epithelial and mesenchymal proteins, and β‐actin as a loading control, along with molecular size markers (representative of n = 3 independent experiments). For original images, see Fig. . (D) Representative imaging of DIC, RFP, immunofluorescence staining (green) of epithelial and mesenchymal proteins and nuclear DAPI (blue) during EMT (TGFβ) and MET (TGFβ withdrawal) in E‐cadherin ‐RFP/Py2T cells ( n = 3 independent experiments). Scale bars, 50 μm.

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Imaging, Expressing, Control, Fluorescence, Two Tailed Test, Western Blot, Immunofluorescence, Staining

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: TGFβ promotes 3D mammosphere growth. (A) Mammosphere size after treatment or not of 4000 EMTimage cells with TGFβ1, BMP7, or BMP4 for 4 days in a 96‐well round bottom low‐attachment plate to obtain single spheres per well. Graph bars are colour‐coded (red, TGFβ1 (5 ng·mL −1 ); dark blue, BMP7 (100 ng·mL −1 ); light blue, BMP4 (100 ng·mL −1 )) and show average values and SD from n = 13 biological replicates, each with technical triplicates, and P ‐values (**** P < 0.0005) after two‐tailed paired Student’s t ‐test. (B) Representative DIC and RFP fluorescence overlay images of E‐cadherin ‐RFP/Py2T cells from passage 1, in three consecutive 3D cultures ( n = 4 independent experiments). Scale bar, 100 μm. (C) Numbers of mammospheres in the three consecutive passages of the mammosphere cells. The cells were treated under the same conditions as in panel A. Graph bars are colour‐coded (red or blue) and show average values and SD from n = 3 biological replicates, each with technical triplicates, and P ‐values (**** P < 0.0005) after two‐tailed paired Student’s t ‐test. (D) Immunoblot of E‐cadherin ‐RFP/Py2T cells cultured under 3D conditions and collected after stimulation with TGFβ1 (5 ng·mL −1 ) or BMP7 (100 ng·mL −1 ) for 3 and 5 days, followed by analyses for RFP, and epithelial, mesenchymal and stem cell proteins, as well as β‐actin as a loading control, along with molecular size markers (representative of n = 3 independent experiments). For original images, Fig. .

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Two Tailed Test, Fluorescence, Western Blot, Cell Culture, Control

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: TGFβ increases EMTimage stem cell frequency. (A) Schematic drawing of the experiment. Upon TGFβ‐induced EMT of E‐cadherin ‐RFP/Py2T cells (marked with a red circle) cultured under 2D conditions, RFP low mesenchymal cells (marked by light pink triangle) were cultured in 3D. In the absence of TGFβ in 3D, the mammosphere cells underwent partial MET and became enriched in RFP high cells (marked by red polka‐dotted hexagon). In the presence of TGFβ in 3D, the mammospheres remained in an RFP low state and grew larger (marked by light pink striped hexagon). Note that the same colour‐coded symbols are used in panels B‐E. (B‐E) 2D cells were pre‐treated with 5 ng·mL −1 TGFβ1 (red symbols) or 100 ng·mL −1 BMP7 (blue symbols) for 7 days and then analyzed (2D), or trypsinized and seeded in 3D cultures in the absence or presence of 5 ng·mL −1 TGFβ1 or 100 ng·mL −1 BMP7 for 5 days (3D). Representative DIC and RFP fluorescence overlay images of E‐cadherin ‐RFP/Py2T cells in 2D and 3D culture ( n = 3 biological replicates). Scale bar, 100 μm. (C) ELDA measuring sphere‐forming frequency upon TGFβ1 or BMP7 stimulation for 7 days. The number of wells devoid of spheres (fraction nonresponding) is plotted against the number of plated cells per well (from 200 to 1 cell). Average stem cell frequency values are fitted into straight lines. Steeper slopes indicate higher frequencies of sphere‐forming cells. A table indicates average stem cell frequency per condition and associated P ‐values ( n = 3 biological replicates, technical octaplicates; ** P < 0.01, *** P < 0.001, **** P < 10 ‐6 ) after χ 2 ‐test followed by P ‐value test performed to assess goodness of fit. (D) Immunoblot of E‐cadherin ‐RFP/Py2T cells cultured and stimulated as explained in panel B, as indicated, and analyzed for RFP, and epithelial, mesenchymal and stem cell proteins, as well as β‐actin as a loading control, along with molecular size markers (representative of n = 3 independent experiments). For original images, Fig. . (E) Relative expression of the indicated genes normalized to Gapdh in E‐cadherin ‐RFP/Py2T cells cultures as explained in panel B, shown as average values and SD from n = 3 biological replicates, each with technical triplicates, and P ‐values (* P < 0.05, ** P < 0.01, *** P < 0.001) after two‐tailed paired Student’s t ‐test. Graph bars are colour‐coded as explained in panels A and B.

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Cell Culture, Fluorescence, Western Blot, Control, Expressing, Two Tailed Test

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: Partial EMT, EpCAM low /CD51 + and EpCAM low /CD51 + /CD61 + cells, are preferentially induced by TGFβ under 3D culture conditions. (A) EMT‐score analysis by flow cytometry of cell surface proteins in the indicated two biological conditions and with detailed analysis in Fig. . EpCAM high or EpCAM low populations were gated. Three biological replicates were used for the analysis. (B) Pie charts illustrating the indicated colour‐coded cell populations as percentage of the total under the two biological conditions used. The cell type analysis was performed by flow cytometry of the indicated surface proteins in the EpCAM low population. Detailed analysis is shown in Fig. . (C) Impact of TGFβ on EMT‐scores under 3D culture conditions. The data are identical to those of panel B ( n = 3 biological replicates). Triple‐negative (TN) cells represent early EMT and correspond to EpCAM low /CD51 ‐ /CD61 ‐ /CD106 ‐ ; triple‐positive (TP) cells represent complete EMT and correspond to EpCAM low /CD51 + /CD61 + /CD106 + . (D) The EpCAM low /triple‐positive cell population generated after TGFβ stimulation under 3D conditions is graphed relative to the control (no TGFβ) condition, which is normalized to 1. The data source is identical to that in panels B and C ( n = 3 biological replicates). (E) Partial EMT‐score analysis by flow cytometry in the indicated two biological conditions. CD24 low cells were gated first, and CD44 and CD104 cell surface expression was analyzed using the indicated fluorescently‐conjugated antibodies. Three biological replicates were used for the analysis. (F) The percent of CD24 low /CD44 high /CD104 high cells is graphed for cells responding to TGFβ under 3D culture conditions. The data source is identical to that of panel E ( n = 3 biological replicates).

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Generated, Control, Expressing

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: 3D mammosphere growth and invasion under the influence of TGFβ. (A) Schematic drawing of the experiment as in Fig. . Mammospheres were placed on adherent conditions to measure cell migration. (B) Representative DIC and fluorescence microscopy overlay images for the indicated proteins (green), RFP (red) and nuclei (DAPI, blue) of E‐cadherin ‐RFP/Py2T mammospheres and migrating cells. Mammospheres were generated from 2D cells that were pre‐treated with 5 ng·mL −1 TGFβ1 for 7 days and then trypsinized and seeded in 3D cultures in the absence (3D control) or presence of 5 ng·mL −1 TGFβ1 (3D TGFβ) for 5 days, and finally transferred to 2D culture on glass chambers ( n = 3 biological replicates). Scale bar, 100 μm. (C) Violin plots quantifying RFP intensity of 2D control (epithelial) and 2D TGFβ‐treated (EMT) cells as calibration controls, and in migratory cells emanating from mammospheres. Each point represents a single cell. The total cell number was n = 60 and significance ( P ‐value) was assessed using two‐tailed paired Student’s t ‐test. (D) Representative DIC and fluorescence microscopy overlay images of migratory cells emanating from mammospheres under different TGFβ1 concentrations ( n = 2 biological replicates). Scale bar, 100 μm. (E) Mammosphere size after treatment with different TGFβ1 concentrations. Data show average values and SD from n = 6 biological replicates per condition and significance ( P ‐value) assessed using two‐tailed paired Student’s t ‐test. (F) Percentage of mammospheres exhibiting migratory cells emanating from spheres at the indicated time points and at different TGFβ1 concentrations. Data show average values from triplicate determinations. (G) Quantification of invasive area around the mammospheres after stimulation with the indicated TGFβ1 concentrations. DIC and fluorescence microscopy overlay images of two representative mammospheres (0 and 5 ng·mL −1 TGFβ1). Data show average values and SD from triplicate ( n = 3) determinations and associated significance ( P ‐value) assessed by two‐tailed paired Student’s t ‐test. (H) Percentage of migratory/non‐migratory cells emanating from mammospheres under the indicated conditions. The TGFβ type I receptor kinase inhibitor LY2157299 was used at 2.5 μ m with DMSO as vehicle. A representative from n = 3 independent experiments is shown. (I) TGFβ1 concentration was measured by ELISA in the conditioned medium of the indicated conditions. Data show average values with SD from triplicate ( n = 3) determinations and associated significance ( P ‐value) assessed by two‐tailed paired Student’s t ‐test.

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Migration, Fluorescence, Microscopy, Generated, Control, Two Tailed Test, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: TGFβ selects for pro‐stemness over pro‐invasive phenotypes during cancer cell epithelial–mesenchymal transition

doi: 10.1002/1878-0261.13215

Figure Lengend Snippet: EMTimage breast tumour analysis in mice. (A) Schematic drawing of the experiment as in Fig. , with mammospheres injected orthotopically in the mammary fat pads of mice. (B) Quantification of primary tumour volume 6 weeks after orthotopic injection of the indicated cells into 2 distinct sites of a mammary fat pad in each mouse. Data show average values and SD from n = 7 (2D control, 3D control and 3D TGFβ) or n = 8 (2D TGFβ) mice and associated significance ( P ‐value) assessed by two‐tailed paired Student’s t ‐test. (C) Primary tumour and lung metastasis incidence in the indicated conditions. (D) Representative images of lung metastases (out of n = 13 lungs analyzed). Upper, macroscopic images of whole lungs with metastatic nodules (yellow arrowheads) generated by the indicated conditions. Scale bar, 0.5 cm. Lower, H&E staining of single metastatic nodules. Scale bar, 100 μm. (E) Representative images of immunohistochemical staining of E‐cadherin and RFP in metastatic breast tumour in the lung, generated by injection of 3D control cells into mouse mammary fat pads ( n = 3 independent lungs analyzed). The tumour (T), nearby stromal area (demarcated by dotted lines and verified based on extracellular matrix content) and a blood vessel (V) are indicated. Scale bar, 20 μm. (F) The single lung metastasis in only one mouse (see panel C), observed 6 weeks after the injection of 3D TGFβ cells. H&E staining of the single lung metastatic nodule (orange arrowhead) along with immunohistochemistry for the indicated proteins in adjacent serial sections. Scale bar, 50 μm.

Article Snippet: TGFβ1 was measured in the conditioned medium of E‐cadherin ‐RFP/Py2T cells using human TGFβ Quantikine ELISA kit and mouse TGFβ1 Duoset ELISA (R&D Systems Inc., Minneapolis, MN, USA, Cat# DB100B and Cat# DY1679‐05, respectively), according to the manufacturer’s instructions.

Techniques: Injection, Control, Two Tailed Test, Generated, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Enzyme-linked immunosorbent assay (ELISA) analysis of IL-10 concentration in supernatant from Caco-2, MC-38 and Raw 264.7 cells treated with various amounts of PBS, IL-10 -mRNA or IL-10 -mRNA NPs for 24 h. ( B ) Western blot analysis of IL-10 expression in MC-38 cells treated with PBS, IL-10 -mRNA, blank NPs or IL-10 -mRNA NPs for 12 h. IL-10 -mRNA 750 ng/mL. ( C ) Confocal microscopy images of immunofluorescence staining of IL-10 expression in MC-38 cells treated with free IL-10 -mRNA or IL-10 -mRNA NPs. Hoechst (blue) was used to stain the cell nuclei. An Alexa Fluor 647-labeled antibody was used to stain IL-10. ( D ) Schematic illustration of how IL-10 -mRNA NPs directly transfect macrophages, inhibiting the polarization of macrophages to proinflammatory M1 phenotype in the presence of lipopolysaccharide (LPS), an inflammatory stimulator. ( E ) ELISA analysis of anti-inflammatory cytokine IL-10 and proinflammatory cytokine TNF-α and IL-6 in supernatant from RAW 264.7 cells treated with PBS or IL-10 -mRNA NPs (w/wo LPS stimulation) following procedures illustrated in (D). ( F ) Schematic illustration of how IL-10 -mRNA NPs first transfect intestinal epithelial cells, then indirectly induce the repolarization of macrophages from proinflammatory M1 phenotype to anti-proinflammatory M2 phenotype. ( G ) ELISA analysis of IL-10 and TNF-α and IL-6 in supernatant from RAW 264.7 cells treated with supernatant from Caco-2 cells incubated with PBS or IL-10 -mRNA NPs following procedures illustrated in (F). ( H ) Ex vivo images of excised rat gastrointestinal (GI) tract at various time points from 15 min to 6 h post-administration. Rats were orally administered six Cy5-mRNA-RNACaps (L100-55 coated). The left panel shows enlarged images of RNACaps at 1 h and 2 h (images 1–4). Cy5-mRNA: 50 μg per rat. Color scale, 0-255 gray value. ( I ) Confocal microscopy images of intestine sections from rats treated with Cy5-mRNA-RNACaps (purple) for 4 h. Hoechst (blue) was used to stain the cell nuclei. Scale bar, 100 μm. ( J ) Ex vivo images of excised intestines at 6 h post-administration. Rats were orally administered six Cy5-mRNA-RNACaps (L100 coated). Color scale, 0-255 gray value. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in ( E ) and ( G ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data in (A, B, C, E, G, H, I, J) are representative of n = 3 independent experiments. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: In Vitro, Ex Vivo, Imaging, In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Expressing, Confocal Microscopy, Immunofluorescence, Staining, Labeling, Incubation

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for oral administration of IL-10 -mRNA-RNACaps to acute colitis rat models. IL-10 -mRNA: 25 μg in 3 RNACaps per rat. Acute colitis was induced in rats by providing free access to drinking water supplemented with 6.0% (w/w) dextran sulfate sodium (DSS) for 8 days. Rats were treated with RNACaps on day 2, 5 and 8. ( B and C ) Relative body weight (B) and disease activity index (DAI) (C) of healthy (plain water-treated), DSS-treated or DSS plus IL-10 -mRNA-RNACap-treated rats were monitored daily. ( D ) Quantification of colon length on day 8. ( E to J) Colon tissue protein expression of IL-10 (E), tumor necrosis factor-alpha (TNF-α) (F), interleukin-1β (IL-1β) (G), IL-6 (H), IL-17A (I) and monocyte chemoattractant protein-1 (MCP-1) (J) by ELISA. ( K to P ) Quantification of protein expression in blood of IL-10 (K), TNF-α (L), IL-1β (M), IL-6 (N), IL-17A (O) and MCP-1 (P) by ELISA. ( Q ) H&E staining images of the colon tissue sections. Dashed box indicates inset. Scale bars, 500 μm and 400 μm. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in (B to P). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n = 5 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for the oral administration of IL-10 -mRNA-RNACaps in acute colitis rat models. Rats were given free access to drinking water supplemented with 8.0% (w/w) DSS for 10 days to induce colitis. Afterward, plain water was provided, and rats were treated with RNACaps ( IL-10 -mRNA: 25 μg in 3 RNACaps per rat.) on days 11, 14 and 17 or sulfasalazine (SSZ, standard therapy, 100 mg/kg/day) daily. ( B and C ) Relative body weight (B) and DAI (C) of healthy (plain water-treated), DSS-treated, DSS plus IL-10 -mRNA-RNACap-treated or DSS plus SSZ-treated rats were monitored daily. ( D ) Quantification of colon length on day 17. ( E to J ) Quantification tissue protein expression of IL-10 (E), TNF-α (F), IL-1β (G), IL-6 (H), IL-17A (I) and MCP-1 (J) by ELISA. ( K to P ) Quantification of protein expression in blood of IL-10 (K), TNF-α (L), IL-1β (M), IL-6 (N), IL-17A (O) and MCP-1 (P) by ELISA. ( Q ) H&E staining images of the corresponding colon tissue sections. Dashed box indicates inset. Scale bars, 100 μm and 400 μm. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in (B to P). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n = 5 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for oral administration of IL-10 -mRNA-RNACaps to rats for safety assessment. IL-10 -mRNA: 25 μg in 3 RNACaps per rat. ( B and C ) Analysis of blood chemistry, including aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) and complete blood count analysis, including white blood cell count (WBC), neutrophil (NE) (B), lymphocyte (LY), red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT) (C). ( D ) The rats were euthanized at the end of the study, and the indicated organs were sectioned and stained with H&E. Scale bars, 100 μm and 400 μm. ( E and F ) Cytokine concentration in the serum 6 h following oral administration of Fluc -mRNA-RNACap ( Fluc -mRNA: 25 μg in 3 RNACaps per rat) to healthy rats were measured using ELISA and Luminex. Cytokines measured include IL-1ra, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, and IL-10 (E) or IL-12p70, IL-13, IL-17A, IL-18, IFN-γ, TNF-α, GM-CSF, and VEGF (F). Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by unpaired two-tailed Student’s t-test in (B, C, E, F). * P < 0.05, *** P < 0.001. n = 3 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: In Vivo, Cell Characterization, Concentration Assay, Staining, Enzyme-linked Immunosorbent Assay, Luminex, Two Tailed Test

Journal: bioRxiv

Article Title: Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

doi: 10.1101/2023.05.16.540918

Figure Lengend Snippet: A. Schematic representation and immunostaining images of blastocysts and ICMs at stages E3.5 and E4.5. GATA6 and GATA4 are used as markers for PrE fate (green), and NANOG and SOX2 are used as markers for EPI fate (magenta) at stages E3.5 and E4.5, respectively. B. Quantification of total cell numbers in the ICM from blastocysts and isolated ICMs at stage E3.5, blastocysts and isolated ICMs at stage E4.5, and isolated ICMs cultured in vitro for 24 hours from stage E3.5 to E4.5. n=33, 30, 40, 21, 31 embryos for the different groups, respectively. Independent samples t-test between E3.5 blastocysts and E3.5 ICMs, p =0.106. One-way ANOVA between E4.5 Blastocysts, E4.5 ICMs, and E3.5 ICMs+24hr, p =0.145. C. Representative time-lapse imaging of ICMs isolated from E3.5 blastocysts expressing PrE-specific H2B-GFP ( Pdgfrα H2B-GFP , green) and ubiquitous H2B-mCherry ( R26-H2B mCherry , magenta), out of total 8 datasets from 3 independent experiments. Time is indicated in hh:mm, t=00:00 corresponds to start of live-imaging at stage E3.5+3hours, following completion of immunosurgery. D. Schematic representation of single-cell tracking of EPI and PrE cells from isolated ICMs from (C). Line plots indicating radial distances of all cells from one representative ICM until E4.0 stage. Colour of the line indicates cell fate – PrE, green and EPI, magenta. Shaded regions show spatial dispersion as mean ±SD of cell position along ICM radial axis. The geometric centroid of the ICM is considered as d=0.0 and ICM outer surface is considered as d=1.0 to normalise cell position across samples. Time-series plots for cell position were smoothed using a rolling average. E. Quantification of sorting score for isolated ICMs between stage E3.5 and E4.0. Data from n=8 ICMs. F. Line plots for radial cell position versus time from tracking of PrE and EPI cell movements in isolated ICMs. Time-series plots for cell position were smoothed using a rolling average. Cell tracking data pooled from n=160 PrE cells and n=133 EPI cells from 8 ICMs. G. Schematic diagram for analysis of PrE and EPI cell movements. Cell displacement is measured along the radial axis between consecutive timepoints and classified as inward or outward movement depending on the direction of displacement. H. Polar plots indicating preferential direction of cell movements among PrE and EPI. Cell position is plotted along radial axis, time is plotted along angular axis. Measurements are binned according to initial radial cell position and time. The mean displacement of each interval is plotted, colour indicates direction of movement. Scale bars 20μm. ns, non-significant

Article Snippet: Primary antibodies against GATA6 (R&D systems, AF1700), GATA4 (R&D systems, BAF2606), SOX2 (Cell Signaling, 23064), bi-phosphorylated myosin regulatory light chain (ppMRLC) (Cell Signaling, 3674), and Laminin (Novus Biologicals, NB300-14422) were diluted at 1:200.

Techniques: Immunostaining, Isolation, Cell Culture, In Vitro, Imaging, Expressing, Single Cell Tracking, Dispersion, Cell Tracking Assay

Journal: bioRxiv

Article Title: Apical-driven cell sorting optimised for tissue geometry ensures robust patterning

doi: 10.1101/2023.05.16.540918

Figure Lengend Snippet: A. Immunofluorescence image of a 3x blastocyst at stage E3.75 showing laminin distribution around PrE cells. White dotted line, ICM-cavity interface. White arrowhead marks GATA6-expressing nucleus of a PrE cell enriched for laminin expression. B. Immunofluorescence image of a 3x blastocyst at stage E3.75 showing PKCλ+ζ distribution in PrE cells. White arrowhead marks leading edge of a PrE cell with PKCλ+ζ localisation. C. Immunofluorescence image of an E3.75 ICM showing PKCλ+ζ localisation in PrE and EPI cells. White dotted lines mark cell boundaries. Yellow line indicates the line segments from cell inner edge (towards ICM centroid) to cell outer edge (towards ICM-fluid interface) along which fluorescence intensity is measured. D. Line plots for normalised fluorescence intensity of PKCλ+ζ in individual inside cells from E3.75 isolated ICMs. Colour of the line indicates GATA6-expression level of the cell. n=260 cells from 32 ICMs. Each of the thin lines corresponds to measurement from one cell. Bold line and shaded region indicate mean±SD of aPKC intensity for GATA6-high and GATA6-low cells. E. Schematic description of polarisation index. Polarisation index is calculated as the ratio between mean aPKC intensity at 1/4 th distance from outer edge and mean aPKC intensity at 1/4 th distance from inner edge. Boxplots for comparison of the polarisation index in PrE (GATA6 high) versus EPI cells (GATA6 low). GATA6 expression level is categorised as high or low by thresholding the bimodal distribution of GATA6 fluorescence intensity. Colour of the line indicates GATA6-expression level of the cell. n=136 GATA6-high and 124 GATA6-low cells from 32 ICMs. One-way ANOVA, p =6.03e -20 . F. Scatterplot of polarisation index of cells versus radial distance of the cell from the ICM centroid. Colour of the datapoint indicates GATA6-expression level of the cell. Black dotted line, linear regression with Pearson’s R=0.079, p =0.205. n=260 cells from 32 ICMs. G. Immunofluorescence images of control and Gö6983-treated E4.0 isolated ICMs and quantification of sorting score. n=16,24 ICMs for control and Gö6983-treated ICMs respectively. Independent samples t-test, p = 8.01e -04 H. Immunofluorescence images of representative WT, Prkci +/+ ;Prkcz -/- , and Prkci +/- ;Prkcz -/- E4.5 blastocysts and quantification of number of ectopic PrE cells in E4.5 blastocysts from each group. n= 25, 17, 12 blastocysts for WT, Prkci +/+ ;Prkcz -/- , and Prkci +/- ;Prkcz -/- respectively. Mann-Whitney U test, p = 2.43e -04 , 6.36e -04 Scale bars 20μm. ns, non-significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001

Article Snippet: Primary antibodies against GATA6 (R&D systems, AF1700), GATA4 (R&D systems, BAF2606), SOX2 (Cell Signaling, 23064), bi-phosphorylated myosin regulatory light chain (ppMRLC) (Cell Signaling, 3674), and Laminin (Novus Biologicals, NB300-14422) were diluted at 1:200.

Techniques: Immunofluorescence, Expressing, Fluorescence, Isolation, Comparison, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence imaging of immune synapses between H1299 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. H1299 lung cancer cells are pretreated with phosphate- buffered saline (PBS) or DAC prior to coculture with γδ T cells. Quantifications of immune synapses per cancer cell on eight randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). Scale bar: 100 μm. p value is calculated by the Mann-Whitney test. ( B ) A scatter plot of DAC-induced surface proteomes in H1299 (y-axis) and A549 (x-axis) human lung cancer cells following daily treatment of 100 nM DAC for 72 hours and culture in drug-free medium for 3 days (D3R3). ICAM-1 is among the top upregulated surface proteins by DAC in both cells. ( C ) Western blot analyses of ICAM-1 protein expression in mock-treated vs. DAC-treated human lung cancer cells. D3: daily treatment of 100 nM decitabine for 72 hours. D3R3: daily treatment for 72 hours, followed by a 3-day rest period in drug-free medium. β-actin: loading control. ( D ) Immunofluorescence staining of ICAM-1 and immune synapse molecules (e.g., LFA-1, LAT) at immune synapses formed between γδ T cells and DAC-treated H1299 lung cancer cells. Scale bar: 10 μm. ( E ) Representative flow cytometric dot plot showing H1299 lung cancer cells with CRISPR-knockout of ICAM1 (KO-ICAM1) subject to γδ T cell killing for 2 hours. The effector to target (E: T) ratio is 3:1. Lung cancer cells are pre-treated with mock, DAC alone, γδ T cells alone or a combination of DAC and γδ T cells. The X-axis denotes surface ICAM1 levels. Y-axis represents signal intensities of propidium iodide. ( F ) Bar graphs showing percent cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with CRISPR-knockout of ICAM-1 subject to γδ T cell killing for 2 hours. Cell death is measured by Annexin V and propidium iodide apoptosis assays (mean ± SEM, n = 3). Statistical significance is determined by one-way ANOVA test. ( G ) Representative flow cytometric dot plot showing H1299 lung cancer cells with a Tet-on expression system of ICAM1 (OV-ICAM1) subject to γδ T cell killing for 2 hours. Doxycycline (1 μg/mL) is added 24 hours prior to coculture to induce ICAM-1 protein expression. Cell death is measured by Annexin V (x-axis) and propidium iodide (y-axis) apoptosis assays. ( H ) Bar graphs showing cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with ICAM-1 over-expression subject to γδ T cell killing for 2 hours. E:T ratio is 3:1. Cell death is measured by Annexin V and propidium iodide apoptosis assays. Statistical significance is determined by one-way ANOVA test (* p < 0.05, ** p < 0.01, ***, p < 0.001). ( I ) Immunofluorescence imaging of immune synapses between H1299 KO-ICAM1 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. Scale bar: 100 μm. Quantifications of immune synapses per cancer cell on six randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). p value is calculated by the Mann-Whitney test.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Imaging, Staining, Saline, MANN-WHITNEY, Western Blot, Expressing, Control, CRISPR, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Sequencing results of the KO-ICAM1 lung cancer cells are aligned against the reference sequence of the ICAM1 genome locus. Alignment gaps are denoted as hyphens (-) to mark the lost (knockout) regions of the edited ICAM1 genome locus.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Sequencing, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence staining of F-actin (red), ICAM-1 (green), pTyr (phosphotyrosine, white) at immune synapses between γδ T cells and DAC-pretreated H1299 lung cancer cells at D3R3. Accumulation of F-actin beneath the cell membrane is noted in DAC-pretreated lung cancer cells. DAPI: 4′,6-diamidino-2-phenylindole, as a nuclear counterstain. Scale bar: 10 μm. ( B ) Representative immunofluorescence images of the interfaces between γδ T cells and H1299 lung cancer cells (parental vs. ICAM-1 knockout (KO-ICAM1)). Signals of F-actin (red) in the periphery of H1299 cancer cells are shown in two- and-a-half-dimensional (2.5D) images in the lower panels. Scale bar: 10 μm. ( C ) Dot plots of signal intensities of F-actin (left panel) and ICAM-1 (right panel) from five pTry-positive immune synapses between γδ T cells and H1299 lung cancer cells (parental or KO-ICAM1). p value is calculated by two-way ANOVA test. ( D ) Immunofluorescence images of immune synapses between γδ T cells (marked with T) and H1299 lung cancer cells (marked with C) stained for ICAM-1 (green), F-actin (Red) and phosphotyrosine (pTyr, white). Lung cancer cells (parental or KO-ICAM1) are pretreated with PBS (Mock) or 100 nM DAC and cocultured with γδ T cells at D3R3. ( E ) Dot plots of F-actin signal intensities at immune synapses between γδ T cells and H1299 cells. H1299 cells are pretreated with PBS (Mock), DAC alone or combination of DAC pretreatment (D3R3) and 1 μg/mL Cyto B (cytochalasin B, an inhibitor of actin filament polymerization) for 1.5 hours prior to coculture with γδ T cells (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (***, p < 0.001; ****, p < 0.0001). ( F ) Representative immunofluorescence images of immune synapses (pTyr staining) between γδ T and H1299 cells pretreated with PBS (Mock), DAC alone, and combination of DAC and Cyto B. Blow-up images of the square areas for each treatment are shown in the lower panels. Arrows denote immune synapses between γδ T and H1299 cells. Scale bar: 100 μm (upper) and 20 μm (lower panels). ( G ) Dot plots showing numbers of immune synapses per cancer cell on eight randomly taken high power fields for H1299 cells pretreated with PBS (Mock), DAC, and combination of DAC and Cyto B (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (*, statistical significance).

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Staining, Membrane, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Parental or ICAM-1 knockout (KO-ICAM1) H1299 cells are pretreated daily with PBS (Mock) or 100 nM DAC for 72 hours followed by 3-day drug-free culture before coculture with γδ T cells. Signal intensities of each protein (F-actin, red; ICAM-1, green; phosphotyrosine, pTyr, white) along the immune synapse area are graphed on the right. DAPI: 4’,6-diamidino-2-phenylindole, as nuclear counterstain. Scale bar: 10 μm.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Visualization of multi-omics data (i.e., mRNA-seq, Omni-ATAC-seq, and MethylationEPIC arrays) for DAPK3 , EVPLL, and TUBE1 in H1299 lung cancer cells. ( B ) Promoter methylation status and mRNA expression levels of the ICAM1 gene measured by Infinium MethylationEPIC arrays (left panels) and mRNA-seq (right panels) in human lung cancer cells treated without and with DAC 100 nM DAC for 3 days followed by a 3-day drug-free culture. ( C ) Open chromatin regions in the promoter areas of the ICAM1 gene in human lung cancer cells upon 100 nM DAC treatment analyzed by Omni-ATAC-seq. The green bar represents a CpG island. ( D ) Validation of Omni-ATAC-seq by quantitative real-time PCR on transposase-accessible chromatin at the ICAM1 promoter of human lung cancer cells subject to daily treatment of 100 nM DAC treatment for 3 days, followed by a 3-day drug-free culture. Experiments are performed in triplicates, and data are presented as mean ± SD. p value was calculated by unpaired t test (*, p < 0.05). ( E ) IPA Network analysis of mRNA expression changes in human lung cancer cells treated by DAC reveals coordinated changes of the immune-related surface molecules and the cytoskeleton-associated genes. ( F ) IPA upstream regulator analysis of mRNA expression changes in human lung cancer cells treated by DAC. T cell effector cytokines such as TNF-α and IFN-γ may enhance DAC-induced expression changes of immune-related molecules and ICAM-1 in lung cancer cells. TP53 is a potential master regulator for cancer cytoskeleton reorganization essential for DAC-potentiated γδ T cell killing.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Biomarker Discovery, Methylation, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Diagram of transcription factor binding sites at the ICAM1 promoter derived from the ENCODE ChIP-seq data ( https://www.encodeproject.org ). Visualizations of ATAC-seq peaks at the ICAM1 promoter in PC9 and CL1-5 lung cancer cell lines subject to DAC treatment are shown above. ( B ) Promoter methylation status and mRNA expression levels of putative transcription factors (i.e., RELB, NFKB2, STATS, and RUNX3) at the ICAM1 promoter in A549, H1299, PC9, and CL1-5 lung cancer cells. Dot and line plots represent methylation levels (β values) of promoter probes measured by Infinium MethylationEPIC arrays. The promoter probes with β values greater or equal to 0.5 at baseline (Mock) are shown. Bar graphs represent relative mRNA expression levels based on normalized FPKM measured by mRNA-seq.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Binding Assay, Derivative Assay, ChIP-sequencing, Methylation, Expressing

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is an epithelial cytokine expressed in alveolar epithelium and airway basal cells in human healthy and asthmatic lungs. (A) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in the LungMAP single-cell human lung atlas. Uniform manifold projection (UMAP) plots show the clustering of 347,970 lung cells (10 single-cell datasets, 148 normal human lung samples from 104 donors: adult, child, and adolescent). Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B and C) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in epithelial cells from human healthy (B) and asthmatic (C) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (B; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (C; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas and downloaded from https://asthma.cellgeni.sanger.ac.uk .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Generated, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Single-cell RNA-seq analysis of IL33 and TSLP expression in human lungs and gating strategy for analysis of mouse lung epithelial cells by flow cytometry. (A and B) Single-cell RNA-seq analysis of IL33 and TSLP expression in epithelial cells from human healthy (A) and asthmatic (B) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (A; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (B; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas , and downloaded from https://asthma.cellgeni.sanger.ac.uk . (C) Gating strategy of Epcam + epithelial cells and CD31 + endothelial cells in the lung of a naïve WT mouse. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with two distinct rat IgG1 isotype controls (rat IgG1 clone eBRG1, D, red; rat IgG1 clone RB40.34, E, red) for the anti-TL1A antibody (rat IgG1, MAB7441, clone 293327). Double staining was performed with antibodies against RAGE (D, green) or IL-33 (E, green). Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Control, Immunohistofluorescence, Staining, Double Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is expressed in mouse alveolar epithelium at steady state. (A) Visualization of Tnfsf15 (TL1A) expressing cells in the LungMAP single-cell mouse lung atlas. UMAP plots show the clustering of 95,658 lung cells (17 samples from late developmental stage to postnatal day 28). The different cell types in the lungs of naïve mice are indicated on the left. Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B) Single-cell RNA-seq analysis of Tnfsf15/TL1A and Il33 gene expression in mouse lung epithelium. UMAP plots show clustering and cell type annotation of 12,536 mouse lung epithelial cells (seven samples from the emergence of the alveolus to postnatal day 28) . The number and percentage of epithelial cells expressing Tnfsf15/TL1A , Il33 , or both are indicated on the right. Results are visualized using ShinyCell and are based upon data obtained by and downloaded from http://www.lungmap.net . (C) Flow cytometry analysis of cell surface TL1A expression on live CD31 + CD45 − endothelial cells and Epcam + CD31 − CD45 − epithelial cells in the lung of a naïve wild type C57BL/6J mouse at steady state. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with antibodies against TL1A (D and E) and RAGE (D) or IL-33 (E) proteins. A tyramide signal amplification (TSA)-based immunofluorescence method was used to detect TL1A-expressing cells in situ. Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Expressing, Generated, RNA Sequencing, Gene Expression, Flow Cytometry, Immunohistofluorescence, Staining, Amplification, Immunofluorescence, In Situ

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: High throughput proteomic analyses of lung ILC2s stimulated ex vivo with IL-33 and/or TL1A. (A) Flow cytometry of cultured lung ILC2s ex vivo. Representative histograms of ST2, CD90.2, Sca-1, CD25, ICOS, KLRG1, and DR3 expression at the surface of cultured ILC2s, 3 days after ILC2 cell isolation from the lung and ex vivo culture in the presence of IL-2. Phenotypic analysis was performed on live Lin – CD45 + cells. (B–D) Large-scale label-free proteomic analyses of mouse lung ILC2s after ex vivo overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plots of IL-33-stimulated ILC2s (B) or TL1A-stimulated ILC2s (C) compared with non-stimulated cells (NS; in culture with IL-2 alone). Volcano plot of IL-33/TL1A-stimulated ILC2s compared to IL-33-stimulated cells (D). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS and IL33 stimulation; n = 3 for TL1A and IL33/TL1A stimulations), using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Representative examples of proteins found modulated in each comparison are shown in color. (E) Flow cytometry of cultured lung ILC2s after 14 h of co-stimulation with IL-33 and TL1A in the presence of IL-2 (ILC2 culture used in ). Intracellular cytokine staining revealed that >99% of ILC2s co-expressed IL-9 and IL-13 intracellularly. Phenotypic analysis was performed on live Lin − CD45 + CD90.2 + cells.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: High Throughput Screening Assay, Ex Vivo, Flow Cytometry, Cell Culture, Expressing, Cell Isolation, Quantitative Proteomics, Comparison, Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A synergizes with IL-33 to induce an IL-9-producing ILC9 phenotype in lung ILC2s. (A and B) Large-scale label-free proteomic analyses of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice and cultured with IL-2 prior to overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plot of IL-33/TL1A-stimulated ILC2s (ILC9 cells) compared with nonstimulated cells (NS; in culture with IL-2 alone) (A). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS; n = 3 for IL33/TL1A stimulation) using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Examples of proteins modulated in both IL-33/TL1A-stimulated ILC2s and IL-33-stimulated ILC2s are shown in blue. Proteins shown in red are representative of molecules specifically modulated in IL-33/TL1A-stimulated ILC2s (A). Heat-map of fold changes of selected proteins in three independent biological replicates (B). (C–K) Analysis of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice , and cultured with IL-2 prior to 14 h stimulation with rIL-2 ± rIL-33 ± rTL1A. Flow cytometry analysis of live Lin − CD45 + cells (C, E, and J), frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) (D and K), and MFI fold change of IL-9 in ILC2s (E), after cytokines treatment and restimulation by PMA, ionomycin, and brefeldin A (4 h, C–E) or brefeldin A (4 h, J and K). Concentration of IL-9 secreted by ILC2s, measured by ELISA (F). Relative STAT5 mRNA expression levels measured by real-time qPCR (G). Samples were normalized to the expression of HPRT and are shown relative to IL-2-stimulated ILC2s. Immunoblot analysis of activated phosphorylated STAT5 (pSTAT5) and α-tubulin (H) or β-actin (I); Arrowheads indicate the migration of the protein of interest; cropped images. Cultured ILC2s were treated with rIL-2 + rIL-33 + rTL1A and increasing doses of a STAT5 inhibitor (STA5i, CAS 285986-31-4) or control vehicle (DMSO) (I–K). Numbers inside outlined areas (C) indicate percent of cells in the relevant gate. Each symbol represents an individual biological replicate (D–G and K). Data are pooled from six (D and E), six to eight (F) or three (G and K) independent experiments, or are representative of six (C and E) or three (H–J) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (D–G and K): ns not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Isolation, Cell Culture, Quantitative Proteomics, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Migration, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s ex vivo. (A) Analysis of cultured lung ILC2s 14 h after ex vivo stimulation by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Flow cytometry analysis of live Lin − CD45 + cells and frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) after cytokine treatment and incubation with brefeldin A (4 h), without restimulation by PMA and ionomycin. Numbers inside outlined area indicate percent of cells in the relevant gate and data are representative of eight independent experiments. (B) Concentration of IL-9 secreted by ILC2s treated with rIL-2 (20 ng/ml) and various concentrations of rIL-33 and rTL1A measured by ELISA. (C and D) MFI of nuclear factor IRF4 (C) and flow cytometry (D) of ILC2s 14 h after ex vivo stimulation of cultured ILC2s by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Numbers inside outlined areas (D) indicate percent of cells in the relevant gate and data are representative of three independent experiments. (E) Immunoblot analysis of JunB and α-tubulin14 h after cytokine stimulation of lung ILC2s; Arrowheads indicate the migration of the protein of interest; cropped image. Data are representative of three independent experiments. (F–H) Relative mRNA expression levels by real time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed relative to IL-2-stimulated ILC2s (F) or relative to HPRT mRNA quantity (G and H). (I and J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-33 + rTL1A ± rIL-2 ± rIL-7 ± rTSLP. Frequency of IL-9 high ILC2s (Lin − CD45 + CD90.2 + cells), after cytokines treatment and re-stimulation by PMA, ionomycin and brefeldin A (4 h, I). Concentration of IL-9 secreted by ILC2s, measured by ELISA (J). (K) Concentration of IL-9 (ELISA) secreted by ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rIL-4 ± rTGF-β. Each symbol represents an individual biological replicates with n = 2–5 independent experiments (A–C and F–K). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (A, C, and F–J) or Dunnett’s (B and K) multiple-comparisons tests: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. In H, all significant P values are annotated with stars, all other comparisons are not significant. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Ex Vivo, Cell Culture, Flow Cytometry, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Migration, Expressing

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A induce phenotypic changes in cultured lung ILC2s at the protein and mRNA levels. (A–J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rTL1A. MFI of the indicated cell surface markers determined by flow cytometry (A, B, D, and E). Relative mRNA expression levels of various genes (C and F–I), including genes characteristic of ILC1s or ILC3s (I), determined by real-time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed as relative to HPRT mRNA quantity. Concentration of IL-5 or IL-13 in cell supernatants, measured by ELISA assay (J). Each symbol represents an individual biological replicate from independent experiments (A–J). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test (B, E, and J) or one-way ANOVA followed by Tukey’s multiple-comparisons test (A, C, D, and F–I): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. In I, all significant P values are annotated with stars, all other comparisons are not significant.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Cell Culture, Ex Vivo, Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A cooperates with IL-33 for induction of IL-9 high ILC2s in vivo. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B) Gating strategy of IL-9 high IL-5 + IL-13 + ILC2s. (C–I) Flow cytometry of IL-5 + IL-13 + ILC2s gated on live ILCs (Lin − CD45 + CD90.2 + cells) (C) and IL-9 high ILC2s gated on live IL-5 + IL-13 + ILC2s (E), frequency of lung IL-5 + IL-13 + ILC2s among live ILCs (D), IL-9 high ILC2s among live IL-5 + IL-13 + ILC2s (F), and IL-9 high IL-13 + ILC2s among live ILCs (G) or IL-9 high ILCs (H), and concentration of IL-9 in BAL fluids (ELISA assay, I) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate and data are representative of two independent experiments (C and E). Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (D) or Dunnett’s (F, G, and I) multiple-comparisons tests: ns, not significant, ** P < 0.01, **** P < 0.0001. (J) Frequency of lung eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells, at day 7 after a single i.n. exposure to rIL-33 or rIL-33 plus rTL1A. Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test: * P < 0.05. (K and L) Multiphoton imaging (K) and intravital microscopy (L) of whole lungs of INFER IL-9 fluorescent reporter mice, with detection of IL-9-eGFP + ILC2s (green) and staining of blood vessels (red) and collagen fibers (blue), 16–18 h after a single i.n. administration of IL-33/TL1A combination (1 μg rIL-33 plus 5 μg rTL1A). To increase the numbers of lung IL-9 high ILC2s accessible to in vivo imaging, the single i.n. exposure to IL-33/TL1A combination was performed after prior expansion of lung ILC2s by repeated i.p. injections of IL-33 (K and L). Multiphoton image (K) is a 3D reconstitution of stitched images (7 × 7 tiles and 181 z-stack). Time-lapse images (L) illustrate the migratory behavior of IL-9-eGFP + ILC2s. Time in h/min/s. Scale bars: K, 300 μm; L, 20 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Imaging, Intravital Microscopy, Staining, In Vivo Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s in vivo. (A) Gating strategy and representative flow cytometry plots of live lung ILCs (live Lin − CD45 + CD90.2 + cells), live lung IL-5 + IL-13 + ILC2s (live IL-5 + IL-13 + ILCs) and live lung IL-9 high ILC2s (live IL-9 high IL-5 + IL-13 + ILC2s) in vivo in wild type (WT) C57BL/6J mouse, 14 h after a single i.n. administration of rIL-33 (1 μg) and rTL1A (5 μg). (B) Verification of the absence of contamination of the IL-5 + IL-13 + ILC2s and IL-9 high ILC2s populations by TCR + cells (T cells and NKT cells) using anti-TCRβ and anti-TCRγδ antibodies. (C) Confirmation of the expression of IL-5 and IL-13 in live Lin − CD3/TCR − NK1.1 − CD45 + CD90.2 + lung ILCs using antibodies against CD3/TCR and NK1.1 with a different fluorescence from the Lin cocktail (CD4, CD19, CD45R, CD11b, CD11c, Ter119, Ly6G, FcεRI). (D and E) Frequency of lung IL-9 high Lin − cells among live CD45 + cells (D), and flow cytometry of IL-9 high IL-13 + ILC2s (live IL-9 high IL-13 + Lin − CD45 + CD90.2 + cells) (E) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate. (F) Frequency of lung IL-9 high Lin − cells among live CD45 + cells of WT mice pretreated with six daily i.p. injections of rIL-33 (days 1–6) prior to one i.n. injection of PBS or rIL-33 and/or rTL1A (day 7). Flow cytometry analyses were performed on day 8. (G) Frequency of IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αIL-2 mAb) or without (Iso, isotype control mAb) IL-2 blockade. (H and I) Analysis of IL-9 and TL1A release in BAL fluids by ELISA at different time points after the third exposure to A. alternata in a chronic exposure model (repeated i.n. administration of 12.5 μg A. alternata at days 0, 3, and 6). Each symbol represents an individual mouse and data are pooled from two (D and G) or three (F, H, and I) independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (G) or one-way ANOVA followed by Dunnett’s multiple-comparison test (D, F, H, and I): * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Expressing, Fluorescence, Injection, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s accumulate around blood vessels after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by multiphoton imaging in the cleared lung of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. 360° rotation of a 3D static representation at a frame rate of 25 fps (500 frames per 20 sec).

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s migrate along collagen fibers after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by lung intravital multiphoton imaging of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A functions as an epithelial alarmin rapidly released after allergen exposure. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B–F) Analysis of TL1A and IL-33 release in BAL fluids after a single allergen exposure. TL1A (B and E), IL-33 (C and F), and LDH (D) levels in BAL fluids were determined by ELISA (B, C, E, and F) or LDH (D) assays, 15 min (B–D) or at different time points (E and F) after a single i.n. administration of A. alternata extract (12.5 μg). Each symbol represents an individual mouse and data are pooled from two independent experiments (B–F). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (B–D) or Dunnett’s (E and F) multiple-comparisons tests: ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G–K) Analysis of TL1A release in cell supernatants after exposure of TL1A-expressing cells to A. alternata or bee venom phospholipase A2 (PLA2). U2OS epithelial cells transfected with a mouse TL1A-Flag expression vector (mTL1A-Flag vector) or control vector were analyzed by indirect immunofluorescence microscopy with anti-mTL1A and anti-Flag antibodies (G). Scale bar, 20 μm. TL1A (H and J) and LDH (I and K) levels in cell supernatants were determined by ELISA (H and J) or LDH cytotoxicity assays (I and K) 15 min after treatment with A. alternata extract ( A. alternata , H and I) or 1 h after treatment with bee venom PLA2 (J and K). NT, not treated. Each symbol represents an individual biological replicate and data are pooled from three independent experiments (H–K). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (treatment versus NT): ** P < 0.01, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Control, Immunofluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A is important for early induction of IL-9 high ILC2s after allergen exposure. (A) Treatment schedule of naïve WT mice. (B) IL-9 mRNA levels in the lungs analyzed by qPCR at different time points after a single allergen exposure. Data are expressed as relative to IL-9 mRNA levels in mice treated with PBS. (C–H) Flow cytometry and frequency of IL-9 high Lin − cells among live CD45 + cells (C and D) and IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) (E and F), flow cytometry (G), and MFI of IRF4 expression in ILC2s (H), in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αTL1A mAb) or without (Iso, isotype control mAb) TL1A blockade. Numbers inside outlined areas indicate the percent of cells in the relevant gate (C, E, and G) and data are representative of two (G) or three (C and E) independent experiments. Each symbol represents an individual mouse and data are pooled from three (D and F) or two (B and H) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (B) or unpaired two-tailed Student’s t tests (D, F, and H): ns, not significant, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: ILC9 cells have an increased capacity to initiate IL-5-dependent allergic airway inflammation. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice by a single i.v. adoptive cell transfer of classical IL-33-activated ILC2s (ILC2) or IL-33/TL1A-activated ILC2s (ILC9). (B–H) Flow cytometry (B and D) and frequency of eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells from BALF (C and F) or lung (E and G), and number of Red5 + ILC2s or ILC9s in total lung of mice (H), at day 7 after a single i.v. adoptive transfer of 5 × 10 5 ILC2s or ILC9s in separate host mice. Adoptively transferred ILC2s and ILC9s were prepared from Rag2 −/− mice ( Il5 +/+ cells) (B–E) or Red5 mice ( Il5 −/− cells) (F–H). Control mice received an intravenous injection of PBS. Red5 + cells indicate the activity of the Il5 promoter. Each symbol represents an individual mouse and data are representative (B and D) or pooled (C and E–H) from two independent experiments. (I–K) Live imaging of ILC2s and ILC9 cells in the lung. Lung intravital microscopy was performed 1–4 h after adoptive transfer of 6 × 10 5 of each cell type in the same host (green, classical IL-33-activated ILC2s-CFSE + ; red, IL-33/TL1A-activated ILC9 cells-CTO + ) (I). Imaging of the migratory behavior of ILC2s and ILC9 cells in the lung (J) and cell quantification from lung intravital microscopy data (K). Time-lapse images, 2 h after adoptive cell transfer (J). A maximum intensity projection of stitched images (2 × 2 tiles and 18 z-stack) is shown (K). Time in h/min/s. Scale bars: J, 20 μm; K, 100 μm. Lung intravital microscopy data are representative (J and K) or analyzed (K) from three adoptive transfer experiments on four mice. Data are expressed as mean (±SEM) with P values determined by paired two-tailed Student’s t test (K) or one-way ANOVA followed by Tukey’s multiple-comparisons test (C and E–H): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Adoptive Transfer Assay, Control, Injection, Activity Assay, Imaging, Intravital Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Adoptively transferred ILC2s and ILC9s are equally recruited to the lung and exhibit an ameboid-like mode of migration. IL-33-activated ILC2s (CFSE/green), IL33/TL1A-activated ILC9s (CTO/red), blood vessels (Evans Blue/dark blue), and collagen fibers (second harmonic generation/light blue) were observed by lung intravital multiphoton imaging 2 h after intravenous adoptive transfer (6 × 10 5 cells). Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Migration, Imaging, Adoptive Transfer Assay

Journal: Stem Cell Research & Therapy

Article Title: Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell-based in vitro disease modeling: convergence of schizophrenia- and autism-related cellular phenotypes

doi: 10.1186/s13287-020-01980-5

Figure Lengend Snippet: Establishment and molecular characterization of NPC lines and mature neuronal cultures. Investigation of target genes KHSRP and LRRC7. a Changes in gene expression patterns in NPCs and neurons derived from the case-parent trio. b , c NPCs and neurons derived from hiPSCs by the hippocampal neuronal differentiation protocol were investigated by immunofluorescence staining and visualized by confocal fluorescent microscopy. Immunocytochemical staining shows Nestin/Sox2 ( b ) and Map 2/Prox1 ( c ) positivity in these established neural cell types. Scale bars = 100 μm. d , e Immunofluorescence staining for KHSRP and LRRC7 in neurons. KHSRP ( d ) shows nuclear and cytoplasmic localization, while LRRC7 ( e ) localized postsynaptically in neurons. Scale bars = 50 μm

Article Snippet: The samples were then incubated for 1 h at room temperature with antibodies anti-SOX2 (monoclonal/mouse, 1:20 dilution; MAB2018, R&D Systems, Minneapolis, USA) and anti-Nestin (polyclonal/rabbit, 1:250 dilution; ab92391, Abcam, Cambridge, UK) or for overnight at 4 °C with antibodies anti-PROX1 (polyclonal/rabbit, 1:500 dilution; ab101851, Abcam, Cambridge, UK) and anti-MAP 2 (monoclonal/mouse, 1:500 dilution; M1406, Sigma/Merck, Darmstadt, Germany or polyclonal/rabbit, 1:1000 dilution; ab5622, Millipore, MA, USA).

Techniques: Gene Expression, Derivative Assay, Immunofluorescence, Staining, Microscopy

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Gene Expression, Derivative Assay, Staining

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Derivative Assay, Biomarker Discovery, Microarray, Western Blot, Control, Immunofluorescence, Membrane, Marker

Journal: bioRxiv

Article Title: An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii

doi: 10.1101/562587

Figure Lengend Snippet: A, B) Microscopic images of T. gondii parasites showing localization of an HA tagged TgTPx1/2 (red) with a mitochondrial marker SPTP-SOD2-GFP (green) and an apicoplast marker ACP (green). C) A Western blot analysis for total cell lysate of parasites stably expressing TgTPx1/2-HA using anti-HA antibodies. Full-length Western blot is represented in Supplementary Fig. S3. D, E) Fluorescence images of parasites stably expressing TgTPx1/2N 1-50 -EGFP (green) with a mitochondrial marker SPTP-SOD2-DsRed (red) and apicoplast maker, ACP (red). Scale bar, 2µm. Numbers (in black) at the top right corner of the DIC+Merge panel indicates the number of parasites inside the vacuole.

Article Snippet: The proteins were transferred to PVDF membrane, blocked for an hour with 5% BSA-Tris Buffered Saline (TBS) and probed with primary antibodies anti-HA (C29F4) Rabbit monoclonal antibody (mAb) (Cell Signaling Technology®) or mouse raised anti-GFP antibodies (Roche) for 2 hours.

Techniques: Marker, Western Blot, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii

doi: 10.1101/562587

Figure Lengend Snippet: A) SignalP 3.0-HMM and MitoProt scores for the wildtype TgTPx1/2 and the mutants TgTPx1/2(R24A) and TgTPx1/2(L17A,L27A). Immunofluorescence images of the parasites expressing B, C) TgTPx1/2(R24A) (red/green) and D, E) TgTPx1/2(L17A,L27A) (green/red). Here ACP is used to label the apicoplast (green) while Mitotracker Red (red) is used for labelling the mitochondrion of T. gondii . Scale bar, 2µm. Numbers (in black) at the top left corner of the DIC panel indicates the number of parasites inside the vacuole. F) A Western blot analysis for the whole parasite lysates of wildtype TgTPx1/2, TgTPx1/2(R24A) and TgTPx1/2(L17A,L27A) using anti-HA antibodies. Full-length Western blot is represented in Supplementary Fig. S3.

Article Snippet: The proteins were transferred to PVDF membrane, blocked for an hour with 5% BSA-Tris Buffered Saline (TBS) and probed with primary antibodies anti-HA (C29F4) Rabbit monoclonal antibody (mAb) (Cell Signaling Technology®) or mouse raised anti-GFP antibodies (Roche) for 2 hours.

Techniques: Immunofluorescence, Expressing, Western Blot

Journal: Nature Neuroscience

Article Title: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

doi: 10.1038/s41593-024-01615-5

Figure Lengend Snippet: a , Immunofluorescence (IF) images of primary cortical neurons immunostained for glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2) simultaneously with PSD95 to show that the cultures are devoid of glial cells or oligodendrocytes, respectively. DAPI for nuclei; PSD95 for excitatory neurons. Magnification, ×40. Scale bars, 50 μm. b , TurboID-PSD95 was cloned without (top row) and with (bottom row) its 5′ and 3′ UTRs and lentivirally expressed in primary cortical neurons. White dashed boxes are zoomed in areas in black&white images. DAPI for nuclei; MAP2 for dendrites; Flag for each TurboID. % dendritically localized TurboID-PSD95 is quantified by co-localization with MAP2 signal in ImageJ. 3 different areas of images per replicate ( n = 3). Magnification, ×20. Scale bars, 50 μm. Significance was derived from biological replicates, showing the center line at mean. c , IF images of TurboID-PSD95-transduced neurons immunostained for DAPI (blue, for nuclei), PSD95 (red, for endogenous PSD95) and TurboID-PSD95 (cyan, detected by Flag). Magnification, ×60. Scale bar, 50 μm. d , IF images show the expression of a presynaptic marker, Synaptophysin (cyan), and TurboID-PSD95 (red, detected by Flag antibody) in primary cortical neurons transduced with TurboID-PSD95. DAPI (blue) marker for nuclei. Three zoomed in regions are marked by the white boxes. Magnification, ×60. Scale bar, 10 μm. e , IF images show TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 or Pan-TurboID after 30 minutes of biotin incubation. DAPI (blue, nuclei); MAP2 (green, dendrites); Flag (red, TurboID); and Streptavidin (cyan, biotinylated proteins). Magnification, ×20. Scale bars, 50 μm. f , Western blots stained for Flag and β-Actin from Pan-TurboID and TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin shown to indicate the relative expression levels of TurboID proteins. Quantifications of TurboID protein levels normalized to β-Actin are shown on the right ( n = 3); relative levels are not significant by two-tailed, paired Student’s t -test. g , Western blots stained for streptavidin signal in inputs (‘in’) and streptavidin pulldowns (‘pd’) from Pan-TurboID or TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. h , Streptavidin pulldowns shown for dendritic (SHANK3, GKAP, NLGN1 and HOMER1) and negative control (GAPDH) proteins from TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. Flag signal indicates self-biotinylation of each construct. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by TurboID-PSD95 in each condition is calculated by dividing the signal in the pulldown lane by that of the input lane, after each is adjusted to total, and quantifications are shown as bar graphs ( n = 3). P values: Flag = 0.58, SHANK3 = 0.0061, GKAP = 0.018, NLGN1 = 0.00052, HOMER1 = 0.021, GAPDH = 0.42. i , Streptavidin pulldowns shown for dendritic (BAIAP2 and DLGAP3) and nuclear (TBR1, H4 and H2AX) proteins from Pan-TurboID and TurboID-PSD95-transduced neurons in the presence (+) of exogenous biotin. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by each TurboID is calculated as in (h) ( n = 3). P values: BAIAP2 = 0.0052, DLGAP3 = 0.0035, TBR1 = 0.0063, H4 = 0.018, H2AX = 0.0037. j , Phosphorylation of EEF2, eIF2α, ERK1/2 and IRE1 and total levels of ATF4 and CHOP are shown in resting (rest), activated (DHPG, Dep) and stressed (Sodium arsenite (NaAsO 2 )) cells by using phospho-specific and total antibodies. The amount of phosphorylated or total protein is shown in the bar graphs, calculated by dividing the phosphorylated signal to total and β-Actin for the phosphorylated proteins and by dividing the total to β-Actin for ATF4 and CHOP ( n = 3). Significance was calculated with respect to rest. P values: P-EEF2 (DHPG = 0.0088, Dep = 0.0023, NaAsO 2 = 0.039), P-eIF2α (DHPG = 0.018, Dep = 0.0034, NaAsO 2 = 0.028), P-ERK1/2 (DHPG = 0.015, Dep = 0.0067, NaAsO 2 = 0.00084), P-IRE1 (DHPG = 0.06, Dep = 0.37, NaAsO 2 = 0.0027), ATF4 (DHPG = 0.038, Dep = 0.42, NaAsO 2 = 0.016), CHOP (DHPG = 0.044, Dep = 0.18, NaAsO 2 = 0.024). k , Quantitative PCR (qPCR) results shown for immediate early genes, Arc , Fos and Jun . The fold changes for each gene are calculated by first normalizing to the house-keeping gene β-Actin in each condition, then dividing the value of each condition by that of the resting state ( n = 3). l , Dendritic spine size in resting and KCl-depolarized neurons are measured using the Keyence microscope. Red squares are examples of spines that are counted ( n = 3, 12 spines from each biological replicate are counted as technical replicates). Significance was derived from the biological replicates using the two-tailed, unpaired Student’s t -test. Box plots show the min and max, with the center line at median. Magnification, ×100. Scale bars, 5 μm. m , Fluo-4-AM staining in resting, KCl-depolarized and DHPG-depolarized cells. Fluo4-AM was loaded in resting cells and measurements were taken at indicated time points after Fluo4-AM removal. In depolarized cells, the dye was loaded during silencing. After silencing, fluorescence was measured during stimulus at 10, 30 and 60-minute time points for the KCl treatment and at 10-minute for the DHPG-induced activation. Fluorescence was also measured 60 minutes after the stimulus removal (60′post KCl and 60′post DHPG). Circles represent data from 2 biological and 3 technical replicates. Below: Examples of Fluo4-AM fluorescence are shown in resting, 10-minute KCl-treated and 10-minute DHPG-treated neurons. Fluo4-AM loading (45 minutes) was performed during the last 45 minutes of the silencing step prior to stimulus addition for the KCl and DHPG treatment and simultaneously for the resting neurons. Imaging was performed 10 minutes after the stimulus was added. Scale bars, 50 μm. (b,f,h-k,m) Data are mean ± s.d. Significance was calculated using the two-tailed, paired Student’s t -test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Article Snippet: Puromycin (1:3,000, mouse, Kerafast, EQ0001, RRID: AB_2620162), Flag (1:3,000, mouse, Sigma-Aldrich, F1804, RRID: AB_262044), β-Actin antibody (1:2,500, mouse, Sigma-Aldrich, A1978, RRID: AB_476692), RPL10A (1:1,000, rabbit, Abcam, ab174318), MAP2 (1:2,500, guinea pig, Synaptic Systems, 188004, RRID: AB_2138181), GFAP (1:500, rabbit, Abcam, ab7260, RRID: AB_305808), OLIG2 (1:500, rabbit, Proteintech, 13999-1-AP, RRID: AB_2157541), PSD95 (1:500, mouse, Millipore, MABN68, RRID: AB_10807979), Synaptophysin (1:300, mouse, Abcam, ab8049, RRID: AB_2198854), SHANK3 (1:500, mouse, Novus, NBP1-47610, RRID: AB_10010567), GKAP (1:500, rabbit, Novus, NBP1-76911, RRID: AB_11017331), NLGN1 (1:200, mouse, Novus, NBP2-42192), HOMER1 (1:1,000, rabbit, Proteintech, 12433-1-AP, RRID: AB_2295573), GAPDH (1:5,000, mouse, Thermo Fisher Scientific, AM4300, RRID: AB_2536381), BAIAP2 (1:500, rabbit, Proteintech, 11087-2-AP, RRID: AB_2063075), DLGAP3 (1:500, rabbit, Proteintech, 55056-1-AP, RRID: AB_10858793), TBR1 (1:500, rabbit, Proteintech, 20932-1-AP, RRID: AB_10695502), H4 (1:1,000, mouse, Abcam, ab31830, RRID: AB_1209246), H2A.X (1:1,000, rabbit, Proteintech, 10856-1-AP, RRID: AB_2114985), EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2332, RRID:AB_10693546), P-EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2331, RRID: AB_10015204), eIF2α (1:1,000, rabbit, Cell Signaling Technology, 9722, RRID: AB_2230924), P-eIF2α (1:1,000, rabbit, Cell Signaling Technology, 3398, RRID: AB_2096481), p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 4695, RRID: AB_390779), P-p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 9101, RRID: AB_331646), P-IRE1 (1:500, rabbit, Novus, NB100-2323SS, RRID: AB_10145203), IRE1 (1:500, rabbit, Novus, NB100-2324SS, RRID: AB_10000972), CHOP (1:1,000, mouse, Cell Signaling, 2895T, RRID: AB_2089254), ATF4 (1:1,000, rabbit, Cell Signaling Technology, 11815S, RRID: AB_2616025), MPHOSPH (1:300, rabbit, Biorbyt, orb100446), KCNJ9 (1:300, rabbit, LSBio, LS-C352416), KCNJ9 (1:300, mouse, Antibodies Incorporated, 75-445, RRID: AB_2686912), eIF4G2 (1:1,000, rabbit, Cell Signaling Technology, RRID: AB_10622189 and rabbit, Cell Signaling Technology, RRID: AB_2261993), NSUN3 (1:250, rabbit, LSBio, LS-C163024), MTF1 (1:300, rabbit, Novus, NBP1-86380, RRID: AB_11011361), ZFP64 (1:300, rabbit, Proteintech, 17187-1-AP, RRID: AB_2218826) and KATNBL1 (1:250, rabbit, Proteintech, 24795-1-AP, RRID: AB_2879730).

Techniques: Immunofluorescence, Clone Assay, Derivative Assay, Expressing, Marker, Transduction, Incubation, Western Blot, Staining, Two Tailed Test, Negative Control, Construct, Isolation, Real-time Polymerase Chain Reaction, Microscopy, Fluorescence, Activation Assay, Imaging

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A and B: bulk RNA-seq of aortic tissues from control and AD mice (n=3 per group). Volcano plot displaying differentially expressed genes in aortas between the two groups. Cthrc1 was significantly up-regulated in AD mouse models. (B) KEGG enrichment analysis of differentially expressed genes in aortic tissues from control and AD mice. (C) C to F: scRNA-seq data of 4 samples from control and AD mice. Violin plots showing nFeature, nCount, percent.mt and expression level of the housekeeping gene beta-actin (Actb) after quality control of 4 samples. (D) UMAP plot of 82,326 single cells from 4 samples colored according to the 15 clusters. (E) Heatmap showing expression signatures of the marker genes in each cell type. (F) Heatmap showing expression signatures of the top differentially expressed genes in 2 fibroblast subclusters. (G) UMAP plots showing CTHRC1 expression in fibroblasts from control individuals and patients with ascending thoracic aortic aneurysm (GSE155468). (H) H to J: CTHRC1 protein levels in vitro (NIH3T3 cells (H), human umbilical vein endothelial cells (HUVECs) (I), and MOVAS cells(J), incubated with Ang-II (10^-6 M) and BAPN (0.2 mM). (K) bulk RNA sequencing of aortas in male and female mice. PCA of gene profiles of transcriptome between different sexes. (L) Volcano plot displaying differentially expressed genes in aortic tissues from control and AD female mice (n=3 per group). Cthrc1 was also significantly up-regulated in AD female mouse models. (M) KEGG enrichment analysis of the differentially expressed genes in AD female mouse models. (N) Immunohistochemical staining for CTHRC1 in female mouse aortas. The orange arrows indicate CTHRC1 expression in adventitial fibroblasts of the dissected aortas. (O) Female mouse serum CTHRC1 content in control and AD mice (n=6). (an unpaired two-tailed Student’s t-test, **** P ≤ 0.0001.)

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: RNA Sequencing, Control, Expressing, Marker, In Vitro, Incubation, Immunohistochemical staining, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A schematic diagram illustrating the experiment design. Single-cell RNA sequencing was applied to 4 samples from C57BL/6 mice that were infused with saline or simultaneously administrated by Ang-II/BAPN for 14 days. (B) UMAP plot of the 82,326 single cells from the 4 samples outlined in (A), showing the identification of 9 main clusters. (C) The proportion of 9 cell clusters in control and AD mice. (D) UMAP plot of the 9,043 fibroblasts with 2 subclusters. (E) UMAP plots showing comparison of fibroblast clusters or Cthrc1 expression between the AD and control group. (F) Immunohistochemical staining for CTHRC1 in mouse aortas. The orange arrows indicating CTHRC1 expression in adventitial fibroblasts of the dissected aortas. (G) Representative images showing immunoblotting analysis of CTHRC1 levels in control and AD mice (n=6). (H) Mouse serum CTHRC1 content in control and AD groups (n=6). Data are presented as mean ± SEM. (Student’s t test, ** P ≤ 0.01.) (I) Heatmap of expression levels of genes related to vascular smooth muscle cell contraction and extracellular matrix disassembly between control and AD mice. (J) Scatterplot showing gene expression level in the adventitia between A+ Pos and A+ Neg samples. Red: upregulated genes in the adventitia of aortas from patients prone to aorta rupture, blue: downregulated genes. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: RNA Sequencing, Saline, Control, Comparison, Expressing, Immunohistochemical staining, Staining, Western Blot, Gene Expression

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Experiment design. 6-week-old male Cthrc1 -/- mice (n=16) and wide type (WT) mice (n=18) were treated with saline or Ang-II/BAPN for 14 days. (B) Representative images of immunoblotting analysis of CTHRC1 levels in aortas from the mice in A. Bottom: quantification of CTHRC1 expression in aortas. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01.) (C) Representative ultrasound images of aorta in mice (scale bar, 1mm). (D) Measurements of maximum aortic diameter of mice in A (n=6-9 per group). (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01, *** P ≤ 0.0001.) (E) Representative pictures of whole aortas from the mice in A. The yellow arrows indicate the dissected sites of vascular lesions. (F) Quantification of AD incidence in the whole aorta, ascending, thoracic and abdominal aortas from WT and Cthrc1 -/- mice. (Fisher’s exact test, * P ≤ 0.05.) (G) Kaplan-Meier survival rates analysis of the mice in A. (Kaplan-Meier survival rates analysis, * P ≤ 0.05, ns, no significance.) (H) Representative images of histological staining with hematoxylin and eosin in aortic sections from the mice in A. Right: measurements of aortic wall thickness of aortas (scale bar, 50 μm). (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01.) (I) Representative images of Elastica van Gieson staining in aortic sections from the mice in A. Right: measurement of numbers of elastin breaks per vessel (scale bar, 100 μm). (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, *** P ≤ 0.0001). (J) UMAP plot of the 28,700 single cells from WT and Cthrc1 -/- mice challenged with Ang-II/BAPN for 14 days, displaying the identification of 8 major clusters. (K) The proportion of 8 cell clusters in WT and Cthrc1 -/- mice infused with Ang-II/BAPN. (L) Upper: UMAP plot of fibroblast subclusters in the WT group and Cthrc1 -/- group respectively. Bottom: the proportion of FB1 and FB2. (M) KEGG enrichment analysis of differentially expressed genes in aortic tissues from WT and Cthrc1 -/- mice analyzed by bulk-RNA seq data. (N) Heatmap showing the expression levels of genes related to vascular smooth muscle cell contraction and extracellular matrix disassembly. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Saline, Western Blot, Expressing, Comparison, Staining, RNA Sequencing

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Immunohistochemical staining for CTHRC1 in aortas from the Cthrc1 -/- and WT mice. The orange arrows indicate CTHRC1 expression in adventitial fibroblasts of the dissected aortas, with no positive staining observed in Cthrc1 -/- group. (B) Alican blue staining of aortas from the Cthrc1 -/- and WT mice. (C) C to F: bulk RNA-seq of aortic tissues from WT and Cthrc1 -/- mice. GSEA showing the inhibition of extracellular matrix disassembly and the activation of vascular smooth muscle contraction. (D) : D to F: Violin plots illustrating gene expressions of MMPs (D), ADAMs (E) and ADAMTSs (F) between WT and Cthrc1 -/- mice infused with Ang-II/BAPN. MMPs: matrix metalloproteinases; ADAMs: a disintegrin and metalloproteinases; ADAMTSs: ADAMs with a thrombospondin motif. (G) G to P: 6-week-old female Cthrc1 -/- mice (n=16) and WT mice (n=18) were treated with saline or Ang-II/BAPN for 14 days. Representative ultrasound images of aorta in female mice (scale bar, 1mm). (H) Measurements of maximum aortic diameter of mice in G (n=8 per group). (a Welch ANOVA test followed by a post hoc analysis using the Tamhane T2 method, *** P ≤ 0.001; ** P ≤ 0.01.) (I) Representative pictures of whole aortas from the female mice. The yellow arrows indicate the dissected sites of vascular lesions. (J) Quantification of AD incidence in the whole aorta, ascending, thoracic and abdominal aortas from WT and Cthrc1 -/- female mice. (Fisher’s exact test, * P ≤ 0.05.) (K) Kaplan-Meier survival rates analysis of the four groups of female mice. (Kaplan-Meier survival rates analysis, ns, no significance.) (L) Immunohistochemical staining for CTHRC1 in aortas from the female mice. The orange arrows indicate CTHRC1 expression in adventitial fibroblasts of the dissected aortas from the WT female mice, with no positive staining observed in Cthrc1 -/- female mice (scale bar, 50 μm). (M) Representative images of histological staining with hematoxylin and eosin in aortic sections from the female mice (scale bar, 50 μm). (N) Measurements of aortic wall thickness of aortas. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, *** P ≤ 0.001; **** P ≤ 0.0001.) (O) Representative images of EVG staining in aortic sections from the female mice (scale bar, 50 μm). (P) Measurements of numbers of elastin breaks per vessel. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01; *** P ≤ 0.001.) (Q) Q to S: bulk RNA-seq of aortic tissues from WT and Cthrc1 -/- female mice. PCA of gene profiles of transcriptome between different sexes. (R) KEGG enrichment analysis of differentially expressed genes in aortic tissues from WT and Cthrc1 -/- female mice. (S) Heatmap showing the expression levels of genes related to vascular smooth muscle cell contraction.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Immunohistochemical staining, Staining, Expressing, RNA Sequencing, Inhibition, Activation Assay, Saline, Comparison

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Comparison of the total number of cell-cell interactions and the interaction strength from the scRNA-seq data of control and AD mice. (B) Circle plot showing differential cell-cell interaction numbers and strength between fibroblast, SMC, EC, MoMaphDC, Schwann, B cell and T cell clusters predicted by CellChat. Each circle represents one cell cluster, edges between circles represent intercellular signaling between cell clusters, and edge thickness reflects interaction number or strength. Red edges represent increased interaction number or strength and blue represent decreased interaction number or strength in AD mice versus control mice. (C) Identification of up-regulated signaling by comparing the communication probabilities mediated by ligand–receptor pairs from fibroblasts to SMCs in AD mouse models. (D) D to H: scRNA-seq of the two aortas from WT and Cthrc1 -/- mice infused with Ang-II/BAPN for 14 days were pooled per group. The proportion of major cell types including SMCs, ECs and FBs in Ang-II/BAPN-infused WT and Cthrc1 -/- mice. (E) Bar diagram showing the percentage of 4 subclusters in aortic SMCs from the two groups of mice. (F) Boxplot illustrating the differential gene expression of contractile genes in aortic SMCs of the two mouse groups. (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.0001. ns, no significance.) (G) Barplot depicting the numbers of differential expressed genes in SMC subclusters of the two mouse groups. (H) Gene Ontology enrichment analysis of down-regulated genes in fibromyocytes of the two mouse groups. (I) Immunofluorescence staining of α-SMA and SM22α expression in aortas from Ang-II/BAPN-infused WT and Cthrc1 -/- mice. DAPI: 4’,6-diamidino-2-phenylindole (scale bar: 100 μm). (J) Quantification of α-SMA and SM22α positive area in aortas. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, **** P ≤ 0.00001.) (K) K to L: Incubating the murine aortic smooth muscle cells (MOVAS) with rhCTHRC1 in vitro. Gene Ontology enrichment analysis of up-regulated genes in MOVAS cells treated with rhCTHRC1 by bulk RNA-seq. (L) Heatmap showing the expression levels of genes related to metallopeptidase activity, activation of MAPKK activity and positive regulation of smooth muscle cell proliferation in MOVAS cells treated with rhCTHRC1 or Vehicle. (M) Immunoblotting analysis of the protein levels of α-SMA, SM22α, and p-ERK1/2 in MOVAS with or without rhCTHRC1 treatment. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Comparison, Control, Gene Expression, Immunofluorescence, Staining, Expressing, In Vitro, RNA Sequencing, Activity Assay, Activation Assay, Western Blot

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A to G: scRNA-seq data of aortas from WT and Cthrc1 -/- mice challenged with Ang-II/BAPN for 14 days. Violin plots showing data characteristics on nFeature, nCount and percent.mt after quality control of the two groups. (B) The violin plot showing the expression level of the housekeeping gene beta-actin (Actb) between the two groups. The relative stability of Actb expression indicates consistency. (C) UMAP plot of 82,326 single cells from the samples colored according to the 14 clusters. (D) Heatmap showing expression signatures of the marker genes in each cell type. (E) UMAP plot of 1,962 SMCs colored according to the identified 4 SMC subclusters. (F) Heatmap showing the scaled mean expression of signature genes in 4 SMC subclusters. (G) Gene Ontology enrichment analysis of up-regulated genes in contractile SMCs of the two groups of mice. (H) Immunoblotting analysis of α-SMA and SM22α expression in aortas from Ang-II/BAPN-infused WT and Cthrc1 -/- mice (n=6). (I) Quantification of α-SMA and SM22α protein expression levels. (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, *** P ≤ 0.001, **** P ≤ 0.0001.) (J) J and K: whole transcriptome analysis of MOVAS cells treated with vehicle and rhCTHRC1. A volcano plot displaying differentially expressed genes. Upward expression of Dcn , Adam9 , Angpt2 and Mmp8 gene in the presence of rhCTHRC1. (K) KEGG enrichment analysis of up-regulated genes in aortas from Ang-II/BAPN-infused WT and Cthrc1 -/- mice.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Control, Expressing, Marker, Western Blot, Comparison

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Workflow of the immunoprecipitation (IP) of CTHRC1 identified by mass spectrometry (MS). (B) Venn diagram of intersection analyses of two independent assays for potential receptors of CTHRC1 on MOVAS cells. Right: Scores of candidate binding proteins identified by MS. (C) Co-IP-blotting of CTHRC1 with ADAM9 receptor on MOVAS cells. (D) D to E: whole transcriptome analysis of MOVAS cells incubated with rhCTHRC1, knockouting Roas26 (control) or Adam9 ( Adam9 -KO). Gene Ontology enrichment analysis of down-regulated genes in Adam9 -KO MOVAS cells treated with rhCTHRC1. (E) Heatmap showing the expression levels of genes associated with extracellular matrix disassembly and contraction. (F) Immunoblotting analysis of ADAM9, α-SMA, SM22α, and p-ERK1/2 expression in MOVAS cells, knockouting Roas26 or Adam9 . (G) Immunoblotting analysis of ADAM9, α-SMA, SM22α, and p-ERK1/2 expression in MOVAS cells with or without treatment of ADAM9 inhibitor (ADAM9i). (H) H to I: Comprehensive profiles of genes in MOVAS cells with treatment of ADAM9i analyzed by bulk RNA-seq. Gene Ontology enrichment analysis of down-regulated genes in MOVAS cells treated with ADAM9i. (I) Heatmap showing the expression levels of metalloprotein genes in MOVAS MOVAS cells in the presence of rhCTHRC1cells treated with ADAM9i or DMSO. (J) Interaction of CTHRC1 with ADAM9 in human aortic smooth muscle cells (HASMCs) confirmed by Co-IP-blotting. (K) Western blotting analysis of ADAM9, α-SMA, SM22α, and p-ERK1/2 expression in HASMCs with treatment of ADAM9i. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Immunoprecipitation, Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Incubation, Control, Expressing, Western Blot, RNA Sequencing

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Coomassie staining revealing the protein content in the samples coimmunoprecipitated with CTHRC1. Red box indicates the potential interactors. (B) The representative peptide mass spectrum of ADAM9 receptor on VSMCs. (C) Immunoblotting analysis of ADAM9 in MOVAS incubated with rhCTHRC1 for 0, 3, 6, 12, and 24 hours. (D) Violin plot of Adam9 gene expression in MOVAS cells with treatment of vehicle or rhCTHRC1. (E) Heatmap depicting the gene expression of ADAMs in control and AD mice. (F) F and G: transcriptome analysis of gene expression profiles in MOVAS cells without or with rhCTHRC1, knockouting Roas26 or Adam9 . A volcano plot of differentially expressed genes. The expression levels of Agt , Mmp2 and Mmp9 are down-regulated. (G) KEGG enrichment analysis of down-regulated genes in MOVAS cells in the presence of rhCTHRC1, knockouting Roas26 or Adam9 . (H) Relative mRNA levels of α-SMA and SM22α in MOVAS cells, without or with rhCTHRC1, knockouting Roas26 or Adam9 . (a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.) (I) I to K: Comprehensive profiles of genes in MOVAS cells without or with treatment of ADAM9i by bulk RNA-seq. A volcano plot of differentially expressed genes. Egr1 , Mmp2 and Agt are marked with down-regulated expression. (J) KEGG enrichment analysis of down-regulated genes in MOVAS cells in the presence of rhCTHRC1, with or without ADAM9i. (K) Heatmap showing the expression levels of contraction-related genes in MOVAS cells without or with treatment of ADAM9i, incubated with rhCTHRC1.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Staining, Western Blot, Incubation, Gene Expression, Control, Transcriptome Wide Gene Expression, Expressing, Comparison, RNA Sequencing

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) Amino acid sequence homology of immunogen with human or mouse CTHRC1 protein. (B) The affinity, specificity, and efficacy of anti-CTHRC1 Ab confirmed by ELISA (left) and immunoblotting (right) analysis. (C) C to E: mRNA sequencing results illustrating the transcriptomic profiles of aortas from WT mice treated with IgG Isotype and anti-CTHRC1 Ab (n=3 per group). PCA of transcriptome profiles in aortic tissues between the two groups. (D) Gene Ontology enrichment analysis of down-regulated genes between the two groups (anti-CTHRC1 Ab versus IgG Isotype). (E) Heatmap showing the expression levels of genes related to vascular smooth muscle cell contraction and extracellular matrix disassembly. (F) F to K: WT female mice were intraperitoneally injected with anti-CTHRC1 Ab or IgG Isotype under saline or Ang-II/BAPN administration (n=10 per group). Kaplan-Meier survival rates analysis of the four groups of female mice. (Kaplan-Meier survival rates analysis, ns, no significance.) (G) Upper: Representative ultrasound images of aorta in female mice (scale bar, 1mm). Bottom: Measurements of maximum aortic diameter of mice (n=6 per group). (Mann Whitney test, *** P ≤ 0.001.) (H) Representative pictures of whole aortas in female mice treated with IgG Isotype or anti-CTHRC1 Ab. (I) Quantification of AD incidence in the whole aorta, ascending, thoracic and abdominal aortas in female mice. (Fisher’s exact test, * P ≤ 0.05.) (J) Representative images of histological staining with hematoxylin and eosin in aortic sections from the female mice. (K) Representative images of EVG staining from the female mice injected with anti-CTHRC1 Ab or IgG Isotype.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Injection, Saline, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) The sequences of heavy and light chains from anti-CTHRC1 monoclonal antibody (anti-CTHRC1 Ab), confirmed by variable region cloning, expressing and sequencing. (B) Molecular docking analysis showing the interaction between antibody-CTHRC1 antigen and CTHRC1-ADAM9 receptor at the molecular level. (C) Experiment design. WT mice were intraperitoneally injected with anti-CTHRC1 Ab or IgG Isotype at a dose of 5 μg/g body weight under saline or Ang-II/BAPN administration (n=10 per group). (D) Kaplan-Meier survival rates analysis of the mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration. (Kaplan-Meier survival rates analysis, * P ≤ 0.05, ns, no significance.) (E) Representative ultrasound images of aorta in mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration (scale bar, 1mm). (F) Measurements of maximum aortic diameter of mice in E (n=6-9 per group). (Student’s t-test, ** P ≤ 0.01.) (G) Representative pictures of whole aortas in mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration. The yellow arrows indicate the dissected sites of vascular lesions. (H) Quantification of AD incidence in the whole aorta, ascending, thoracic and abdominal aortas from the mice in E. (Mann-Whitney test, ** P ≤ 0.01.) (I) Representative images of histological staining with hematoxylin and eosin in aortic sections from the mice in E. Bottom: Measurements of aortic wall thickness of aortas (scale bar, 50 μm; scale bar, 200 μm). (Student’s t-test, **** P ≤ 0.00001.) (J) Representative images of Elastica van Gieson staining in aortic sections from the mice in E. Bottom: Numbers of elastin breaks per vessel. (scale bar, 50 μm; scale bar, 200 μm). (Student’s t-test, **** P ≤ 0.00001.) (K) K to O: scRNA-seq of the aortic tissues from WT mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration (n=2 per group). UMAP plot of the 42,623 single cells, displaying the identification of 8 major clusters. (L) The proportion of major cell types including SMCs, ECs and FBs in WT mice treated with anti-CTHRC1 Ab or IgG Isotype under Ang-II/BAPN administration (M) Bar diagram showing the percentage of 4 SMC subclusters in aortic SMCs of the two groups of mice. (N) Boxplot illustrating the differential expression of contractile genes in aortic SMCs of the two groups of mice. (*** P ≤ 0.001, ns, no significance.) (O) Gene Ontology enrichment analysis of differentially down-regulated genes in fibromyocytes of the two groups of mice. (P) Immunoblotting analysis and quantification of α-SMA and SM22α expression in aortas from the WT mice treated with IgG Isotype or anti-CTHRC1 Ab under Ang-II/BAPN infusion (n=6-9). (Student’s t-test, **** P ≤ 0.00001.) See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Cloning, Expressing, Sequencing, Injection, Saline, MANN-WHITNEY, Staining, Western Blot

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A to E: scRNA-seq of aortic tissues in healthy controls and human aortic dissection (n=2 per group). Experiment design of scRNA-seq. (B) Dotplot showing the expression level of CTHRC1 in fibroblasts of the aortas from AD patients and controls. Dash line in each group represents its mean expression value. (C) The proportion of 7 major cell clusters in AD patients and controls. (D) Boxplot displaying the differential gene expression of contractile genes in aortic SMCs of the controls and AD patients. (E) Gene Ontology enrichment analysis of differentially up-regulated genes in fibromyocytes from AD patients versus controls. (F) Heatmap showing the expression levels of genes related to vascular smooth muscle cell contraction and extracellular matrix disassembly in aortic tissues from AD patients and controls by bulk RNA-seq. (*** P ≤ 0.001.) (G) Immunoblotting analysis of CTHRC1, ADAM9, p-ERK1/2 expression levels in human aortas (n=6). (H) Quantification of CTHRC1, ADAM9, p-ERK1/2 protein expression levels in G. (Student’s t-test, ** P ≤ 0.01, **** P ≤ 0.0001.) (I) Immunofluorescence staining of α-SMA and SM22α expression in human aortas (scale bar, 500 μm). (J) Laboratory tests for human serum CTHRC1, cTnT, and D-dimer contents in control individuals, acute myocardial infraction (AMI) and AD. (Student’s t-test, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns, no significance.) (K) Graphical abstract illustrating an important role of CTHRC1 acts as a candidate circulating biomarker and therapeutic target for aortic dissection through CTRHC1-ADAM9-ERK axis. See also .

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Dissection, Expressing, Gene Expression, RNA Sequencing, Western Blot, Immunofluorescence, Staining, Control, Biomarker Assay

Journal: bioRxiv

Article Title: CTHRC1 is a new therapeutic target and serum diagnostic biomarker for aortic dissection

doi: 10.1101/2025.04.19.649636

Figure Lengend Snippet: (A) A to E: scRNA-seq data of aortas from human healthy controls and AD patients (n=2 per group). Violin plots showing data characteristics on nFeature, nCount and percent.mt after quality control of the two groups. (B) Violin plot showing the expression level of the housekeeping gene beta-actin (ACTB) of the four samples. The relative stability of ACTB expression indicates consistency. (C) UMAP plot of 45,501 single cells from 4 samples colored according to 7 different cell clusters. (D) UMAP plot of SMCs colored according to the identified 4 SMC subclusters. (E) Bar graphs depicting the numbers of differentially expressed genes in SMC subclusters in healthy controls and AD patients. (F) Gene Ontology enrichment analysis of down-regulated genes in contractile SMCs of the aortas. (G) KEGG enrichment analysis of differentially expressed genes in aortas between the two group. (H) Representative imaging results of contrast-enhanced computed tomography (CT) scanning of patients with aortic dissection, coronary angiography (CAG) of patients with acute myocardial infarction (AMI). The yellow and blue arrows indicate lesion sites in aortas and coronary arteries. (I) Clinical tests for serum biomarkers (Myoglobin, CK-MB, hsCRP and BNP) between AMI or AD patients. (Student’s t-test, * P ≤ 0.05, ** P ≤ 0.01, *** P≤ 0.001, ns, no significance.) (J) A corresponding ELISA kit developed by our team for detecting serum CTHRC1 levels. (K) CTHRC1 point-of-care testing (POCT) equipment, providing rapid and reliable results from a single drop of in less than 10 minutes. (L) The process of assessing chest pain during ambulance transport or in the ER.

Article Snippet: Slides were incubated with Mouse CTHRC1 primary antibody (AF5960, R&D System) at 4°C overnight, then HRP-conjugated anti-Sheep IgG secondary antibody (HAF016, R&D System) were added the next day at room temperature for 1 hour.

Techniques: Control, Expressing, Imaging, Computed Tomography, Dissection, Enzyme-linked Immunosorbent Assay