Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: a, Outline of the studies. b, mRNA levels of Cdkn2a, Cdkn1a, Il6, Mmp3 and Lmnb1, normalized to Actb and Tub mRNA (n = 6 for each group). c, Representative SA-β-gal staining (SA-β-gal+ SnCs in arrows; n = 3 per group/three images per n), EdU (green, EdU negative non-proliferating SnCs in arrows; n = 3 for each group/5–6 images per n) and Hoechst labelled nuclei (blue). Scale bar, 100 μm. d–g, Quantification of the percentage of SA-β-gal+ cells (d), EdU+ cells (e), Lamin B1 levels, with around 1,000 cells (f) and HMGB1+ cells (n = 3 per group; at least 100 cells were counted) in MDFs 3 or 6 days after culturing in young or old mouse serum (g). h, Bioluminescence from p16-3MR-expressing cells in non-senescent MDFs cultured in serum from either young or old mice for 3 (n = 9 for each group) or 6 days (n = 6 for each group). i, Representative SA-β-gal staining (left; + cells are marked with arrows; n = 3 for each group/5–6 images per n) and percentage of SA-β-gal+ cells (right). j, Gene expression of senescence and SASP markers in human renal epithelial cells cultured with young or old human plasma for 3 or 6 days (d3 or d6; n = 8 per group). k, IL-6 level secreted by human renal epithelial cells treated with young or old human plasma for 6 days (n = 8 per group). l, Pearson correlation of secreted IL-6 levels by human renal epithelial cells treated with young or old human plasma and IL-6, MMP-3 and HMGB1 levels in human plasma (Pearson correlation coefficient and P are shown in the figures). Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using a two-tailed t-test with a Welch’s correction (b,d–i,k) (*P < 0.05; **P < 0.01) and two-way ANOVA followed by two-stage step-up method by Benjamini, Krieger and Yekutieli, FDR < 0.05 (*q < 0.05; ** q < 0.01) (j). Rel, relative. Scale bars, 100 μm.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

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

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: (a) mRNA levels for Cdkn2a and Cdkn1a and SASP factors Il6, Mmp3 and Laminb1, normalized to Actb mRNA, determined by RT–PCR (n = 8 for young or old serum treatment for 3 days; n = 4 for young + old (50/50) serum treatment for 3 days). (b) Representative EdU (green; EdU negative non-proliferating SnCs in arrows), HMGB1 (red; SnCs marked by HMGB1 nuclear loss with arrows), Hoechst labelled nuclei (blue) visualized by fluorescence microscopy (3–6 images per n) and SA-β-gal staining (3–7 images per n) and (c) quantification of EdU-positive SnCs in MDFs 3 days after culturing in young, old or young + old (50/50) mouse serum (n = 4 for each group). (d) Bioluminescence from 3MR-expressing cells (Renilla luciferase assay) in non-senescent MDFs from cultured in young (4-month-old), old (32-month-old) or young+old (50/50) mouse serum for 6 days (A.U.) (n = 4 for young or old mouse serum; n = 6 for young+old mouse serum). Data are means ± s.e.m. of biologically independent samples. Statistical significance was tested using one-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons with *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars, 100 μm. Rel, relative.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Reverse Transcription Polymerase Chain Reaction, Fluorescence, Microscopy, Staining, Expressing, Luciferase, Cell Culture

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: a, Experimental setup for heterochronic blood exchange. b, Luminescence images of young p16-3MR mice receiving young blood from C57BL/6J mice (YY) and young p16-3MR mice receiving old blood from C57BL/6J mice (YO) 14 days after blood exchange (left) and quantification of the luminescence (right) (in arbitrary units, a.u.) (n = 7, 4, 3, 4 or 4 mice). c, Ratio of circulating SASP proteins (>1.5-fold) of old mouse blood (n = 7) normalized to young mouse blood (n = 3) measured by antibody array. d, Gene expression of the senescence and SASP markers in lung, hippocampus, heart, skeletal muscles (gastrocnemius (GA) and tibialis anterior (TA)), kidney and liver (n = 6 for YY; n = 7 for YO). Whisker plots represent the 10th and 90th percentiles and the line corresponds to the median. e, Representative images of SA-β-gal staining in skeletal muscle (n = 4 for YY and n = 5 for YO; 3–6 images per mouse) and quantification of SA-β-gal+ cells per area of YY and YO mice. Scale bar, 50 μm. f, Representative images of SA-β-gal staining (n = 8 mice for each group/7–10 images per mouse) and HMGB1 immunohistochemistry (n = 5 for YY; n = 7 for YO; 6–10 images per mouse) and quantification of SA-β-gal+ cells per area (n = 8 per group) of kidney sections of YY and YO mice. Scale bars, 100 μm. g, Representative SA-β-gal staining in liver (n = 9 for YY; n = 8 for YO; 7–10 images per mouse) and quantification of SA-β-gal+ cells per area in YY and YO mice and γ-H2AX foci in a hepatocyte (n = 6 per group; 5–8 images per mouse). this experiment was performed three independent times. Scale bar, 100 μm. Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using a two-tailed t-test with a Welch’s correction (b,e–g) with *P < 0.05; **P < 0.01 and multiple Mann–Whitney tests with a two-stage linear step-up procedure by Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05 (c,d).

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Ab Array, Expressing, Muscles, Whisker Assay, Staining, Immunohistochemistry, Two Tailed Test, MANN-WHITNEY

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: (a) Representative luminescence images of young p16-3MR mice (3-month-old) receiving blood (22-month-old) from old C57BL/6J mice treated with vehicle (YO+Veh) or ABT263 (YO+ABT) 14 days after blood exchange (left) and quantification of the luminescence (right) (A.U.) (n = 4 mice for YO+Veh; n = 3 mice for YO+ABT). Each data point represents an individual mouse. (b) Representative EdU (green; EdU negative non-proliferating SnCs with arrows), HMGB1 (red; SnCs marked by nuclear loss with arrows), and Hoechst labeled nuclei (blue) visualized by immunostaining (n = 4 for each group / 5–8 images per n) and (c) SA-β-gal staining in MDFs cultured in Veh- or ABT-treated old mouse serum for 3 days (n = 4 for veh-treated old mice serum treated; n = 3 for ABT-treated old mouse serum; 3–6 images per n). Quantification of (d) EdU + and (e) SA-β-gal + MDFs. (f) mRNA levels for Cdkn2a and Cdkn1a and SASP factors Il6 and Mmp 3 days after culturing in Veh- or ABT-treated old mouse serum, determined by RT–PCR (n = 4 for each group). Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using two-tailed Student’s t test (a, d-e) (exact P value was shown in the figures) and multiple t tests with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05 (f). Scale bars are shown in each image. Rel, relative.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: In Vivo, Labeling, Immunostaining, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: (a) Relative protein expression ratio (< 0.7-fold) of SASP proteins in plasma from DQ-treated C57BL/6J old mice (DQ; n = 4) normalized to vehicle treated C57BL/6J old mice (Veh; n = 3), measured by antibody array. Each data point represents an individual mouse. Additional SA-β-gal images of (b) kidney and (c) liver in young C57BL/6J mice receiving old C57BL/6J mice treated with Veh (YO+Veh) or DQ (YO+DQ). (d) Representative EdU (green; EdU negative non-proliferating SnCs in arrows), HMGB1 (red; SnCs marked by HMGB1 nuclear loss in arrows), and Hoechst labeled nuclei (blue) visualized by fluorescence microscopy (n = 4 for each group / at least 7 images per n) and (e) SA-β-gal staining in MDFs cultured in Veh- or DQ-treated old mice serum for 3 days (n = 4 for veh-treated old mice serum treated; n = 5 for DQ-treated old mice serum / at least 6 images per n). Quantification of (f) EdU + (n = 4 for Veh-treated old mice serum treated; n = 3 for DQ-treated old mice serum treated) and (g) SA-β-gal + (n = 4 per group) MDFs. (h) mRNA levels for Cdkn2a and Cdkn1a and SASP factors Il6 and Mmp3 3 days after culturing in Veh- or DQ-treated old mice serum, determined by RT–PCR (n = 4 for Veh-treated old mice serum treated; n = 5 for DQ-treated old mice serum treated). Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using multiple t test with two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05; **q < 0.01 (a, h) and two-tailed Student’s t test (f-g) with *, P < 0.05. Scale bars are shown in each image.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Expressing, Ab Array, Labeling, Fluorescence, Microscopy, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: (a) Additional SA-β-gal images of kidney (left) and liver (right) in young C57BL/6J mice receiving old C57BL/6 blood treated with vehicle (YO+Veh) or ABT263 (YO+ABT). (b) HMGB1 immunohistochemistry (brown staining of HMGB1 re-localized to cytoplasm of kidney cells with arrows) (n = 5 per group; 10–15 images per mice). (c) Immunohistochemical staining for KIM-1 (n = 6 for YO+Veh; n = 4 for YO+ABT; 4–5 images per mice) and LTL (n = 5 per group; 5–7 images per mice) on kidney tissues and (d) quantification of KIM-1 + area (%). (e) Serum concentration of KIM-1 (n = 6 per group), (f) blood urea nitrogen (n = 12 for YO+Veh; n = 9 for YO+ABT) and creatine (n = 4 per group). (g) Representative images of Sirius Red (n = 6 for YO+Veh; n = 5 for YO+ABT; 10–15 images per mice) and Masson Trichrom staining and desmin immunohistochemistry (n = 6 mice for YO+Veh; n = 5 mice for YO+ABT; 10–15 images per mice) in livers. Arrows indicate collagen deposition. (h) Quantifications of fibrotic area, as % of area occupied by Sirius Red stain, and desmin + area (n = 6 for YO+Veh; n = 5 for YO+ABT). (i) Quantification of mRNAs encoding Col1a1, Col3a1, Col4a1 and Col4a2 in the liver (n = 6 per group). (j) Oil Red O + area (%) indicated as adiposity index (n = 6 per group; 5–9 images per mice). (k) Serum analyses for ALT (n = 8 for YO+Veh; n = 9 for YO+ABT) and bilirubin (n = 9 per group). All data are expressed as means± s.e.m. of biologically independent samples. A two-tailed t test with a Welch’s correction (d-f, h, j-k; *, P < 0.05) and multiple t test with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05; **q < 0.01 (i). Scale bars, 100 μm. Rel, relative.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Immunohistochemistry, Staining, Immunohistochemical staining, Concentration Assay, Two Tailed Test

Journal: Nature metabolism

Article Title: Systemic induction of senescence in young mice after single heterochronic blood exchange

doi: 10.1038/s42255-022-00609-6

Figure Lengend Snippet: a, Study design used in b–g. b, Venn diagram showing the overlap between up-regulated circulating SASP proteins identified in old versus young mice (>1.5-fold) and down-regulated proteins in vehicle (Veh)-treated versus DQ-treated old mice (<0.7-fold). c, List of overlapping proteins in b. d,e, Senescence/SASP-associated gene expression in kidney (d) and liver (e) of young mice receiving old blood treated with Veh (YO+Veh; n = 4) or DQ (YO+DQ; n = 6). f,g, Representative SA-β-gal staining (n = 5 for YO+Veh; n = 7 for YO+DQ; 10–15 images per mouse) and quantification of SA-β-gal+ cells per area in kidney (f) and liver (g) (n = 5 for YO+Veh; n = 8 for YO+DQ; 7–10 images per mouse). h, Study design used in i–p. i, Venn diagram showing the overlap between up-regulated circulating SASP proteins in old versus young mice (>1.5-fold) and down-regulated proteins in Veh-treated versus ABT263-treated old mice (<0.7-fold). j, List of overlapping proteins in i. k–l, Senescence/SASP-associated gene expression in kidney and liver of young mice receiving old blood treated with Veh (YO+Veh) or ABT263 (YO+ABT) (n = 6 per group). m,n, Representative images of SA-β-gal staining in kidney (n = 8 for YO+Veh; n = 7 for YO+ABT; 6–10 images per mouse) and quantification of SA-β-gal+ cells per area (m) and HMGB1+ tubular cells (n). Scale bar, 500 μm. o,p, Images of SA-β-gal staining in liver (n = 5 for YO+Veh; n = 6 for YO+ABT; ten images per mouse) and quantification of SA-β-gal+ cells per area (o), γ-H2AX foci (n = 5 per group; 6–8 images per mouse) and TUNEL+ hepatocytes (n = 4 for YO+Veh; n = 5 for YO+ABT; 5–9 images per mouse) (p). Scale bar, 100 μm. Two independent experiments were performed. Data are means ± s.e.m. of biologically independent samples. Statistical significance was calculated using multiple Mann–Whitney tests with a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 5%, *q < 0.05 (d,e,k,l) and a two-tailed t-test with a Welch’s correction (f,g,m–p) with *P < 0.05; **P < 0.01; ***P < 0.001. Rel, relative.

Article Snippet: MMP-3 and HMGB1 levels were quantified in human plasma by using HMGB1 detection kit (Chondrex, 6010) and MMP-3 quantikine ELISA kit (R&D system, DMP300).

Techniques: Expressing, Staining, TUNEL Assay, MANN-WHITNEY, Two Tailed Test

Journal: Communications Biology

Article Title: Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins

doi: 10.1038/s42003-021-02953-x

Figure Lengend Snippet: a HT3 B3-WT (blue) or HT3 B3-KO (red) cells were subjected to nucleofection (nuc, +) in the absence (−) or presence (+) of 5 ug/ml LPS-EK (LPS), caspase-1 inhibitor, VX765, or lysosomal cysteine protease inhibitor, E64. An aliquot of cells were also not nucleofected (nuc, −). After 3 h, cells were stained with SG and Hoescht 33342 and imaged. Quantification: (# Sytox positive nuclei/# of blue nuclei) × 100. Means ± SD were compared using a two-tailed t -test (* P < 0.05). b Immunoblot of gasdermin D (GSDMD) and gasdermin E (GSDME) cleavage in THP1 cells treated with α-hemolysin (HlA); positive control. HT3 B3-WT /HT3 B3-KO were nucleofected with 0, 1, or 5 µg of LPS and left to recover for 1 h. Open arrowheads: full-length GSDMD and GSDME based on molecular mass. Black arrowheads: cleaved GSDMD. Dashed box: area contrast enhanced for clarity. Note, the positive control for GSDME cleavage is provided in Fig. . c , d Flow cytometry analysis of HT3 B3-WT /HT3 B3-KO cells treated with a lysosomotropic agent, LLOMe ( c ) or 1 µg/ml LPS ( d ). Cells were stained with the lysosomotropic dye, acridine orange (Y-axis), and the cell viability dye, Sytox™ blue (X-axis). Lines indicate the threshold fluorescence levels (gate) to determine each quadrant. Numbers indicate cell percentage in each quadrant. e Schematic representation of each quadrant. Quadrant 1 (Q1) contained live cells positive for lysosomal staining (acridine orange positive, Sytox blue negative). Quadrant 2 (Q2) contained dead cells with positive lysosomal staining (acridine orange positive, Sytox blue positive). Quadrant 3 (Q3) contained dead cells negative for lysosomal staining (acridine orange negative, Sytox blue positive). Quadrant 4 (Q4) contained live cells negative for lysosomal staining (acridine orange negative, Sytox blue negative). Arrows indicate the timing of fluorescence loss for different cell death pathways. If the lysosomal loss occurred prior to plasma membrane permeabilization, then the percentage of cells will increase from Q1 to Q4 to Q3 over time. If the lysosomal loss occurred after plasma membrane loss, then the percentage of cells will increase from Q1 to Q2 to Q3 over time. f , g Graphical representation of the average of three separate experiments indicating the percentage of cells treated with LLOMe ( f ) or nucleofected LPS ( g ) in HT3 B3-WT (blue) and HT3 B3-KO (red) cells. Error bars represent means ± SD. Uncropped immunoblots can be found in Supplementary Fig. .

Article Snippet: The human monocyte cell line THP1-HMGB1-Lucia™ (thp-gb1lc) was obtained from InvivoGen.

Techniques: Protease Inhibitor, Staining, Two Tailed Test, Western Blot, Positive Control, Flow Cytometry, Fluorescence, Membrane

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Scheme of L -arginine-immobilized NHS magnetic beads and the enrichment strategy for arginine-binding proteins. B Volcano plot of arginine- and leucine-binding proteins. C , D GO analysis of arginine-binding proteins in A2780 cells. E Venn diagram showing arginine-binding proteins that were upregulated in both 500 μM arginine-treated A2780 ovarian cancer cells and omentum metastases. F Relative abundance of DDX3X protein captured by arginine or leucine-coupled magnetic beads. G WB detection of DDX3X in A2780 cells with arginine treatment, or lysate from omentum metastasis, and elution after purification with L -leucine- or L -arginine-immobilized NHS magnetic beads. H Molecular docking between DDX3X (HMDB ID: HMDB0000517) and L -arginine (PubChem CID: 6322) performed using Discovery Studio. I Molecular dynamics (MD) simulation was performed using Gromacs. The backbone root mean square deviation (RMSD) of the DDX3X- L -arginine complex over the 100 ns MD simulation was shown. J Surface plasmon resonance analysis for the binding of L -arginine to DDX3X. Data were shown as the mean ± SD. The p value was calculated using an unpaired two-tailed Student’s t -test ( F ). ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Magnetic Beads, Binding Assay, Purification, SPR Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Representative confocal microscopy images and immunofluorescence analysis of DDX3X in A2780 cells treated with arginine at the indicated times. Scale bars, 10 μm. B WB analyses of DDX3X, histone H3, and tubulin protein levels in cytoplasmic and nuclear fractions of A2780 cells under the indicated treatments. C Representative confocal microscopy images of DDX3X immunofluorescence in A2780 cells treated as indicated. Scale bars, 10 μm. D WB analyses of DDX3X, CRM1, histone H3, and tubulin protein levels in the cytoplasmic and nuclear fractions of A2780 cells under the indicated treatments. E Representative confocal microscopy images of Flag immunofluorescence in A2780 shDDX3X cells treated as indicated. Scale bars, 10 μm. F WB analyses of Flag, histone H3, and tubulin protein levels in the cytoplasmic and nuclear fractions of A2780 shDDX3X cells under the indicated treatments. G Quantification of the nuclear-to-cytoplasmic ratio of DDX3X fluorescence intensity in A2780 cells treated with arginine as indicated. H Quantification of the nuclear-to-cytoplasmic ratio of DDX3X fluorescence intensity in A2780 shDDX3X cells treated with arginine as indicated. I Representative images and quantification of immunohistochemical staining of DDX3X in primary tumors and omentum metastases ( n = 20). Scale bars, 50 μm. J Immunohistochemical quantification of nuclear DDX3X in primary tumors and omentum metastases ( n = 20). Scale bars,50 μm. Data were shown as the mean ± SEM. The p value was calculated using one-way ANOVA ( A , G , H ), and unpaired two-tailed Student’s t -test ( I , J ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Confocal Microscopy, Immunofluorescence, Fluorescence, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A GO analysis of differentially expressed genes between DDX3X knockdown and control A2780 cells. B GO analysis of differentially expressed genes between A2780 cells with 100 and 500 μM arginine. C The relative levels of reactive oxygen species (ROS) were measured using the dihydroethidium (DHE) fluorescence method in fresh primary tumor and omental tissues ( n = 5). D Representative image and quantification of immunohistochemical staining of γH2AX and 8-OHdG in primary tumors and omentum metastases ( n = 20). Scale bars, 50 μm. E WB analyses of the protein levels of H2AX, γH2AX, and GAPDH in metastases from mice fed with the indicated diets. F Representative images and quantification of γH2AX immunofluorescence staining in A2780 cells treated with cisplatin and under the indicated treatments. Scale bars, 10 μm. G Representative images and quantification of the comet assay in A2780 cells treated with cisplatin and under the indicated treatments. Scale bars, 10 μm. H Representative images and quantification of immunofluorescence staining of γH2AX in A2780 cells treated with H 2 O 2 (1 mM final concentration) and under the indicated treatments. Scale bars, 10 μm. I Representative images and quantification of comet assay in A2780 cells treated with H 2 O 2 (1 mM final concentration) and under the indicated treatments. Scale bars, 10 μm. Data were shown as the mean ± SEM. The p value was calculated using one-way ANOVA ( F - I ), and unpaired two-tailed Student’s t -test ( C , D ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Knockdown, Control, Fluorescence, Immunohistochemical staining, Staining, Immunofluorescence, Single Cell Gel Electrophoresis, Concentration Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A The bar graph shows the fold changes in DDR gene expression following treatment with 500 μM arginine or DDX3X knockdown. B , C WB analyses for the protein level of ATM, Phospho-ATM (Ser 1981), ATR, Phospho-ATR, and GAPDH in metastasis of mice treated with the indicated diets. D , E WB showing the protein level of DDX3X, ATM, Phospho-ATM (Ser 1981), CHK2, Phospho-CHK2 (Thr68), P53, Phospho-P53 (Ser15), H2AX, γH2AX, and GAPDH in A2780 or HEY A8 cells with indicated treatments. F WB analyses for the protein level of ATM, Phospho-ATM (Ser 1981), H2AX, γH2AX, and GAPDH in A2780 cells with indicated treatments. G – I Representative images and quantification of immunofluorescence staining of γH2AX, comet assay in A2780 cells treated with cisplatin and ATM inhibitors (AZD0156[10 μM] and AZ31[5 μM]). Scale bars, 10 μm. J WB analyses for the protein level of CHK2, Phospho-CHK2 (Thr68), H2AX, γH2AX, and GAPDH in A2780 cells with the indicated treatments. K – M Representative images and quantification of immunofluorescence staining of γH2AX, comet assay in A2780 cells treated with cisplatin and CHK2 inhibitors (CCT241533 [5 μM] and PHI-101[1 μM]). Scale bars, 10 μm. Data were shown as the mean ± SEM from three independent experiments. The p value was calculated using one-way ANOVA ( H , I , L , M ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Gene Expression, Knockdown, Immunofluorescence, Staining, Single Cell Gel Electrophoresis

Journal: Cell Death & Disease

Article Title: Arginine dependency in omental metastasis of epithelial ovarian cancer reveals a therapeutic vulnerability

doi: 10.1038/s41419-026-08606-3

Figure Lengend Snippet: A Experimental scheme illustrating orthotopic ovarian cancer and intraperitoneal ovarian cancer mice with arginase (10 μg/kg, intraperitoneal injection, three times a week) or DDX3X inhibitor (RK-33, 50 mg/kg, intraperitoneal injection, three times a week) administration. B Plasma arginine levels in arginase-injected mice were quantified using a biochemical assay kit at 24 h post-injection. C , D Representative IVIS bioluminescence images and quantitative luminescence analysis of mice with indicated treatments at 30 days after i.o. injection of ID8-Luc cells ( n = 5/group). E Representative images and weight of ovaries from orthotopic ovarian tumor mice with indicated treatments at 60 days after cell injection ( n = 5/group). Scale bars, 1 cm. F Representative IVIS bioluminescence images and quantitative luminescence analysis of mice with indicated treatments at 30 days after i.p. injection of ID8-Luc cells ( n = 5/group). G Kaplan–Meier analysis of mice with i.p. injection of ID8-Luc cells treated as indicated. ( n = 10/group). H – J Representative images of peritoneal metastasis ( H ), total number of metastatic deposits ( I ), and ascites volume ( J ) in mice with indicated treatments at 35 days after i.p. injection ( n = 5/group). K – M Representative images, tumor weight, and size of subcutaneous xenograft tumors in nude mice 30 days after injection of A2780 cisplatin-resistant cells, with the indicated treatments ( n = 5/group). Data were shown as the mean ± SD. The p value was calculated using unpaired two-tailed Student’s t -test ( A ), two-way ANOVA ( D – F , I , J , L ), and one-way ANOVA ( M ). Survival curves were generated with the Kaplan–Meier method and analyzed using the log-rank test ( G ). * p < 0.05, ** p < 0.01.

Article Snippet: Permeabilized cells were then sequentially incubated with a primary rabbit anti-DDX3X antibody (Cat. No. 11115-1-AP, Proteintech, Wuhan, China) and a secondary Goat Anti-Rabbit IgG antibody (Cat. No. AS070, ABclonal, Wuhan, China).

Techniques: Injection, Clinical Proteomics, Quantitative Luminescence, Two Tailed Test, Generated

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a – e Schematic diagrams of the brain of mouse ( a ), and the red box in the cerebral cortex shows the location where the images were taken. Immunofluorescence double labelling ( b , c , 2 double-labelled neurons are indicated as examples in ( b , c )) and quantification ( d , e , n = 6 images from 3 mice) of GDF11 (green, b ) and NeuN (red, b ) or GDF11 (green, c ) and CaMKIIα (red, c ) in the cerebral cortices of the mice aged 3 months (3 M). f Representative images of immuno-electron microscopy (Immuno-EM) of GDF11 labelled with nanogold particles (there are many GDF11 labelled black dots and only some examples are indicated with red arrows) in the cerebral cortex of the mice aged 3 M ( n = 3 mice). Nuc, nucleus; Den, dendrite. g Immunofluorescence double labelling of GDF11 (green, arrow) and GABA (red, double arrowheads) ( n = 3 mice). h Immunofluorescence double labelling of GDF11 (green) together with Olig2 (red, left), GFAP (red, middle), Iba1 (red, middle) in the cerebral cortex (Cx) and Dcx (red, right) in the dentate gyrus (DG) of the mice aged 3 M ( n = 3 mice). The GDF11 negative cells are indicated by arrows in ( h ). i Schematic diagrams of the brain of the marmoset (one aged 62 M and another aged 70 M), and the red box in the cerebral cortex shows the location of the images ( n = 2 marmosets). j – o Immunofluorescence double labelling ( j , m , n , o ) and quantification ( k , l ) of GDF11 (green) together with CaMKIIα (red, j , k , l , 2 double-labelled neurons are indicated as examples in ( j ); n = 8 images from 2 marmosets) or GABA (red, m ), Olig2 (red, n ) or GFAP (red, o ). The GDF11 negative cells are indicated by arrows in ( m , n , q ). p Schematic diagrams of the human brain. The red box in the cerebral cortex shows the location of the images. q – s Immunofluorescence double labelling ( q , male patient aged 24 years (Y) and female patient aged 23Y diagnosed with intractable epilepsy and the focus of epileptic cortices had to be removed surgically) and quantification ( r , s , n = 4 patients, male patient aged 23Y, male patient aged 52Y, female patient aged 54Y and male patient aged 60Y suffered brain injury) of GDF11 (green) together with CaMKIIα (red) in the cerebral cortex of patients and 2 double-labelled neurons are indicated by arrows in ( q ). t Immunofluorescence double labelling of GDF11 (green) together with GABA (red, left), Olig2 (red, middle), GFAP (red, middle) and Iba1 (red, right) in the cerebral cortex of patients ( n = 4 patients). The GDF11 negative cells are indicated by arrows in ( t ). Scale bars, as shown on the images, 30 μm ( b , c ), 250 nm ( f ), 10 μm ( g ), 40 μm ( j , m , n , o ), 20 μm ( h , q , t ). Data are presented as mean ± SEM. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Immuno-Electron Microscopy

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Quantification by qPCR of the relative mRNA of GDF11 in the brain of the WT mice aged 3 M, 9 M or 36 M ( n = 3 mice/group). b Immunofluorescence double labelling of GDF11 (green) and CaMKIIα (red) in the cerebral cortices of the mice aged 3 M, 9 M and 36 M. One GDF11 + CaMKIIα + neuron is indicated by an arrow as an example per group. c Quantification of the average gray value of GDF11 in GDF11 + CaMKIIα + neurons in the cerebral cortices of the mice aged 3 M, 9 M and 36 M (3 M, n = 140; 9 M, n = 160; 36 M, n = 232 cells). d – g Representative images ( d ) and quantification ( e – g ) of the SA-β-Gal + cells in layers 4 and 5 ( d , up, and e , the dashed lines indicate the borders of layers 4 and 5, WT, n = 6; GDF11 f/f , n = 8; GDF11 cKO , n = 6), layer 6a ( d , middle, and f layer 6a is the deep layer cortex near the corpus callosum (CC), WT, n = 8; GDF11 f/f , n = 8; GDF11 cKO , n = 8) of the insular cortex (IC), and layers 2 and 3 of the piriform cortex ( d , down, and g the dashed lines indicate the borders of layers 2 and 3, WT, n = 8; GDF11 f/f , n = 10; GDF11 cKO , n = 10) of GDF11 cKO or GDF11 f/f or WT mice aged 10 M. h–j Representative images ( h ) and quantification of the SA-β-Gal + cells in the cingulate cortex of GDF11 cKO or GDF11 f/f mice aged 10 M ( i , GDF11 f/f , n = 8; GDF11 cKO , n = 6) and 17 M ( j , GDF11 f/f , n = 3; GDF11 cKO , n = 4). Examples of the SA-β-Gal + cells are indicated by double arrowheads in ( d , h ). k A schematic summary on the distribution of the SA-β-Gal + cells in the brain of GDF11 cKO or GDF11 f/f mice aged 10 M and 17 M. l Representative images of double labelling of SA-β-Gal staining (blue) and immunofluorescence of NeuN (fluorescence shown in white) in the insular cortex of GDF11 cKO or GDF11 f/f mice aged 10 M. Examples of the SA-β-Gal + NeuN + neurons are indicated by red arrowheads. m Representative images of double labelling of SA-β-Gal staining (blue) and immunohistochemical staining of CaMKIIα (brown) in the cerebral cortices of GDF11 cKO or GDF11 f/f mice aged 10 M. Examples of the SA-β-Gal + CaMKIIα + ENs are indicated by black arrows. n Survival curves of GDF11 f/f ( n = 35 mice) and GDF11 cKO mice ( n = 15 mice) which died naturally, and log-rank test P value was shown. Median survival is 25 months in GDF11 f/f mice and 22.8 months in GDF11 cKO mice. Scale bars, as shown on the images, 20 μm ( b , d up, m ), 40 μm ( d , middle and down), 50 μm ( h ) and 10 μm ( l ). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. a ( F (2, 6) = 6.672, e 0.0298; 3 M versus 36 M, P = 0.0270), c ( F (2529) = 18.77, P < 0.0001; 3 M versus 9 M, P < 0.0001; 3 M versus 36 M, P < 0.0001; 9 M versus 36 M, P = 0.5477), e ( F (2, 17) = 20.14, P < 0.0001; WT versus GDF11 f/f , P = 0.9950; GDF11 f/f , versus GDF11 cKO , P < 0.0001), f ( F (2, 21) = 4.825, P = 0.0189; WT versus GDF11 f/f , P = 0.9963; GDF11 f/f , versus GDF11 cKO , P = 0.0322) and g ( F (2, 25) = 11.61, P = 0.0003; WT versus GDF11 f/ f, P = 0.4738; GDF11 f/f , versus GDF11 cKO , P = 0.0002). One-way ANOVA with post Tukey multiple comparisons test. i ( P = 0.3427) and j ( P = 0.0280), unpaired two-tailed t test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Staining, Fluorescence, Immunohistochemical staining, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Immunofluorescence image of NeuN (green) in Neuro-2a cells ( n = 6 fields). Scale bar, 40 μm. b PCR of the cell genomes verified successful knockout of the targeted part of exon 2 of GDF11 in Neuro-2a cells (GDF11 KO ) ( n = 3 clones of GDF11 KO cells). c Verification of GDF11 knockout by comparing the mRNA enrichment tracks of GDF11 between GDF11 KO and WT Neuro2a cells by bulk RNA-seq. d Quantification of the relative mRNA of GDF11 in the GDF11 KO and WT Neuro-2a cells by qPCR ( n = 3 biological repeats/group). e , f Western blot ( e ) and Immunofluorescence of GDF11 ( f , scale bar, 40 μm) in GDF11 KO or WT Neuro-2a cells ( n = 3 biological repeats/ group). g , h Representative images ( g ) and quantification ( h , GDF11 KO , n = 13; WT, n = 12 fields) of the SA-β-Gal + cells (blue) in GDF11 KO and WT Neuro-2a cells. All cells are indicated by black stars, and a few representative SA-β-Gal + cells are indicated by black arrows. Scale bar, 50 μm. i Quantification of SA-β-Gal + cells in 3 independent clones of GDF11 KO and WT Neuro-2a cells (GDF11 KO , n = 3; WT, n = 3 clones). j , k Representative images ( j , DAPI, blue) and quantification ( k , GDF11 KO , n = 234 cells; WT, n = 211 cells) of the nuclei of GDF11 KO and WT Neuro-2a cells. Scale bar, 3 μm. l Volcano plot of upregulated (706) and downregulated (411) genes caused by deletion of GDF11 in Neuro-2a cells and revealed by bulk-RNA-seq ( n = 3 clones). m Bulk RNA-seq gene ontology (GO) analysis reveals the top 10 enriched biological processes downregulated by GDF11 deletion in Neuro-2a cells, and the logarithm base 2 of the fold change below −1 was included. n Heatmap of downregulated (11) or upregulated (1) genes involved in “lipid metabolic process” listed in m or “lipid droplets” caused by deletion of GDF11 in Neuro-2a cells, and the logarithm base 2 of the fold change above 1 or below −1 was included. o Representative images of transmission electron microscope (TEM) show the ultrastructure features of GDF11 KO and WT Neuro-2a cells. Cell nucleus (Nuc), lipofuscin (light blue arrows), neurosecretory granules (red double arrowheads) and mitochondrion (brown arrowheads) are indicated as examples. Scale bars, 2 μm. p – r Representative TEM images ( p , lipofuscins, light blue arrows) and quantification of the number (Q, GDF11 KO , n = 20 cells; WT, n = 20 cells) or the area ( r , GDF11 KO , n = 141; WT, n = 85 lipofuscins) of lipofuscins in the GDF11 KO and WT Neuro-2a cells. Scale bars, 500 nm. s – u Representative TEM images ( s , mitochondrion, brown arrowheads; neurosecretory granules, red double arrowheads) and quantification of the number ( t , GDF11 KO , n = 10 cells; WT, n = 10 cells) or the area ( u , GDF11 KO , n = 299; WT, n = 254 mitochondria) of the mitochondria of the GDF11 KO and WT Neuro-2a cells. Scale bars, 500 nm. v Quantification of the number of neurosecretory granules (GDF11 KO , n = 8 cells; WT, n = 10 cells) of the GDF11 KO and WT Neuro-2a cells. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant, d ( P < 0.0001), h ( P < 0.0001), i ( P = 0.0024), k ( P = 0.0030), q ( P = 0.0002), r ( P = 0.0274), t ( P = 0.8009), u ( P < 0.0001), v ( P = 0.0047), unpaired two-tailed t test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Immunofluorescence, Knock-Out, Clone Assay, RNA Sequencing Assay, Western Blot, Transmission Assay, Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Schematic diagrams (left) and representative images (right) of the cingulate gyrus 2 (Cg2), in the prefrontal cortex of GDF11 f/f mice aged 4M-5M, where bilateral focal injection of AAV9-CaMKIIα-Cre-P2A-GFP virus (KO) or AAV9-CaMKIIα-GFP virus (Ctrl) was received at age of 2–3 M and survived for two more months. b Infrared-differential interference contrast (IR-DIC) image (top) and GFP fluorescent image (bottom) of an example of GFP + EN which is undergoing whole-cell patch clamp recording ( n = 64 cells from six mice). c Representative whole-cell recordings in brain slice of a control EN (in Cg2 of GDF11 f/f mice, Ctrl, blue) and a GDF11 deleted-EN (in Cg2 of fGDF11 cKO mice, KO, red) show the firing of action potentials (AP) in response to a series of step current injections. d Examples show typical firing patterns of GFP + EN of fGDF11 cKO mice. e Pie graphs show the percentage of GFP + EN with diverse firing patterns (RS, regular spiking; IS, irregular spiking; IB, intrinsic bursting; RB, repetitive bursting) in WT or KO mice. f Left, plots of the AP frequency as a function of injected currents. Curves are color coded (Ctrl, blue, n = 31 cells from three mice; KO, red, n = 33 cells from three mice). Inset shows the beginning of the curve. Right, plots of the rheobase (Ctrl: 113 ± 16 vs. KO: 81 ± 10 pA, P = 0.049) and slope (Ctrl: 0.18 ± 0.01 vs. KO: 0.30 ± 0.03, P = 0.000) in the two groups (Ctrl, n = 31 cells from three mice; KO, n = 30 cells from three mice). g Left, representative AP waveforms (top) and phase plots (bottom) from Ctrl (blue) or KO (red) group. Right, plots of the AP threshold (Ctrl: −37.9 ± 0.8 vs. KO: −35.0 ± 0.7 mV, P = 0.014), amplitude (AMP) (Ctrl: 85.8 ± 1.6 vs. KO: 78.6 ± 2.2 mV, P = 0.010) and half-width (Ctrl: 0.79 ± 0.03 vs. KO: 0.74 ± 0.03 ms, P = 0.30) in the two groups (Ctrl, n = 29 cells from three mice; KO, n = 24 cells from three mice). h Left-top, representative membrane potential responses to negative current pulses from Ctrl (blue) or KO (red) groups. Plots of the input resistance (Ctrl: 104 ± 10 vs. KO: 214 ± 21 MΩ, P = 0.000), membrane constant (Ctrl: 14.4 ± 1.1 vs. KO: 22.1 ± 2.0 ms, P = 0.003), Sag ratio (Ctrl: 1.18 ± 0.02 vs. KO: 1.27 ± 0.03, P = 0.033), membrane capacitance (Ctrl: 147 ± 11 vs. KO: 95 ± 5 pF, P = 0.000) and RMP (Ctrl: −67.3 ± 1.0 vs. KO: −63.1 ± 0.9 mV, P = 0.004) in the two groups (Ctrl, n = 31 cells from three mice; KO, n = 33 cells from three mice). i Representative whole-cell recordings of mIPSC from the EN in GDF11 f/f mice (Ctrl, blue) and fGDF11 cKO mice (KO, red). j Left, scaled mIPSC examples in the two groups. Right, plots of rising time (Ctrl: 0.65 ± 0.04 vs. KO: 0.85 ± 0.06 ms, P = 0.005) and decay time (Ctrl: 4.44 ± 0.21 vs. KO: 4.69 ± 0.34 ms, P = 0.53) of mIPSCs in the two groups (Ctrl, n = 18 cells from four mice; KO, n = 16 cells from four mice). k , l Cumulative frequency curve of the inter-event-interval ( k ) and amplitude ( l ) of mIPSCs. Insets show the group plots of mIPSC frequency ( k , Ctrl: 34.6 ± 5.2 vs. KO: 4.0 ± 0.9 Hz, P = 0.000) and amplitude ( l , Ctrl: 24.0 ± 1.6 vs. KO: 20.5 ± 1.8 pA, P = 0.16). m – p Recordings of mEPSCs (Ctrl, n = 24 cells from four mice; KO, n = 28 cells from 4 mice) and similar plots as the mIPSCs shown above. Rising time ( n , ctrl: 0.87 ± 0.05 vs. KO: 0.81 ± 0.06 ms, P = 0.46); Decay time ( n , ctrl: 3.54 ± 0.20 vs. KO: 2.98 ± 0.24 ms, P = 0.041); Frequency ( o , Ctrl: 3.66 ± 0.84 vs. KO: 3.13 ± 0.65 Hz, p = 0.82); Amplitude ( p , Ctrl: 14.5 ± 0.8 vs. KO: 14.3 ± 0.9 pA, P = 0.33). q , r Representative traces showing IPSC ( q , left) or EPSC ( r , left) evoked by extracellular electric stimulations for the comparison of paired-pulse ratio (PPR) in GDF11 f/f mice (Ctrl, blue) and fGDF11 cKO mice (KO, red). Group plots of PPR for IPSC ( q , right, Ctrl, n = 7 cells from 3 mice: 0.98 ± 0.07 vs. KO, n = 9 cells from three mice: 1.16 ± 0.20, P = 0.92) and EPSC ( r , right, Ctrl, n = 9 cells from 3 mice: 1.38 ± 0.07 vs. KO, n = 6 cells from three mice: 1.26 ± 0.06, P = 0.24). s Track diagrams in the 3-chamber test (3CT) between the fGDF11 cKO (KO) and GDF11 f/f (Ctrl) mice aged 4–5 M. O object, S1 stranger mouse, S2 new stranger mouse. t Quantification of the exploration time in 3CT (KO, n = 13; Ctrl, n = 13 mice) on objects between the fGDF11 cKO (KO) and GDF11 f/f (Ctrl) mice aged 4–5 M. O1, object 1; O2, object 2. u Quantification of the preference index (S1-O) between the S1 and object in the KO and Ctrl groups (KO, n = 13; Ctrl, n = 13 mice). v Quantification of the preference index (S2-S1) between the S2 and S1 in the KO and Ctrl groups (KO, n = 13; Ctrl, n = 13 mice). w Schematic diagram of the novel object recognition test (NORT) between the GDF11 cKO and GDF11 f/f mice aged 10 M. Red squares indicate the familiar toy while blue triangle indicates a novel toy. x Quantification of the percentage of exploration time (GDF11 cKO , n = 9; GDF11 f/f , n = 6 mice) on the familiar or a novel toy in the GDF11 cKO and GDF11 f/f mice aged 10 M. y Quantification of the novel object discrimination index ((novel-familiar)/(novel + familiar)) between the familiar or a novel toy in the GDF11 cKO and GDF11 f/f mice aged 10 M (GDF11 cKO , n = 9; GDF11 f/f , n = 6 mice). Data are presented as mean ± SEM. Whisker boxplots in ( f , h ) represent the median and interquartile range; whiskers represent 1.5× interquartile range. * P < 0.05, ** P < 0.01 and “ns” represents not significant. f (Rheobase/Slope), h (Input resistance/Membrane constant/Sag ratio/Capacitance), j (Rising time), k , n (Decay time), o – q Mann–Whitney U test. g , h (RMP), j (Decay time), l , n (Rising time), r , u ( P = 0.0118), v ( P = 0.0128), x (GDF11 f/f : Familiar versus Novel, P = 0.0331; GDF11 cKO : Familiar versus Novel, P = 0.0188) and y ( P = 0.0254), unpaired two-tailed t test. t (Ctrl: O1 versus O2, P = 0.3210; KO: O1 versus O2, P = 0.2200), two-way ANOVA with post Sidak’s multiple comparisons test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Injection, Virus, Patch Clamp, Slice Preparation, Control, Membrane, Comparison, Whisker Assay, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Schematic diagrams of the cingulate gyrus 2 (Cg2), in the prefrontal cortex of GDF11 f/f mice aged 4–5 M, where bilateral focal injection of AAV9-CaMKIIα-Cre-P2A-GFP virus (KO) or AAV9-CaMKIIα-GFP virus (Ctrl) was received at age of 2–3 M and survived for two more months. b UMAP of the clustered 16 cell types in snRNA-seq of the Cg2 in both 3 KO mice and 3 control mice (Ctrl) aged 4–5 M. c Violin chart of the relative mRNA of GDF11 by snRNA-seq in KO-GFP + , KO-GFP - , Ctrl-GFP + or Ctrl-GFP - EN. The KO-EN were divided into KO-GFP + and KO-GFP - groups whereas “Ctrl-EN” were divided into Ctrl-GFP + and Ctrl-GFP − groups. d and e , Heatmap shows the average transcription of downregulated and upregulated ageing-related genes ( d ) and SASP-related genes ( e ) in snRNA-seq of KO-GFP + , KO-GFP − , Ctrl-GFP + or Ctrl-GFP − EN. f Confocal images (Left) and 3D-reconstruction (Right) of representative EN from Ctrl (Top) or KO (Bottom) groups. Dendrites and soma are presented in red, and axons are in blue. Scale bar, 50 μm. g , h Plots of the number of intersections of dendrites ( g ) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice) and the group data showing the number of total dendrite intersections ( h , Ctrl: 448 ± 28 vs. KO: 346 ± 36, P = 0.028). i – k Group data show the total number of apical dendrite intersections ( i , Ctrl: 238 ± 17 vs. KO: 181 ± 18, P = 0.036), the total length of apical dendrites ( j , Ctrl: 3.77 ± 0.28 vs. KO: 2.83 ± 0.34 mm, P = 0.044), and the apical branch orders against the averaged dendrite length ( k , branch order 1, Ctrl: 445 ± 28 vs. KO: 403 ± 22 μm, P = 0.26; branch order 2, Ctrl: 115 ± 3 vs. KO: 93 ± 8 μm, P = 0.017; order 3, Ctrl: 91 ± 4 vs. KO: 70 ± 6 μm, P = 0.007; branch order 4, Ctrl: 72 ± 6 vs. KO: 56 ± 7 μm, P = 0.12) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). l – n Group data comparing the number of total basal intersections ( l , Ctrl: 207 ± 15 vs. KO: 162 ± 22, P = 0.11), total basal dendrite length ( m , Ctrl: 2.73 ± 0.18 vs. KO: 2.16 ± 0.29 mm, P = 0.11) and the basal branch orders against the averaged dendrite length ( n , branch order 1, Ctrl: 102 ± 4 vs. KO: 102 ± 8 μm, P = 0.98; branch order 2, Ctrl: 82 ± 3 vs. KO: 82 ± 9 μm, P = 0.32; order 3, Ctrl: 69 ± 8 vs. KO: 59 ± 2 μm, P = 0.25) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). o , p Plots of the axon distance from soma against the number of intersections ( o ) in the two groups (Ctrl, n = 11 cells from three mice; KO, n = 11 cells from three mice). Group data show the number of total axon branches intersections ( p , Ctrl: 239 ± 17 vs. KO: 190 ± 28, P = 0.15). q Confocal examples of dendritic spines (red arrows indicate the big mushroom spines while yellow arrows point to small mushroom spines) in the two groups. Scale bar, 5 μm. r , s Group data show total spine density per 10 μm ( r , Ctrl: 6.28 ± 0.23 vs. KO: 1.61 ± 0.13/10 μm, P = 0.000) and mushroom spine diameter ( s , Ctrl: 0.66 ± 0.01 vs. KO: 0.80 ± 0.02 μm, P = 0.000) in two groups (Ctrl, n = 68 dendrites from 16 cells; KO, n = 70 dendrites from 16 cells). t Plots of spine density against the mushroom spine diameter in the two groups (Ctrl, n = 16 cells from three mice; KO, n = 16 cells from three mice). u A schematic summary: GDF11 deletion results in hyperexcitability of the EN as reflected by an enhancement in their firing frequency (due to increased input resistance and elevated RMP) and a decrease in mIPSC frequency. In addition, GDF11 deletion in the EN prunes and shortens their apical dendrites, reduces their dendritic mushroom spine density while enlarges its size. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. h , i , j , k , l , m , n , p , unpaired two-tailed t test; r , s , Mann–Whitney U test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Injection, Virus, Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a , b SnRNA-seq GO analysis reveals the top ten enriched biological processes of upregulated ( a ) or downregulated ( b ) in the KO-GFP + EN in comparison with the KO-GFP - EN, and the EN were obtained from the Cg2 of the “KO” mice and the “Ctrl” mice aged 4–5 M. c Volcano plot shows upregulated and downregulated DEGs in the KO-GFP + EN in comparison with the Ctrl-GFP + EN. Some of the top upregulated and downregulated genes were annotated. c , d FC fold change. P value was calculated using Wilcox test and adjusted for multiple testing using Benjamini–Hochberg correction. d Volcano plot shows upregulated and downregulated DEG in the KO-GFP + EN in comparison with the KO-GFP - EN. Some of the top upregulated and downregulated genes were indicated. e UMAP visualization highlights the distribution and the transcription of Cdkn1a/p21 in the identified cell types in snRNA-seq. f Dot plot representing the frequency and average transcription of Cdkn1a/p21 in the identified cell types in snRNA-seq. g , h Relative mRNA of Cdkn1a/p21 ( g ) or p53 ( h ) among four types of EN: Ctrl-GFP - , Ctrl-GFP + , KO-GFP - and KO-GFP + by snRNA-seq. i Heatmap of upregulated (10) and downregulated (6) genes involved in “cellular senescence” caused by deletion of GDF11 in Neuro-2a cells, and the logarithm base 2 of the fold change above 1 or below −1 was included. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant. a , b Hypergeometric test with Benjamini and Hochberg (BH) correction. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Comparison

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a , b Genetic strategy for generation of p21 f/f mice ( a ) and CaMKIIα-Cre; GDF11 f/f ; p21 f/f mice ( b ) to selectively delete both GDF11 and p21 in CaMKIIα + neurons through Cre/Loxp system. c – g Representative images ( c ) and quantification ( d – g ) of the SA-β-Gal + cells in the cingulate cortex ( c , up, and d , n = 4 per group), layers 4 and 5 ( c , middle, and e GDF11 f/f , n = 4; GDF11 cKO , n = 3; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 5), layer 6a ( c middle, and f layer 6a is the deep layer cortex near the corpus callosum (CC), GDF11 f/f , n = 5; GDF11 cKO , n = 4; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 4) of the insular cortex (IC), and layers 2 and 3 of the piriform cortex ( c down, and g the dashed lines indicate the borders of layers 2 and 3, GDF11 f/f , n = 8; GDF11 cKO , n = 4; CaMKIIα-Cre; GDF11 f/f ;p21 f/f , n = 8) of CaMKIIα-Cre; GDF11 f/f ;p21 f/f or GDF11 cKO or GDF11 f/f mice aged 17 M. Examples of the SA-β-Gal + cells are indicated by double arrows. Scale bars, as shown on the images, 50 μm ( c , up and middle) and 20 μm ( c , middle and down). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01. d ( F (2, 9) = 72.52, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0006; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0004), e ( F (2, 9) = 78.16, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0020; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001), f ( F (2, 10) = 49.87, P < 0.0001; GDF11 f/f versus GDF11 cKO , P < 0.0001; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0347) and g ( F (2, 17) = 102.8, P < 0.0001; GDF11 f/f versus GDF11 cKO , P = 0.0227; GDF11 cKO versus CaMKIIα-Cre;GDF11 f/f; p21 f/f , P < 0.0001; GDF11 f/f versus CaMKIIα-Cre;GDF11 f/f ;p21 f/f , P = 0.0001), One-way ANOVA with post Tukey multiple comparisons test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques:

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: a Quantification by qPCR of the relative p21 mRNA in the GDF11 KO and WT Neuro-2a cells ( n = 3 clones). b – e Immunofluorescence representative images ( b ) and quantification of the number of p21 + cells per field ( c , n = 5 fields/group), the proportion of p21 + cells ( d , n = 6 fields/group) or the average gray value of p21 per cell ( e , GDF11 KO , n = 420 cells; WT, n = 280 cells) in the GDF11 KO and WT Neuro-2a cells. Scale bar, 25 μm. Examples of the p21 + cells are indicated by double arrowheads. f – h The same snRNA-seq data were used, as described in Fig. . f Rank for regulons in the EN based on regulon specificity score (RSS). The EN were obtained from the Cg2 of the “KO” mice and the “Ctrl” mice aged 4–5 M. g Regulons activity analysis based on area under the curve (AUC) in the identified cell types in snRNA-seq of the “KO” mice and the “Ctrl” mice aged 4–5 M. The activity of regulon Smad3 (highlighted in red) is high in the EN. h Cytoscape network visualization of genes including GDF11, Cdkn1a (p21), Smad2, Smad3 (highlighted in red) and their transcription factors (TFs, yellow). i – m Representative images ( i and l ) and quantification by densitometry of western blot analysis of Smad2 ( j ), phosphorylated Smad2 (pSmad2, k ) and Smad3 ( m ) in the total protein extracted from the GDF11 KO and WT Neuro-2a cells ( n = 3 biological repeats/group). n ChIP-qPCR assessment of the enrichment of Smad2 at the promoter of Cdkn1a/p21 in the GDF11 KO and WT Neuro-2a cells ( n = 3 biological repeats/group). o A proposed working model for loss of GDF11 on cellular senescence. Loss of GDF11 upregulates pSmad2, enhances nuclear entry of Smad2/3 tricomplex and then Smad2 binds to the promoter of p21 and promotes the pro-senescence factor p21 transcription, and eventually causes cellular senescence. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and “ns” indicates not significant. a ( P = 0.0037), c ( P = 0.0033), d ( P = 0.0157), e ( P < 0.0001), j ( P = 0.6648), k ( P = 0.0040) and m ( P = 0.0299), unpaired two-tailed t test. n (IgG: WT versus GDF11 KO , P = 0.57; Smad2: WT versus GDF11 KO , P < 0.001), two-way ANOVA with Sidak’s test. Source data are provided with this paper.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: Clone Assay, Immunofluorescence, Activity Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: GDF11 slows excitatory neuronal senescence and brain ageing by repressing p21

doi: 10.1038/s41467-023-43292-1

Figure Lengend Snippet: Evidence of both in vitro (left) and in vivo (right) indicates that growth differentiation factor 11-Smad2/3-p21 pathway acts as a brake on excitatory neuronal senescence and brain ageing.

Article Snippet: Mouse GDF11 and GDF8 expression plasmids (pMs-GDF11-Flag-Myc or pMs-GDF8-Flag-Myc) with C-terminal 3Flag and Myc tag were designed and produced by Origene.

Techniques: In Vitro, In Vivo

Journal: bioRxiv

Article Title: PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

doi: 10.1101/2025.06.14.659731

Figure Lengend Snippet: (a) HCT116 cells stably expressing DDX6-GFP were plated in 96-well plates, treated with 280 compounds at 10µM concentrations, and subjected to high-content imaging using the CQ1 confocal quantitative imaging system. (b) The analyzed images consist of four channels: (i) Bright-field image for cellular morphology, (ii) Mitochondrial network, (iii) Processing body, and (iv) Nucleus. Merged composite demonstrates spatial relationships between these subcellular compartments. Scale bar: 10μm. (c) Mitochondrial channels were processed through Cellpose 3.0 to generate a curated dataset containing over 400,000 high-quality single-cell images. (d) A contrastive clustering framework was implemented for unsupervised feature extraction, followed by UMAP dimensionality reduction to identify compounds with analogous mechanism-of-action (MOA) profiles through cluster localization analysis. (e) Quantitative analysis of P-body formation followed by drug treatment. (f) Mechanistic evaluation of lead compounds via imaging analysis.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP) and EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382).

Techniques: Stable Transfection, Expressing, Imaging, Extraction

Journal: bioRxiv

Article Title: PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

doi: 10.1101/2025.06.14.659731

Figure Lengend Snippet: (a) A simulation model of intracellular p-body was constructed to generate synthetic p-body distributions with ground truth annotations. (b) A YOLO-v7 architecture trained on synthetic datasets was implemented for automated identification and quantitative analysis of P-body formation. (c) Example of P-body detection, achieving >95% agreement with manual annotations . (d) P-body numbers per cell in the time course under different drug treatment groups. (e) DDX6-GFP expression level (a.u.) per cell under different drug treatment groups. Error bars represent the STD of three independent analyses for d and e. (f) Quantitative analysis of P-body numbers at 6 hours post-treatment across different drug groups. (g) Quantitative analysis of DDX6 expression level (a.u.) at 6 hours post-treatment across different drug groups. The p -values were determined using the two-tailed Mann–Whitney test for f and g. The statistical significance compared with DMSO was indicated as *** P < 0.001; ** P < 0.01; * P < 0.05; ns, no significant difference. Data points that lay outside the 15% - 85% range were deemed outliers and excluded from the statistical analysis. (h, i) Mechanism of Action (MOA) profiling for drugs in Groups 1 and 3.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP) and EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382).

Techniques: Construct, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: PB-scope: Contrastive learning of dynamic processing body formation reveals undefined mechanisms of approved compounds

doi: 10.1101/2025.06.14.659731

Figure Lengend Snippet: (a) HCT116 cells were knocked down using JAK1 and JAK2 siRNA, and immunostained for P-body components DDX6 (magenta) and EDC4 (green). The nuclei were visualized with DAPI (blue). Scale bar, 10μm. (b) Quantification of P-body number per cell across three experimental groups. Statistical significance determined by an unpaired t-test was indicated as *** P < 0.001. (c) Model of JAK/STAT signaling pathway-mediated P-body regulation. JAK is activated when cytokines or growth factors bind to their respective receptors, leading to receptor dimerization, JAK and STAT phosphorylation, and subsequent transcriptional regulation. Inhibition of the pathway by knockdown of JAK1/2 leads induction of P-body formation. The JAK inhibitors identified in this work that modulate P-body formation are shown in the right panel.

Article Snippet: As primary antibodies, we used DDX6 rabbit polyclonal antibody (Proteintech, 14632-1-AP) and EDC4 mouse monoclonal antibody (Santa Cruz Biotechnology, sc-376382).

Techniques: Phospho-proteomics, Inhibition, Knockdown

Journal: Journal of Virology

Article Title: Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II

doi: 10.1128/jvi.00719-23

Figure Lengend Snippet: Metabolites derived from Bacillus subtilis against African swine fever virus evaluated in PAMs. (A) Schema of the experimental design for the screening in PAMs. (B) The role of metabolites of B. subtilis in ASFV infection. The dot represents the mean, the black dotted line represents the 90% inhibition ratio, the red dots represent the metabolites of the four B. subtilis strains with the highest inhibition efficiency against ASFV proliferation. (C) The function of the metabolites of the four B. subtilis strains assessed using western blot analysis. (i) ASFV p72 and porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins were detected. (ii) The band intensity of ASFV p72 proteins. (D) The metabolites from the four B. subtilis strains showing the highest inhibition efficiency against ASFV proliferation were estimated using a hemadsorption assay. (E) The metabolites from the four B. subtilis strains showing the highest viral inhibition efficiency were estimated by immunofluorescence staining. ASFV p72 proteins were stained with CoraLite488-conjugated Goat Anti-mouse IgG (green); nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (blue). (i) Stained cells were examined using the EVOS FL Auto system. (ii) Mean fluorescence intensity in PAMs treated with different media. AU, arbitrary units. Data were analyzed using a two-tailed Student’s t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: After blocking with 1% BSA for 1 h at 37°C, PAMs were incubated with an ASFV p72 monoclonal antibody preserved in our laboratory at 37°C for 2 h. After washing three times with phosphate buffer solution (PBS) for 5 min each, the PAMs were incubated with CoraLite488-conjugated Goat Anti-mouse IgG(H + L) (Proteintech) at 37°C for 1 h. Samples were visualized using the EVOS FL Auto system (Thermo Fisher Scientific). . HAD assay The HAD assay was performed to detect the titer of ASFV in the cell supernatant and tissue homogenate samples, as previously described ( 33 ).

Techniques: Derivative Assay, Virus, Infection, Inhibition, Western Blot, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: Journal of Virology

Article Title: Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II

doi: 10.1128/jvi.00719-23

Figure Lengend Snippet: Oral administration of four B. subtilis strains with the highest inhibition efficiency prevents ASFV occurrence in pigs. (A) Schema of the experimental design for assessing the activity of B. subtilis strains against ASFV infection in pigs. The biologics of four B. subtilis strains and fermentation broth were orally administered throughout the experimental cycle for 38 days, including 10 days before and 28 days after infection. The biologics consisted of 109 CFUs of the four B. subtilis strains (2.5 × 108 CFU each) and 8 mL of metabolites from the four B. subtilis isolates (2 mL each) for each pig daily. The pigs were challenged with 500 HAD50 ASFV by oral administration on day 0. Daily observations and swab collection were conducted after ASFV infection. (B) Daily body temperature of pigs after ASFV infection. (C) Survival rate of pigs after ASFV infection. Virus shedding after ASFV infection. Daily viral genomic DNA copies were determined from nasal (D), oral (E), and rectal (F) swabs of pigs from different groups after infection. (G) Viral genomic DNA copies in the blood of pigs at 0, 7, 14, 21, and 28 dpi. (H) Viral genomic DNA copies in the heart, liver, spleen, lung, kidney, SLN, ILN, and MLN of pigs at death or up to 28 dpi. (I) Viral titer in the heart, liver, spleen, lung, kidney, SLN, ILN, and MLN of pigs at death or up to 28 dpi. (J) Hematoxylin and eosin staining assay showing subtle pathological changes in the spleen, kidney, and ILN of pigs. Red arrows indicate lesions in tissue samples. (K) Immunohistochemistry using antibodies to ASFV p72 proteins. SLN, submaxillary lymph node; ILN, inguinal lymph node; MLN, mesenteric lymph node. Data were analyzed using a two-tailed Student’s t-test (****P < 0.0001).

Article Snippet: After blocking with 1% BSA for 1 h at 37°C, PAMs were incubated with an ASFV p72 monoclonal antibody preserved in our laboratory at 37°C for 2 h. After washing three times with phosphate buffer solution (PBS) for 5 min each, the PAMs were incubated with CoraLite488-conjugated Goat Anti-mouse IgG(H + L) (Proteintech) at 37°C for 1 h. Samples were visualized using the EVOS FL Auto system (Thermo Fisher Scientific). . HAD assay The HAD assay was performed to detect the titer of ASFV in the cell supernatant and tissue homogenate samples, as previously described ( 33 ).

Techniques: Inhibition, Activity Assay, Infection, Virus, Staining, Immunohistochemistry, Two Tailed Test

Journal: Journal of Virology

Article Title: Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II

doi: 10.1128/jvi.00719-23

Figure Lengend Snippet: Cellular and humoral immunity before and after ASFV infection. (A) Ratio of porcine positive CD4+ and CD8+ T lymphocytes at 5 and 10 days after oral administration with B. subtilis or vehicle in pigs without ASFV infection. Porcine IL-4 (B), IL-1β (C), IL-8 (D), IFN-β (E), IFN-γ (F), and IFN-α (G) levels. (H) Ratio of porcine positive CD4+ and CD8+ T lymphocytes at 7 and 14 days after oral administration with B. subtilis or vehicle in pigs infected with ASFV. Porcine IL-4 (I), IL-1β (J), IL-8 (K), IFN-β (L), IFN-γ (M), and IFN-α (N) levels. Data were analyzed using a two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: After blocking with 1% BSA for 1 h at 37°C, PAMs were incubated with an ASFV p72 monoclonal antibody preserved in our laboratory at 37°C for 2 h. After washing three times with phosphate buffer solution (PBS) for 5 min each, the PAMs were incubated with CoraLite488-conjugated Goat Anti-mouse IgG(H + L) (Proteintech) at 37°C for 1 h. Samples were visualized using the EVOS FL Auto system (Thermo Fisher Scientific). . HAD assay The HAD assay was performed to detect the titer of ASFV in the cell supernatant and tissue homogenate samples, as previously described ( 33 ).

Techniques: Infection, Two Tailed Test

Journal: Journal of Virology

Article Title: Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II

doi: 10.1128/jvi.00719-23

Figure Lengend Snippet: Administration of B. subtilis mixed with pig feed reduces ASFV infection and prevents disease occurrence in pigs. (A) Schema of the experimental design. Powders of B. subtilis contain 2 × 109 CFU from the four B. subtilis strains (5 × 108 CFU each) administered to each pig daily. Mixed pig feed was administered throughout the experimental cycle for 38 days, including 10 days before infection and 28 days after infection. The pigs were challenged with 500 HAD50 ASFV via oral administration on day 0. (B) Daily body temperature of pigs after ASFV infection. (C) Survival rate of pigs after ASFV infection. Virus shedding was detected after ASFV infection. Daily viral genomic copies from nasal (D), oral (E), and rectal (F) swabs of pigs after infection. (G) Viral genomic copies in the blood samples of pigs at 0, 7, 14, 21, and 28 dpi. (H) Viral genomic copies in the heart, liver, spleen, lung, kidney, SLN, ILN, and MLN of pigs at death or up to 28 dpi. (I) Viral titer in the heart, liver, spleen, lung, kidney, SLN, ILN, and MLN of pigs at death or up to 28 dpi. (J) Hematoxylin and eosin staining of the tissue samples of the spleen, kidney, and ILN of pigs. Red arrows indicate lesions in tissue samples. (K) Immunohistochemistry of the antibodies of ASFV p72 proteins. SLN, submaxillary lymph node; ILN, inguinal lymph node; MLN, mesenteric lymph node. Data were analyzed using a two-tailed Student’s t-test (****P < 0.0001).

Article Snippet: After blocking with 1% BSA for 1 h at 37°C, PAMs were incubated with an ASFV p72 monoclonal antibody preserved in our laboratory at 37°C for 2 h. After washing three times with phosphate buffer solution (PBS) for 5 min each, the PAMs were incubated with CoraLite488-conjugated Goat Anti-mouse IgG(H + L) (Proteintech) at 37°C for 1 h. Samples were visualized using the EVOS FL Auto system (Thermo Fisher Scientific). . HAD assay The HAD assay was performed to detect the titer of ASFV in the cell supernatant and tissue homogenate samples, as previously described ( 33 ).

Techniques: Infection, Virus, Staining, Immunohistochemistry, Two Tailed Test

Journal: Journal of Virology

Article Title: Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II

doi: 10.1128/jvi.00719-23

Figure Lengend Snippet: Screening of small-molecule metabolites from B. subtilis for their activity against ASFV replication. (A) Schema of the experimental design of protein precipitation of the fermentation broth of four B. subtilis strains. (B) Viral genomic copies were detected for the assessment of the anti-ASFV activity of untreated and treated fermentation broth from four B. subtilis strains. (C) Schematic diagram of the study design for screening small-molecule metabolites. (D) The activity of small-molecule metabolites of B. subtilis against ASFV infection. The dot represents the mean, the black dotted line represents the 90% inhibition ratio, and the red dots represent the eight small molecules with the highest inhibition efficiency against ASFV proliferation. (E) The function of the eight small molecules against ASFV replication was assessed using western blot analysis. (i) ASFV p72 and porcine GAPDH proteins were detected. (ii) The band intensity of ASFV p72 proteins. (F) Viral titer was detected for evaluating the function of eight different small molecules against ASFV infection. (G) Measurement of the half-maximal inhibitory concentration of the eight small molecules. The black dotted line represents the 50% inhibition ratio. (H) Biological characterization of the eight small-molecule metabolites from B. subtilis strains. Data were analyzed using a two-tailed Student’s t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: After blocking with 1% BSA for 1 h at 37°C, PAMs were incubated with an ASFV p72 monoclonal antibody preserved in our laboratory at 37°C for 2 h. After washing three times with phosphate buffer solution (PBS) for 5 min each, the PAMs were incubated with CoraLite488-conjugated Goat Anti-mouse IgG(H + L) (Proteintech) at 37°C for 1 h. Samples were visualized using the EVOS FL Auto system (Thermo Fisher Scientific). . HAD assay The HAD assay was performed to detect the titer of ASFV in the cell supernatant and tissue homogenate samples, as previously described ( 33 ).

Techniques: Activity Assay, Infection, Inhibition, Western Blot, Concentration Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II

doi: 10.1128/jvi.00719-23

Figure Lengend Snippet: Arctiin inhibits ASFV replication at the mid-stage of infection. (A) Schematic diagram of the study design. (B) Viral genomic copies at different stages of infection after pre-treatment, co-treatment, and post-treatment with arctiin. (C) Viral titers were measured using the hemadsorption assay. (D) Western blot analysis. (i) ASFV p72 and porcine GAPDH proteins were detected. (ii) The band intensity of ASFV p72 protein. (E) Viral genomic copies were detected at the time of addition of arctiin after infection. (F) Western blot analysis of proteins at the time of addition of arctiin after infection. (i) ASFV p72 and porcine GAPDH proteins were detected. (ii) The band intensity of ASFV p72 protein. (G) Viral titers were detected at the time of addition of arctiin after infection. (H) Antiviral activities at the time of addition of arctiin after infection were analyzed by immunofluorescence staining. The ASFV p72 proteins were stained with CoraLite488-conjugated Goat Anti-mouse IgG (green); nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (blue). (i) Stained cells were examined using the EVOS FL Auto system. (ii) Mean fluorescence intensity in PAMs. AU, arbitrary units. Data were analyzed using a two-tailed Student’s t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: After blocking with 1% BSA for 1 h at 37°C, PAMs were incubated with an ASFV p72 monoclonal antibody preserved in our laboratory at 37°C for 2 h. After washing three times with phosphate buffer solution (PBS) for 5 min each, the PAMs were incubated with CoraLite488-conjugated Goat Anti-mouse IgG(H + L) (Proteintech) at 37°C for 1 h. Samples were visualized using the EVOS FL Auto system (Thermo Fisher Scientific). . HAD assay The HAD assay was performed to detect the titer of ASFV in the cell supernatant and tissue homogenate samples, as previously described ( 33 ).

Techniques: Infection, Western Blot, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: Journal of Virology

Article Title: Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II

doi: 10.1128/jvi.00719-23

Figure Lengend Snippet: Arctiin inhibits ASFV infection by targeting the ATP-binding domain of viral topoisomerase II. (A) Native-PAGE assay to explore the interaction between arctiin and viral proteins. (i) Native-PAGE followed by silver staining to analyze protein bands showing a mobility shift between the lysed proteins from PAMs infected with ASFV at 8 hpi and arctiin. (ii) The results obtained for arctiin combined with four viral proteins by LC-MS/MS analysis. (B) Binding dynamics of the four viral proteins, P1192R, CP204L, NP419L, and C129R, to arctiin as assessed by a BLI binding assay. (C) Binding dynamics between arctiin and viral topoisomerase II. (D) Binding dynamics between ATP and viral topoisomerase II. (E) Binding dynamics between genistein and viral topoisomerase II. (F) Arctiin combined with the ATP-binding site of viral topoisomerase II, as assessed using molecular docking analysis. (G) ATP combined with the ATP-binding site of viral topoisomerase II, as assessed using molecular docking. (H) Genistein combined with the ATP-binding site of viral topoisomerase II, as assessed using molecular docking. (I) Binding dynamics of ATP, arctiin, and genistein with deletion of the ATP-binding domain (1–247 amino acids) of viral topoisomerase II, as assessed using a BLI binding assay. (J) Competitive decatenation assays performed to validate the effect of arctiin and genistein on the catalytic activity of viral topoisomerase II. (i) The decatenation products are detected. (ii) The band intensity of decatenation products. Novobiocin was used as a positive control.

Article Snippet: After blocking with 1% BSA for 1 h at 37°C, PAMs were incubated with an ASFV p72 monoclonal antibody preserved in our laboratory at 37°C for 2 h. After washing three times with phosphate buffer solution (PBS) for 5 min each, the PAMs were incubated with CoraLite488-conjugated Goat Anti-mouse IgG(H + L) (Proteintech) at 37°C for 1 h. Samples were visualized using the EVOS FL Auto system (Thermo Fisher Scientific). . HAD assay The HAD assay was performed to detect the titer of ASFV in the cell supernatant and tissue homogenate samples, as previously described ( 33 ).

Techniques: Infection, Binding Assay, Clear Native PAGE, Silver Staining, Mobility Shift, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Positive Control

Journal: Journal of Virology

Article Title: Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II

doi: 10.1128/jvi.00719-23

Figure Lengend Snippet: Arctiin and genistein inhibit ASFV infection and reduce mortality in pigs. (A) Schematic diagram of the study design. Arctiin (2 mg/kg), genistein (2 mg/kg), and arctiin (2 mg/kg) and genistein (2 mg/kg) were orally administered for 38 days, from 10 days before infection to 28 days after infection for each pig, daily. The pigs were challenged with 500 HAD50 ASFV by oral administration on day 0, and daily observations and swab collection were conducted after ASFV infection. (B) Daily body temperature of pigs after ASFV infection. (C) Survival rate of pigs after ASFV infection. Virus shedding after ASFV infection. Daily viral genomic DNA copies from the nasal (D), oral (E), and rectal (F) swabs of pigs from different groups after infection. (G) Viral genomic DNA copies in the blood of pigs at 0, 7, 14, 21, and 28 dpi. (H) Viral genomic DNA copies in the heart, liver, spleen, lung, kidney, SLN, ILN, and MLN of pigs at death or up to 28 dpi. (I) Viral titer in the heart, liver, spleen, lung, kidney, SLN, ILN, and MLN of pigs at death or up to 28 dpi. (J) Hematoxylin and eosin staining of the tissue samples of the spleen, kidney, and ILN of pigs. Red arrows indicate lesions in tissue samples. (K) Immunohistochemistry of the antibodies of ASFV p72 proteins. SLN, submaxillary lymph node; ILN, inguinal lymph node; MLN, mesenteric lymph node. Data were analyzed using a two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: After blocking with 1% BSA for 1 h at 37°C, PAMs were incubated with an ASFV p72 monoclonal antibody preserved in our laboratory at 37°C for 2 h. After washing three times with phosphate buffer solution (PBS) for 5 min each, the PAMs were incubated with CoraLite488-conjugated Goat Anti-mouse IgG(H + L) (Proteintech) at 37°C for 1 h. Samples were visualized using the EVOS FL Auto system (Thermo Fisher Scientific). . HAD assay The HAD assay was performed to detect the titer of ASFV in the cell supernatant and tissue homogenate samples, as previously described ( 33 ).

Techniques: Infection, Virus, Staining, Immunohistochemistry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Estradiol Attenuates the Severity of Primary Toxoplasma gondii Infection-Induced Adverse Pregnancy Outcomes Through the Regulation of Tregs in a Dose-Dependent Manner

doi: 10.3389/fimmu.2018.01102

Figure Lengend Snippet: Expression of Foxp3 and programmed death-1 (PD-1) at the maternal–fetal interface of mice assessed by immunofluorescence staining. Nonpregnant and pregnant mice at G5 and G15 were injected with Toxoplasma gondii tachyzoites or PBS, respectively. All the animals were sacrificed at 3 days post infection (nonpregnant, G8 and G18). (A) Single staining of Foxp3 + cells. Blue, DAP1; red, Foxp3. Scale bars, 50 µm. (B) Single staining of PD-1 + cells. Blue, DAP1; green, PD-1. Scale bars, 50 µm. (C) Double staining of PD-1 and Foxp3. Blue, DAP1; green, PD-1; red, Foxp3. Scale bars, 50 µm. White arrows indicate PD-1 + Foxp3 + cells.

Article Snippet: In the case of single staining of Foxp3 and PD-1, the slides were incubated with anti-Foxp3 antibody (1:100, Servicebio, Wuhan, China) and anti-PD-1 antibody (1:100, proteintech, Wuhan, China), respectively, overnight at 4°C.

Techniques: Expressing, Immunofluorescence, Staining, Injection, Infection, Double Staining

Journal: Frontiers in Immunology

Article Title: Estradiol Attenuates the Severity of Primary Toxoplasma gondii Infection-Induced Adverse Pregnancy Outcomes Through the Regulation of Tregs in a Dose-Dependent Manner

doi: 10.3389/fimmu.2018.01102

Figure Lengend Snippet: Toxoplasma gondii infection in early pregnancy induced lower levels of programmed death-1 (PD-1) expression on Tregs than that in late pregnancy. The percentage of PD-1 + cells in CD4 + T cells (A) and the mean fluorescence intensity (MFI) of PD-1 expression on CD4 + T cells (B) in spleens and inguinal lymph glands of the nonpregnant and pregnant mice (G8, G18) with T. gondii tachyzoite or PBS injection, respectively. The gating strategy for the identification of the mouse PD-1 + CD4 + T cell population and the isotype control are shown in Figure S1B in Supplementary Material. Data are expressed as the means ± SD of five mice for each group from one experiment representative of two independent experiments. Significance was analyzed by one-way ANOVA. *** P < 0.001, ** P < 0.01, * P < 0.05. The percentage of PD-1 + cells in CD4 + CD25 + Foxp3 + T cells (C) and the MFI of PD-1 expression on CD4 + CD25 + Foxp3 + T cells (D) in spleens and inguinal lymph glands from the mice as mentioned above. The gating strategy for the identification of the mouse PD-1 + CD4 + CD25 + Foxp3 + T cell population and the isotype control are shown in Figure S1B in Supplementary Material. Data are expressed as the means ± SD of five mice for each group from one experiment representative of two independent experiments. Significance was determined by one-way ANOVA. *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: In the case of single staining of Foxp3 and PD-1, the slides were incubated with anti-Foxp3 antibody (1:100, Servicebio, Wuhan, China) and anti-PD-1 antibody (1:100, proteintech, Wuhan, China), respectively, overnight at 4°C.

Techniques: Infection, Expressing, Fluorescence, Injection, Control

Journal: Frontiers in Immunology

Article Title: Estradiol Attenuates the Severity of Primary Toxoplasma gondii Infection-Induced Adverse Pregnancy Outcomes Through the Regulation of Tregs in a Dose-Dependent Manner

doi: 10.3389/fimmu.2018.01102

Figure Lengend Snippet: Estradiol (E2) inhibited the apoptosis of Tregs and enhanced the programmed death-1 (PD-1) expression on Tregs in a dose-dependent manner. (A) Splenocytes from C57BL/6 mice were treated with E2 ranging from 10 −10 to 10 −6 M for 24, 48, 72, and 96 h, respectively. The apoptotic rate of Tregs was estimated by Annexin V/7AAD analysis gated on CD4 + CD25 + T cells. Data are presented as the means ± SD ( n = 6). (B) Splenocytes were treated with E2 as mentioned above. The percentage of PD-1 + cells in CD4 + CD25 + Foxp3 + T cells was evaluated by flow cytometry. Data are expressed as the means ± SD ( n = 6). (C) Mean fluorescence intensity (MFI) of PD-1 expression on CD4 + CD25 + Foxp3 + T cells. Data are presented as the means ± SD ( n = 6). (D) The levels of IFN-γ, IL-10, and TGF-β1 secretion by mouse splenocytes stimulated with E2 ranging from 10 −10 to 10 −6 M for 96 h. Data are represented as the means ± SD ( n = 6). Significance was analyzed using one-way ANOVA. *** P < 0.001, * P < 0.05, n.s. P > 0.05.

Article Snippet: In the case of single staining of Foxp3 and PD-1, the slides were incubated with anti-Foxp3 antibody (1:100, Servicebio, Wuhan, China) and anti-PD-1 antibody (1:100, proteintech, Wuhan, China), respectively, overnight at 4°C.

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Estradiol Attenuates the Severity of Primary Toxoplasma gondii Infection-Induced Adverse Pregnancy Outcomes Through the Regulation of Tregs in a Dose-Dependent Manner

doi: 10.3389/fimmu.2018.01102

Figure Lengend Snippet: Estradiol (E2) inhibited the apoptosis of Tregs and enhanced the programmed death-1 (PD-1) expression on Tregs through ERα. (A) Splenocytes from C57BL/6 mice were treated with OVA (Control), excreted–secreted antigens (ESA), ESA and E2, ESA and ERα agonist (PPT) with different concentrations (10 −10 , 10 −8 , and 10 −6 M), respectively. The apoptotic rate of CD4 + CD25 + T cells was evaluated by flow cytometry. Data are expressed as the means ± SD ( n = 4) and are representative of two independent experiments. Significance was determined by one-way ANOVA. *** P < 0.001, ** P < 0.01. (B) Splenocytes from C57BL/6 mice were treated with OVA (control), ESA, ESA and E2, and ESA together with E2 and ER antagonist ICI 182,780 (ICI), respectively. The apoptotic rate of CD4 + CD25 + T cells was evaluated by flow cytometry. Data are expressed as the means ± SD ( n = 4) and are representative of two independent experiments. Significance was determined by one-way ANOVA. *** P < 0.001, ** P < 0.01. (C) Representative dot plots and the bar graph show the frequency of PD-1 + cells in Tregs after treating with E2, PPT with different concentrations (10 −10 , 10 −8 , and 10 −6 M) and ESA, respectively. Each bar indicates the mean value ± SD ( n = 4) and is representative of two independent experiments. Significance was determined by one-way ANOVA. *** P < 0.001, ** P < 0.01, * P < 0.05. (D) The mean fluorescence intensity (MFI) of PD-1 expression on CD4 + CD25 + Foxp3 + T cells. Data are expressed as the means ± SD ( n = 4) and are representative of two independent experiments. Significance was determined by one-way ANOVA. ** P < 0.01, * P < 0.05. (E) Representative dot plots and the bar graph show the frequency of PD-1 + cells in Tregs after treating with E2, ICI, E2 and ICI, and ESA, respectively. Data are expressed as the means ± SD ( n = 4) and are representative of two independent experiments. Significance was determined by one-way ANOVA. ** P < 0.01. (F) The MFI of PD-1 expression on CD4 + CD25 + Foxp3 + T cells. Data are expressed as the means ± SD ( n = 4) and are representative of two independent experiments. Significance was determined by one-way ANOVA. * P < 0.05.

Article Snippet: In the case of single staining of Foxp3 and PD-1, the slides were incubated with anti-Foxp3 antibody (1:100, Servicebio, Wuhan, China) and anti-PD-1 antibody (1:100, proteintech, Wuhan, China), respectively, overnight at 4°C.

Techniques: Expressing, Control, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Estradiol Attenuates the Severity of Primary Toxoplasma gondii Infection-Induced Adverse Pregnancy Outcomes Through the Regulation of Tregs in a Dose-Dependent Manner

doi: 10.3389/fimmu.2018.01102

Figure Lengend Snippet: Estradiol (E2) in vivo administration in nonpregnant mice decreased the Toxoplasma gondii infection induced apoptosis rate of Tregs by enhancing the expression of Bcl-2. After a 2-week injection of E2 or PBS (control) into nonpregnant mice, the percentage of apoptotic Tregs in spleens (A) and inguinal lymph nodes (B) of mice was evaluated by flow cytometry. Error bars represent the means ± SD of six mice for each group from one experiment representative of two independent experiments. Significance was determined by the two-tailed Student’s t -test. * P < 0.05. (C–F) The splenocytes were isolated from nonpregnant mice with E2 in vivo administration. The frequency of caspase-3 + cells in CD4 + CD25 + T cells (C) and the mean fluorescence intensity (MFI) of caspase-3 expression in CD4 + CD25 + T cells (D) were measured by flow cytometric analysis. The gating strategy for the identification of the mouse caspase-3 + CD4 + CD25 + T cell population is shown in Figure S5A in Supplementary Material. Data are presented as the means ± SD of six mice for each group from one experiment representative of two independent experiments. Significance was determined by the two-tailed Student’s t -test. ** P < 0.01, * P < 0.05. The percentage of Bcl-2 + cells in CD4 + CD25 + Foxp3 + T cells (E) and the MFI of Bcl-2 expression in CD4 + CD25 + Foxp3 + T cells (F) were measured by flow cytometric analysis. The gating strategy for the identification of the mouse Bcl-2 + CD4 + CD25 + Foxp3 + T cell population is shown in Figure S5B in Supplementary Material. Each bar indicates the mean value ± SD of six mice for each group from one experiment representative of two independent experiments. Significance was determined by the two-tailed Student’s t -test. ** P < 0.01.

Article Snippet: In the case of single staining of Foxp3 and PD-1, the slides were incubated with anti-Foxp3 antibody (1:100, Servicebio, Wuhan, China) and anti-PD-1 antibody (1:100, proteintech, Wuhan, China), respectively, overnight at 4°C.

Techniques: In Vivo, Infection, Expressing, Injection, Control, Flow Cytometry, Two Tailed Test, Isolation, Fluorescence

Journal: Frontiers in Immunology

Article Title: Estradiol Attenuates the Severity of Primary Toxoplasma gondii Infection-Induced Adverse Pregnancy Outcomes Through the Regulation of Tregs in a Dose-Dependent Manner

doi: 10.3389/fimmu.2018.01102

Figure Lengend Snippet: Estradiol (E2) in vivo administration in nonpregnant mice increased the expression of programmed death-1 (PD-1). (A) Overlay of representative histograms showing PD-1 expression on CD4 + T cells from spleens and inguinal lymph nodes of mice with E2 in vivo administration. The related dot plots were shown in Figure S6A in Supplementary Material. The percentage of PD-1 + cells in CD4 + T cells (B) and the mean fluorescence intensity (MFI) of PD-1 expression on CD4 + T cells (C) from spleens and inguinal lymph nodes of mice with E2 in vivo administration. Data are represented as the means ± SD of six mice for each group from one experiment representative of two independent experiments. Significance was determined by one-way ANOVA. *** P < 0.001, ** P < 0.01, * P < 0.05. (D) Overlay of representative histograms showing PD-1 expression on CD4 + CD25 + Foxp3 + T cells from spleens and inguinal lymph nodes of mice with E2 in vivo administration. The related dot plots were shown in Figure S6B in Supplementary Material. The percentage of PD-1 + cells in CD4 + CD25 + Foxp3 + T cells (E) and the MFI of PD-1 expression on CD4 + CD25 + Foxp3 + T cells (F) from spleens and inguinal lymph nodes of mice as mentioned above. Data are represented as the means ± SD of six mice for each group from one experiment representative of two independent experiments. Significance was determined by one-way ANOVA. *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: In the case of single staining of Foxp3 and PD-1, the slides were incubated with anti-Foxp3 antibody (1:100, Servicebio, Wuhan, China) and anti-PD-1 antibody (1:100, proteintech, Wuhan, China), respectively, overnight at 4°C.

Techniques: In Vivo, Expressing, Fluorescence

Journal: bioRxiv

Article Title: LGR5 targeting molecules as therapeutic agents for multiple cancer types

doi: 10.1101/2022.09.01.506182

Figure Lengend Snippet: A. Sections from a CRC tumour resection showing LGR5 and β-catenin expression in - top panels , normal tissue; middle panels , dysplastic tissue; and lower panels , CRC. Blue, DAPI fluorescence showing nuclei. White number in the top right corner of the lower set of panels (showing LGR5 expression and nuclei staining) corresponds to relative levels of LGR5 protein using the scoring criteria applied to all TMAs (see text). Scale bar, 40 μM. B. Relative LGR5 protein expression quantified in healthy colon epithelia and CRC tumour stages I-IV scored. Each dot represents a single scored biopsy on the Bern TMA. Level of significance, p-value , determined by two-tailed t-tests comparing LGR5 expression for colon epithelia with each of the four tumour stages. C. Immunofluorescence using Fl-α-LGR5 and an antibody to β-catenin for - top panels , a healthy liver sample; and bottom right panels , a sample from the Cambridge HCC TMA. Numbers in white correspond to scored LGR5 expression levels using the criteria for evaluating the TMA. Arrowhead, cell clusters within the tumour with high levels of cortical β-catenin and low levels of LGR5 levels. White numbers represent scored values for relative LGR5 protein expression. Scale bar, 40 μM. D. Quantitation of LGR5 protein expression levels in 8 healthy liver resections (Liver) and biopies from the Cambridge HCC TMA (HCC). Level of significant difference, p-value , between the sample sets was determined by two-tailed t-test. E. Quantitation of LGR5 expression levels in biopsies of healthy Fallopian tube, ovarian cancer cases (OvC) and omentum cancer cases (OmC) comprising the Cambridge ovarian cancer TMA. Levels of significance in LGR5 expression between Fallopian tube and OvC samples, p-value , was calculated using two-tailed t-test. There were no significant increases ( ns ) in LGR5 expression between Fallopian tube and OmC sample sets. F. Quantitation of LGR5 expression levels in samples from the Cambridge Brain cancer TMA - healthy brain tissue (Brain), low grade glioma (LGG) and glioblastoma (GBB). There were no significant increases ( ns ) in LGR5 expression between Brain, LGG or GBB samples sets, determined by two-tailed t-test. G. Representative example of relative LGR5 protein expression levels in CD4 + T cells, CD8 + T cells and CD19 + B cells from healthy donor PBMCs determined by flow cytometry. A small number of B cells, <3% of the total population, express low levels of LGR5. H. LGR5 transcript levels normalised to TBP , measured by quantitative RT-PCR, in healthy donor B cells (B-cells; 10 samples), B-ALL cell lines (Cell lines; 9 lines), CD19-enriched cell populations from primary B-ALL cases (ALL primary; 15 samples) and CD19-enriched populations from B-ALL tumour cells maintained as PDX models (ALL-PDX 22 samples). Level of significance, p-value , was determined by two-tailed t-tests comparing LGR5 expression in healthy donor B cells and the other sample sets at the *, p<0 . 001 level of significance. LGR5 transcript levels for the individual samples are shown in Suppl. Fig. 2L .

Article Snippet: Quantitative real-time PCR (qRT-PCR) has been previously described ( ) and used Taqman probes specific for human LGR5 (Life Technology, Hs00969422_m1) and for TBP (Life Technology, Hs00427620_m1) as control housekeeping gene.

Techniques: Expressing, Fluorescence, Staining, Two Tailed Test, Immunofluorescence, Quantitation Assay, Flow Cytometry, Quantitative RT-PCR

Journal: bioRxiv

Article Title: LGR5 targeting molecules as therapeutic agents for multiple cancer types

doi: 10.1101/2022.09.01.506182

Figure Lengend Snippet: A. Western blot analysis of LGR5 protein levels in lysates from pre-B ALL cell lines. B. Indirect immunofluorescence of NALM6 cells using α-LGR5. Scale bar, 5 μM. C. Flow cytometric detection of Fl-α-LGR5 (red histograms) or fluorescent isotype control (grey histograms) association with NALM6 cells after 1 hour incubation at 4°C (top panel) or 37°C (bottom) panel. Numbers represent percentage of cells with detectable fluorescence. D. Flow cytometric detection of Fl-α-LGR5 (red histograms) association with 697, REH or NALM6 cells after 1 hour incubation at 37°C. Grey histograms – analysis with Fl-α-LGR5 pre-incubated with Frag1A. E. Representative western blot analysis of LGR5 protein in CRC cell lines using α-LGR5 and an antibody to vinculin as loading control. F. Relative LGR5 transcript levels normalised to TBP in CRC cell lines measured by qRT-PCR. Error bars indicate standard deviation (SD) for 3 biological replicates except for LoVo cells, 4 biological replicates. G. Indirect immunofluorescence of LGR5 in LoVo cells using α-LGR5. Scale bar, 5 μM. H. Flow cytometric analysis of LoVo and SW480 cells that have been incubated with Fl-α-LGR5 (red plots) or with Fl-α-LGR5 pre-incubated with Frag1A (grey plots) at 37°C for 1 hour.

Article Snippet: Quantitative real-time PCR (qRT-PCR) has been previously described ( ) and used Taqman probes specific for human LGR5 (Life Technology, Hs00969422_m1) and for TBP (Life Technology, Hs00427620_m1) as control housekeeping gene.

Techniques: Western Blot, Immunofluorescence, Control, Incubation, Fluorescence, Quantitative RT-PCR, Standard Deviation

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: (A–F) Upon stimulation with TauO or vehicle for 11 days, astrocytes were examined for HMGB1 translocation and release by immunostaining and flow cytometry analysis, respectively. Arrows point to TauO-induced translocation of HMGB1, a signature of cellular senescence. Scale bars, 50 μm. TauO exposure significantly increased p16 INK4A -positive astrocytes (C), the percentage of SA-β-gal-positive astrocytes (D), and cell-cycle arrest (E) p16 INK4A staining (F) after 4 days of treatment with or without conditional media (CM) from senescent astrocytes in the presence or absence of α-HMGB1 antibody (4 μg/mL). Representative images showing the relative decrease in number of p16 INK4A -positive astrocytes after α-HMGB1 antibody treatment. Scale bar, 50 μm. Data are representative of at least three independent experiments (mean ± SEM). Statistical analyses were measured by unpaired, two-tailed Student’s t test.

Article Snippet: After washing in PBS, the sections were blocked for 1h with 5% goat serum and 5% bovine serum albumin (BSA) in PBS and incubated with rabbit-anti-human p16 INK4A (D3W8G antibody, for human tissues) (1:100; CST #92803) or rabbit anti-mouse p16 INK4A (M-156 antibody, for murine tissues) (1:100; #SC-1207) or anti-HMGB1 antibody (1:100; #ab18256) or anti-γH2AX antibody (1:100; CST #9718) (1:100), mouse-anti-TauO-specific monoclonal antibody clone 2 (TOMA) (1:100) ( ) and chicken-anti-GFAP antibody (1:500; #ab4674) (1:500), AT8 antibody (1:100; Thermo #MN1020), anti-IL6 antibody (1:200; CST #12912) or recombinant Alexa fluor488 anti-NeuN antibody (1:250; # ab190195) in antibody diluent at 4°C overnight.

Techniques: Translocation Assay, Immunostaining, Flow Cytometry, Staining, Two Tailed Test

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: Primary astrocytes were cultured in poly-L-lysine (PLL)-coated plates for 48 h and then pretreated with or without HMGB1 release inhibitors EP (10 mM) and/or GA (250 μM) for 30 min followed by treatment with TauO (0.5 μM) for 11 days. (A–C) HMGB1 release inhibition prevents TauO-induced astrocyte senescence, as shown by the decreased percentage of p16 INK4A -positive cells and p16 INK4A mean fluorescence intensity (MFI) and increased MFI of intracellular HMGB1 by flow cytometry. Representative graphs are shown from a minimum of three to four independent experiments (mean ± SEM). Statistical significance was determined by using one-way ANOVA followed by Tukey’s post hoc test (**p < 0.05; ***p < 0.0001). (D and E) Effect of EP+GA on relative protein levels of p16 INK4A , HMGB1, and RAGE was measured by immunoblotting followed by densitometry quantification; β-actin was used as a loading control. The densitometry bar graph is numbered as in blots. Data are shown as mean ± SEM. Statistical significance was determined by using one-way ANOVA followed by Tukey’s post hoc test (**p < 0.05; ***p < 0.0001). (F) Effect of EP+GA on TauO-induced astrocyte senescence was measured by SA-β-gal staining. Pretreatment with EP (10 mM) + GA (250 μM) attenuated TauO-induced astrocytes senescence-like phenotype as shown by decreased SA-β-gal activity. Scale bar, 50 μm. Data are shown as mean ± SEM from four independent experiments in duplicates. Statistical significance was determined using unpaired, two-tailed Student’s t test. (G–I) Conditioned media from the astrocytes culture were used to measure secreted levels of HMGB1, (H) IL-6, and (I) TNF-α using ELISA showing HMGB1 inhibitors effectively inhibit TauO-induced SASP activity. Data are mean ± SEM from at least three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test. (J) Phosphorylated protein levels of p38 and NF-κB assayed by immunoblotting, followed by densitometry quantification; GAPDH was used as a loading control. Data are shown as mean ± SEM (*p < 0.05; **p < 0.001; ***p < 0.0001).

Article Snippet: After washing in PBS, the sections were blocked for 1h with 5% goat serum and 5% bovine serum albumin (BSA) in PBS and incubated with rabbit-anti-human p16 INK4A (D3W8G antibody, for human tissues) (1:100; CST #92803) or rabbit anti-mouse p16 INK4A (M-156 antibody, for murine tissues) (1:100; #SC-1207) or anti-HMGB1 antibody (1:100; #ab18256) or anti-γH2AX antibody (1:100; CST #9718) (1:100), mouse-anti-TauO-specific monoclonal antibody clone 2 (TOMA) (1:100) ( ) and chicken-anti-GFAP antibody (1:500; #ab4674) (1:500), AT8 antibody (1:100; Thermo #MN1020), anti-IL6 antibody (1:200; CST #12912) or recombinant Alexa fluor488 anti-NeuN antibody (1:250; # ab190195) in antibody diluent at 4°C overnight.

Techniques: Cell Culture, Inhibition, Fluorescence, Flow Cytometry, Western Blot, Control, Staining, Activity Assay, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: (A) Experimental design for 8-week treatment with HMGB1 release inhibitors EP (80 mg/kg) + GA (20 mg/kg) or vehicle (saline) three times per week, beginning at 12 months of age in hTau mice. (B) Images of thioflavin-S staining and quantification of NFTs (tangles per region of interest) in the hippocampus. Scale bars, 200 μm. Data are shown as the mean ± SEM. (C) Representative immunostaining images showing hyperphosphorylated tau (AT8 immunoreactivities) in the hippocampus of hTau mice treated with either saline or EP+GA (scale bars, 50 μm) and quantification of AT8-positive cells per 500 μm 2 . Data are the mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences. (D) Novel-object recognition task was used to measure the impact of EP+GA treatment on memory in hTau mice. Mice treated with EP+GA show significantly higher discrimination index than vehicle-treated mice; heatmap of representative mice from the vehicle and EP+GA treatment group showing the time spent on exploring old or novel objects. Statistical significance was determined using unpaired, two-tailed Student’s t test. (E) Y-maze spontaneous alternation test: percentage of spontaneous alterations were measured before and after 8 weeks of EP+GA or vehicle treatment. Data are the mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences. (F) Immunostaining showing NeuN-positive neuronal cells in the CA3 pyramidal layer of the hippocampus of hTau mice treated with either vehicle or EP+GA. Scale bar, 100 μm. Data are the mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences.

Article Snippet: After washing in PBS, the sections were blocked for 1h with 5% goat serum and 5% bovine serum albumin (BSA) in PBS and incubated with rabbit-anti-human p16 INK4A (D3W8G antibody, for human tissues) (1:100; CST #92803) or rabbit anti-mouse p16 INK4A (M-156 antibody, for murine tissues) (1:100; #SC-1207) or anti-HMGB1 antibody (1:100; #ab18256) or anti-γH2AX antibody (1:100; CST #9718) (1:100), mouse-anti-TauO-specific monoclonal antibody clone 2 (TOMA) (1:100) ( ) and chicken-anti-GFAP antibody (1:500; #ab4674) (1:500), AT8 antibody (1:100; Thermo #MN1020), anti-IL6 antibody (1:200; CST #12912) or recombinant Alexa fluor488 anti-NeuN antibody (1:250; # ab190195) in antibody diluent at 4°C overnight.

Techniques: Saline, Staining, Immunostaining, Two Tailed Test

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: (A) Representative immunostaining images and quantification of TauO-associated senescent astrocytes in the hippocampus. Representative immunostaining showing GFAP (green), p16 INK4A (red), and TauO (magenta) immunoreactivities and DAPI (blue nuclei) in the hippocampus of hTau mice treated with either vehicle or EP+GA. Images display colocalization of p16 INK4A and TauO in GFAP-positive astrocytes. (B–E) The graph showing quantitative analysis of p16 INK4A area load (B), TauO area load (C), p16 INK4A MFI (D), and percentage of p16 INK4A -positive and GFAP-positive cells (E) in the hippocampus of hTau mice treated with vehicle (n = 5 mice) and EP+GA (n = 6 mice). Data are shown as mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences. Scale bars, 200 μm. (F–M) Immunostaining quantification of cytoplasmic HMGB1-positive cells (F and J), number of γH2AX foci (G and K), p16 INK4A -positive cells (H and L), and IL-6-positive cells (I and M) in the cortex of hTau mice treated with vehicle (n = 5 mice) and EP+GA (n = 6 mice). Data are shown as mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences. Scale bar, 20 μm.

Article Snippet: After washing in PBS, the sections were blocked for 1h with 5% goat serum and 5% bovine serum albumin (BSA) in PBS and incubated with rabbit-anti-human p16 INK4A (D3W8G antibody, for human tissues) (1:100; CST #92803) or rabbit anti-mouse p16 INK4A (M-156 antibody, for murine tissues) (1:100; #SC-1207) or anti-HMGB1 antibody (1:100; #ab18256) or anti-γH2AX antibody (1:100; CST #9718) (1:100), mouse-anti-TauO-specific monoclonal antibody clone 2 (TOMA) (1:100) ( ) and chicken-anti-GFAP antibody (1:500; #ab4674) (1:500), AT8 antibody (1:100; Thermo #MN1020), anti-IL6 antibody (1:200; CST #12912) or recombinant Alexa fluor488 anti-NeuN antibody (1:250; # ab190195) in antibody diluent at 4°C overnight.

Techniques: Immunostaining, Two Tailed Test

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After washing in PBS, the sections were blocked for 1h with 5% goat serum and 5% bovine serum albumin (BSA) in PBS and incubated with rabbit-anti-human p16 INK4A (D3W8G antibody, for human tissues) (1:100; CST #92803) or rabbit anti-mouse p16 INK4A (M-156 antibody, for murine tissues) (1:100; #SC-1207) or anti-HMGB1 antibody (1:100; #ab18256) or anti-γH2AX antibody (1:100; CST #9718) (1:100), mouse-anti-TauO-specific monoclonal antibody clone 2 (TOMA) (1:100) ( ) and chicken-anti-GFAP antibody (1:500; #ab4674) (1:500), AT8 antibody (1:100; Thermo #MN1020), anti-IL6 antibody (1:200; CST #12912) or recombinant Alexa fluor488 anti-NeuN antibody (1:250; # ab190195) in antibody diluent at 4°C overnight.

Techniques: Recombinant, Protease Inhibitor, Labeling, Bicinchoninic Acid Protein Assay, Cell Cycle Assay, Staining, Enzyme-linked Immunosorbent Assay, Chromatography, Western Blot, Software

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: (A–F) Upon stimulation with TauO or vehicle for 11 days, astrocytes were examined for HMGB1 translocation and release by immunostaining and flow cytometry analysis, respectively. Arrows point to TauO-induced translocation of HMGB1, a signature of cellular senescence. Scale bars, 50 μm. TauO exposure significantly increased p16 INK4A -positive astrocytes (C), the percentage of SA-β-gal-positive astrocytes (D), and cell-cycle arrest (E) p16 INK4A staining (F) after 4 days of treatment with or without conditional media (CM) from senescent astrocytes in the presence or absence of α-HMGB1 antibody (4 μg/mL). Representative images showing the relative decrease in number of p16 INK4A -positive astrocytes after α-HMGB1 antibody treatment. Scale bar, 50 μm. Data are representative of at least three independent experiments (mean ± SEM). Statistical analyses were measured by unpaired, two-tailed Student’s t test.

Article Snippet: HMGB1 ELISA , Novus Biologicals , Cat#NBP2-62766.

Techniques: Translocation Assay, Immunostaining, Flow Cytometry, Staining, Two Tailed Test

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: Primary astrocytes were cultured in poly-L-lysine (PLL)-coated plates for 48 h and then pretreated with or without HMGB1 release inhibitors EP (10 mM) and/or GA (250 μM) for 30 min followed by treatment with TauO (0.5 μM) for 11 days. (A–C) HMGB1 release inhibition prevents TauO-induced astrocyte senescence, as shown by the decreased percentage of p16 INK4A -positive cells and p16 INK4A mean fluorescence intensity (MFI) and increased MFI of intracellular HMGB1 by flow cytometry. Representative graphs are shown from a minimum of three to four independent experiments (mean ± SEM). Statistical significance was determined by using one-way ANOVA followed by Tukey’s post hoc test (**p < 0.05; ***p < 0.0001). (D and E) Effect of EP+GA on relative protein levels of p16 INK4A , HMGB1, and RAGE was measured by immunoblotting followed by densitometry quantification; β-actin was used as a loading control. The densitometry bar graph is numbered as in blots. Data are shown as mean ± SEM. Statistical significance was determined by using one-way ANOVA followed by Tukey’s post hoc test (**p < 0.05; ***p < 0.0001). (F) Effect of EP+GA on TauO-induced astrocyte senescence was measured by SA-β-gal staining. Pretreatment with EP (10 mM) + GA (250 μM) attenuated TauO-induced astrocytes senescence-like phenotype as shown by decreased SA-β-gal activity. Scale bar, 50 μm. Data are shown as mean ± SEM from four independent experiments in duplicates. Statistical significance was determined using unpaired, two-tailed Student’s t test. (G–I) Conditioned media from the astrocytes culture were used to measure secreted levels of HMGB1, (H) IL-6, and (I) TNF-α using ELISA showing HMGB1 inhibitors effectively inhibit TauO-induced SASP activity. Data are mean ± SEM from at least three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test. (J) Phosphorylated protein levels of p38 and NF-κB assayed by immunoblotting, followed by densitometry quantification; GAPDH was used as a loading control. Data are shown as mean ± SEM (*p < 0.05; **p < 0.001; ***p < 0.0001).

Article Snippet: HMGB1 ELISA , Novus Biologicals , Cat#NBP2-62766.

Techniques: Cell Culture, Inhibition, Fluorescence, Flow Cytometry, Western Blot, Control, Staining, Activity Assay, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: (A) Experimental design for 8-week treatment with HMGB1 release inhibitors EP (80 mg/kg) + GA (20 mg/kg) or vehicle (saline) three times per week, beginning at 12 months of age in hTau mice. (B) Images of thioflavin-S staining and quantification of NFTs (tangles per region of interest) in the hippocampus. Scale bars, 200 μm. Data are shown as the mean ± SEM. (C) Representative immunostaining images showing hyperphosphorylated tau (AT8 immunoreactivities) in the hippocampus of hTau mice treated with either saline or EP+GA (scale bars, 50 μm) and quantification of AT8-positive cells per 500 μm 2 . Data are the mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences. (D) Novel-object recognition task was used to measure the impact of EP+GA treatment on memory in hTau mice. Mice treated with EP+GA show significantly higher discrimination index than vehicle-treated mice; heatmap of representative mice from the vehicle and EP+GA treatment group showing the time spent on exploring old or novel objects. Statistical significance was determined using unpaired, two-tailed Student’s t test. (E) Y-maze spontaneous alternation test: percentage of spontaneous alterations were measured before and after 8 weeks of EP+GA or vehicle treatment. Data are the mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences. (F) Immunostaining showing NeuN-positive neuronal cells in the CA3 pyramidal layer of the hippocampus of hTau mice treated with either vehicle or EP+GA. Scale bar, 100 μm. Data are the mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences.

Article Snippet: HMGB1 ELISA , Novus Biologicals , Cat#NBP2-62766.

Techniques: Saline, Staining, Immunostaining, Two Tailed Test

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: (A) Representative immunostaining images and quantification of TauO-associated senescent astrocytes in the hippocampus. Representative immunostaining showing GFAP (green), p16 INK4A (red), and TauO (magenta) immunoreactivities and DAPI (blue nuclei) in the hippocampus of hTau mice treated with either vehicle or EP+GA. Images display colocalization of p16 INK4A and TauO in GFAP-positive astrocytes. (B–E) The graph showing quantitative analysis of p16 INK4A area load (B), TauO area load (C), p16 INK4A MFI (D), and percentage of p16 INK4A -positive and GFAP-positive cells (E) in the hippocampus of hTau mice treated with vehicle (n = 5 mice) and EP+GA (n = 6 mice). Data are shown as mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences. Scale bars, 200 μm. (F–M) Immunostaining quantification of cytoplasmic HMGB1-positive cells (F and J), number of γH2AX foci (G and K), p16 INK4A -positive cells (H and L), and IL-6-positive cells (I and M) in the cortex of hTau mice treated with vehicle (n = 5 mice) and EP+GA (n = 6 mice). Data are shown as mean ± SEM; unpaired, two-tailed Student’s t test was used to determine the statistical differences. Scale bar, 20 μm.

Article Snippet: HMGB1 ELISA , Novus Biologicals , Cat#NBP2-62766.

Techniques: Immunostaining, Two Tailed Test

Journal: Cell reports

Article Title: Tau oligomer induced HMGB1 release contributes to cellular senescence and neuropathology linked to Alzheimer’s disease and frontotemporal dementia

doi: 10.1016/j.celrep.2021.109419

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: HMGB1 ELISA , Novus Biologicals , Cat#NBP2-62766.

Techniques: Recombinant, Protease Inhibitor, Labeling, Bicinchoninic Acid Protein Assay, Cell Cycle Assay, Staining, Enzyme-linked Immunosorbent Assay, Chromatography, Western Blot, Software

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: The human neuroepithelium exhibits differential temporal dynamics of morphogenesis genes (A) Clustering genes by temporal expression dynamics shows species differences in GO:BP term enrichment. Columns from left to right: far left, TCseq clusters with genes in each cluster plotted with their temporal expression (z-scaled) and color-coded by membership value (degree to which data points of a gene belong to the cluster, pink represents high membership values). The 10 clusters are ordered (top to bottom) based on similarity in expression pattern. Middle left: representative GO:BP term from shared (purple), human-exclusive (black), or gorilla-exclusive (fuchsia) terms for each cluster. Middle right: histograms show the number of enriched (p < 0.05) GO:BP terms found in both species (purple), exclusively in human (black) or gorilla (fuchsia) per cluster. Axis range: 0–8 (cluster 2,5,7,8); 0–15 (cluster 9,10); 0–20 (cluster 1); 0–25 (cluster 3); 0–50 (cluster 4); 0–80 (cluster 6). Far right: weighted arc network graph visualizing interspecies differences in the enrichment/membership of specific GO:BP terms per cluster. The bases of the arc are aligned to both a human (black) and a gorilla (fuchsia) bar from the histogram in the adjacent panel, highlighting the species-specific shifts in expression patterns associated with specific GO:BP terms. Weight/thickness of the arc is dictated by the number of GO:BP terms enriched in a species-exclusive manner “moving” between clusters in the defined pattern. (B) Mean temporal expression pattern (z-scaled) of genes in clusters enriched for “cell morphogenesis”-related GO:BP terms (human clusters 1, 9, 10, and gorilla cluster 3). (C) Temporal expression pattern ( Z -scaled) of SHROOM3 , a gene involved in cell morphogenesis and apical constriction. (D) Immunofluorescent staining of day 5 organoids for SHROOM3 showing strong apical expression in gorilla (G1) neuroepithelium, but not human (H9) at this time point. Scale bar, 40 μm. (E) Immunofluorescent staining of day 5 organoids for OCLN showing expression spread along the apicobasal length of human (IMR-90) progenitor cells (white arrowheads) but more limited apically (yellow arrowheads) in gorilla (G1) progenitor cells. DAPI is shown in blue. Scale bar, 100 μm. (F) Venn diagram summarizing search for cell morphogenesis-related transcription factors with species-specific expression patterns. (G) Mean temporal expression pattern ( Z -scaled) of ZEB2 , showing peak expression earlier in gorilla (G1) than human (H9) organoids. Shaded error bars are SD. See also Figure S5 and .

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Expressing, Staining

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: Decreased ZEB2 leads to expanded NE with delayed transition (A) Mean temporal expression pattern (log normalized transcripts per million) of ZEB2 across the entire time series, showing peak expression earlier in gorilla (G1) than human (H9) organoids. Shaded error bars are SD. (B) Western blot expression time course from PSCs to day 25 human (H9) and gorilla (G1) organoids reveals a premature onset and higher levels of ZEB2 protein expression in gorilla compared to human. This is accompanied by a premature expression of the radial glial marker vimentin, and premature downregulation of the epithelial markers CDH1 and EpCAM in gorilla relative to human. Bottom panel shows quantification of ZEB2 relative to GAPDH (AU, arbitrary units). (C) Immunofluorescent stain for ZEB2 and DAPI in human (H9) and gorilla (G1) organoids at days 3, 5, and 10 showing neuroepithelial buds with nuclear expression (yellow arrows). Note the interspecies difference at day 3 where gorilla organoids already display nuclear expression compared to a weaker stain in most human cells. Insets show higher magnification of the boxed regions. Scale bar, 40 μm. (D) Immunofluorescence image of a day 25 human (H9) organoid showing a mutually exclusively pattern of expression between ZEB2 and the committed radial glia marker, BLBP. Scale bar, 100 μm. (E) Western blot of H9 wild-type (WT) and ZEB2 +/− organoids at day 16 for ZEB2, the tight-junction protein OCLN, the junction components CDH1 and CDH2, the dorsal telencephalic marker EMX1, and the loading control β-actin. The blots show a sizeable increase in CDH1 and OCLN and a decrease in CDH2, whereas EMX1 levels, and thus dorsal telencephalic identity, appears to be largely unaffected. (F) Representative bright field images of day 12 WT and ZEB2 +/− . Insets show higher magnification of the boxed regions, dashed yellow are representative neuroepithelial bud perimeters quantified in (G), dashed turquois are representative neuroepithelial bud thicknesses quantified in (H). Scale bar, 500 μm. (G) Quantification of neuroepithelial bud perimeters of WT (n = 106) and ZEB2 +/− (n = 116) organoid buds from 27 WT and 28 ZEB2 +/− organoids at day 17, Mann-Whitney U test, two-tailed ( ∗∗∗ p = 0.0001) from 3 organoid batches. (H) Quantification of neuroepithelial bud thickness of WT (n = 80) and ZEB2 +/− (n = 119) organoid buds from 26 WT and 31 ZEB2 +/− organoids at day 12, Mann-Whitney U test, two-tailed ( ∗∗∗∗ p < 0.0001) from 3 organoid batches. (I) Quantification of neuroepithelial bud perimeters of two ZEB2 +/− ; iZEB2 colonies treated with and without doxycycline. Colony 1: − Dox (n = 108 buds from 25 organoids), + Dox (n = 70 buds from 18 organoids). Colony 2: − Dox (n = 62 buds from 17 organoids), + Dox (n = 84 buds from 19 organoids) across 3 organoid batches. Mann-Whitney U tests, two-tailed ( ∗∗∗ p = 0.0003 ∗∗∗∗ p < 0.0001). (J) Immunofluorescence images of day 15 WT and ZEB2 +/− organoids showing increased OCLN immunostaining (yellow arrowheads) along the apico-(ZO1) basal (dashed line) axis of progenitor cells and reduced numbers of TBR2 + cells in ZEB2 +/− organoids compared to WT. Scale bar, 100 μm. See also Figure S6 .

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Expressing, Western Blot, Marker, Staining, Immunofluorescence, MANN-WHITNEY, Two Tailed Test, Immunostaining

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: ZEB2 expression and targeting for loss of function, related to Figure 6 A. Representative immunofluorescence image showing ZEB2 expression in SOX2+ progenitor cells in day 10 human (H9) organoid. Scale bar: 50 μm. B. Representative immunofluorescence image showing a salt-and-pepper pattern of ZEB2 expression in the ventricular zone at day 25, after the onset of neurogenesis in human (H9) organoid. DCX (Doublecortin) stains newly born neurons. Scale bar: 100 μm. C. Representative immunofluorescence image of a mature day 60 human (H9) organoid revealing ZEB2 expression in CTIP2+ neurons and absence of ZEB2 staining in the ventricular zone. Scale bar: 100 μm. D. Representative immunofluorescence image of a day 25 human (H9) organoid showing a mutually exclusive pattern of expression between ZEB2 and the radial glia marker protein GLAST. Scale bar: 100 μm. E. Schematic representation of the CRISPR-Cas9n editing strategy, where the first coding exon of the ZEB2 gene (exon 2, NCBI ref sequence NM_014795.4:182-323) was targeted by two nickases (dashed lines) and screening was performed by assaying the drop-off frequency of a HEX-labeled probe, binding to one of the nick sites, relative to a FAM-labeled reference probe binding away from the disrupted region. The exon is marked in orange while introns are marked in purple. F. Example ddPCR 2D scatter-plots of a negative control sample (HEK293 cells), showing only a FAM-HEX double positive (red) and an empty droplet cluster (black) and a positive control sample (HEK293 cells expressing WT Cas9 and ZEB2 guides), showing a FAM-only cluster (blue) in the upper-left quadrant of the 2D plot corresponding to edited alleles. ddPCR 2D scatter-plot of the H9 ZEB2 +/− hESC edited line showing a 1:1 ratio between the WT and edited allele. (G). Representative chromatograms of the ZEB2 alleles in the H9 ZEB2 +/− hESC cells. The CRISPR-Cas9 target region was PCR amplified with a high-fidelity polymerase, the PCR product was blunt-end cloned into the pJET1.2 vector and following purification, plasmids from different colonies carrying the insert were sequenced. Sequencing reveals that the edited allele harbors a 23 bp deletion. H. DNA-PAGE analysis of a short PCR amplicon spanning the CRISPR-Cas9 ZEB2 target site in WT H9 and H9 ZEB2 +/− hESCs. The gel reveals the presence of two bands, corresponding to the WT and the edited allele in H9 ZEB2 +/− hESCs. I. Representative images of karyotype analysis on 20 G-banded metaphase spreads from the H9 ZEB2 +/− hESCs used to generate the stock. The cell line displays normal karyotype. J. RT-PCR analysis for expression of ZEB2, the key pluripotency markers SOX2, NANOG, OCT4 and DPPA5 and the loading control GAPDH . PCR shows that upon a ~50% reduction in ZEB2 mRNA levels the mutant stem cells retain expression of pluripotency markers at comparable levels to WT H9 hESCs. WT and ZEB2 +/− were run on the same gel but not adjacent to each other, the dashed line indicates where the gel was spliced. K. Full length western blot for ZEB2 in WT and ZEB2 +/− organoids at day 15 – loading control was GAPDH L. Box and whiskers plot reporting the quantifications of the number of TBR2+ cells per unit area (TBR2+ cells/mm 2 ) in day 16 WT and ZEB2 +/− organoids. Quantifications were performed by manual counting on n = 52 WT and n = 68 ZEB2 +/− ventricles corresponding to 12 organoids from 2 distinct batches. A two-tailed Mann-Whitney U test was used for statistical comparison ( ∗∗∗∗ p < 0.0001). M. Representative immunofluorescence images of day 55 WT and ZEB2 +/− cerebral organoid buds used for quantifications shown in N. Scale bar: 200 μm. N. Box and whiskers plot reporting the quantifications of the number of TBR2+ cells per unit area (TBR2+ cells/mm 2 ) in day 55 WT and ZEB2 +/− organoids. Quantifications were performed using an automated cell segmentation pipeline on n = 17 WT and n = 17 ZEB2 +/− organoid regions from 3 distinct batches. A two-tailed Mann-Whitney U test was used for statistical comparison (ns, p = 0.1139). O. Plasmid maps of the CRISPR homology-directed repair (HDR) templates used to target the AAVS1 safe-harbor locus in H9 hESC cells – top is the CAG-lox-STOP-lox-ZEB2-GFP-Flag inducible expression construct and bottom is the construct encoding CRE recombinase under the control of a tetracycline responsive promoter and the reverse tetracycline transactivator (rtTA) driven by the CAG promoter. P. UCSC Genome Browser view of the AAVS1 locus and CRISPR-Cas9 targeting strategy of intron 1 of PPP1R12C . The promoter-less splice-acceptor (SA), T2A peptide-linked “gene trap” is such that expression of the promoter-less selection cassette is driven by the endogenous PPP1R12C gene, thus effectively eliminating false-positive background arising from random integration. The panel reports the PCR genotyping strategy – upon successful targeting of the AAVS1 locus, while amplicon 1 is lost due to the size increase following insert integration, amplicons 2 and 3 are gained - see A. Q. PCR gel showing successful genotyping of the two rescue clones used for the experiments shown. R. Representative brightfield images of day 15 ZEB2 +/− ; iZEB2 cerebral organoids treated with and without doxycycline. Scale bar: 100 μm. S. Representative immunofluorescence images of ZEB2 +/− ; iZEB2 treated with and without doxycycline stained for GFP, TBR2 and DAPI. Scale bar: 100 μm T. Box and whiskers plot reporting the quantifications done using an automated cell segmentation pipeline of the number of TBR2+ cells per unit area (TBR2+ cells/mm 2 ) in day 15 ZEB2 +/− ; iZEB2 organoids - colony 1: -Dox (n = 17 organoid regions), +Dox (n = 16 organoid regions); colony 2: -Dox (n = 13 organoid regions), +Dox (n = 13 organoid regions) from three independent batches. Mann-Whitney U tests, two-tailed ( ∗∗ p < 0.01).

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Expressing, Immunofluorescence, Staining, Marker, CRISPR, Sequencing, Labeling, Binding Assay, Negative Control, Positive Control, Amplification, Clone Assay, Plasmid Preparation, Purification, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Western Blot, Two Tailed Test, MANN-WHITNEY, Construct, Selection

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: Modulation of ZEB2 and SMAD signaling in human and gorilla cells, related to Figure 7 A. PCR gel showing successful genotyping of the Hum iZEB2 colony used for all experiments shown based on the PCR genotyping strategy outlined in Figure S6 P. The asterisks mark unspecific bands. B. Transgene induction in Hum iZEB2 cells treated with and without doxycycline for 6 days and assayed by western blot for ZEB2, GFP and β-actin. C. Immunofluorescence images of 6-day induced and uninduced Hum iZEB2 cells stained for ZEB2 and DAPI, showing that doxycyline induction results in ZEB2 expression and nuclear translocation, without adverse effects on its localization due to tagging with GFP. Scale bar: 20 μm D. Immunofluorescence images of 6-day induced and uninduced Hum iZEB2 cells stained for DAPI, CDH1 and CDH2. The data reveal a reduction in CDH1 expression and an increase in CDH2 expression following induction. Scale bar: 50 μm. E. Immunofluorescence images of 6-day induced and uninduced Hum iZEB2 cells stained for GFP, Vimentin and EpCAM. The data reveal a reduction in EpCAM expression and an increase in Vimentin expression following expression of ZEB2-GFP. Scale bar: 50 μm. F. Brightfield images of induced (+ Dox) and uninduced (- Dox) Hum iZEB2 organoids and gorilla (G1) organoids at days 3, 5 and 10, showing indistinguishable tissue architecture between organoids at day 3, while day 5 and 10 organoids show neuroepithelial buds that are smaller and more rounded in gorilla and ZEB2 induced (+ Dox) versus uninduced (- Dox) organoids. Scale bar: 200 μm. G. Western blot of day 5 WT and ZEB2 +/− organoids revealing both decreased ZEB2 and SHROOM3 levels in ZEB2 +/− organoids compared to WT control β-Actin was used as loading control. H. Representative bright-field images of uninduced and induced Hum iZEB2 and gorilla neuroepithelial buds at day 5, used for quantification in Figure 7 H. Scale bar: 100 μm. I. Immunofluorescent staining for ZO1, SOX2, DCX and DAPI showing normal tissue morphology and onset of neurogenesis (DCX+ neurons) in uninduced (- Dox) and induced (+ Dox) Hum iZEB2 organoids at day 10. Scale bar: 100 μm. J. Western blot for ZEB2, CDH1, and CDH2, with β-Actin as loading control, of WT and ZEB2 +/− organoids treated with dual-SMAD inhibitors, or treated with vehicle, for 10 days and assayed at day 12. Note the rescued levels of junctional components CDH1 and CDH2. K. Morphological assessment of WT and ZEB2 +/− organoids treated with dual-SMAD inhibitors, or treated with vehicle, for 10 days and assayed at day 12 by brightfield imaging (left panels) and hematoxylin-eosin staining (right panels). Note the elongated neuroepithelial buds (arrows) in mutant organoids that appear rescued (arrowheads) upon SMAD inhibition. Scale bars: 1 mm (left panels), 50 μm (right panels).

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Translocation Assay, Imaging, Mutagenesis, Inhibition

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet: ZEB2-driven junctional remodeling and apical constriction dictate species-specific timing of NE transition (A) Immunofluorescent staining of uninduced (− Dox) and induced (+ Dox) Hum iZEB2 organoids for GFP and SHROOM3. Note the expression of ZEB2-GFP and apical accumulation of SHROOM3 in induced organoids. Scale bar, 50 μm. (B) Representative bright field images of day 5 Hum iZEB2 and gorilla organoids. Induced (+ Dox) Hum iZEB2 organoids show smaller neuroepithelial buds (arrowheads) that are more round in shape, similar to gorilla (G1), while uninduced (− Dox) show more elongated structures typical of human. Scale bar, 200 μm. (C) Immunofluorescence images through day 5 whole mount Hum iZEB2 uninduced (− Dox), induced (+ Dox) and gorilla (G1) organoids stained for GFP, ZO1, and SOX2. Sparse labeling with viral GFP shows ZEB2 induction triggers the constriction of apical processes (arrows) in progenitor cells, similar to gorilla at day 5. Scale bar, 50 μm. (D) Representative immunofluorescence images through whole mount day 5 uninduced (− Dox), induced (+ Dox) Hum iZEB2 and gorilla (G1) organoids with superimposed individual segmented GFP+ progenitor cells (white) showing their 3D morphology. Note the thinning of apical processes observed upon ZEB2 induction. Scale bar, 10 μm. (E) Immunofluorescent staining for ZO1 on the surface of apical lumens showing the apical surface areas of individual progenitor cells in day 5 Hum iZEB2 uninduced (− Dox), induced (+ Dox) and gorilla (G1) organoids. Perimeters of some individual progenitor cells of day 5 organoids are delineated in white. Scale bar, 10 μm. (F) Quantification of the volume as normalized to surface area of the apical processes of induced (+ Dox) versus uninduced (− Dox) Hum iZEB2 neural progenitor cells on day 5. The apical processes of segmented cells directly below the cell body were used for quantification. Gorilla day 5 measurements from Figure 2 E are included for comparison. Mean apical process volume:surface area ratio: Hum iZEB2 − Dox = 1.11; Hum iZEB2 + Dox = 0.76. ∗∗ p < 0.01, Mann-Whitney U, two-tailed, n (− Dox and + Dox) = 9 cells. Error bars are SD. (G) Quantification of the surface area of individual delineated ZO1 cell perimeters as shown in (E). Gorilla measurements from two cell lines (G1, G2) combined are shown for comparison. Mean apical surface area/cell: Hum iZEB2 − Dox = 9.62 μm 2 , Hum iZEB2 + Dox = 3.08 μm 2 , and gorilla (G1,G2) = 4.50 μm 2 . ∗∗∗∗ p < 0.0001, Mann-Whitney U, two-tailed, n (− Dox) = 180 cells from 8 organoids, n (+ Dox) = 199 cells from 8 organoids, both from 2 independent batches, box and whisker plots show median with min-max values, data points represent individual cells. (H) Quantification of perimeters of neuroepithelial buds from bright field images at days 5 and 10, and overall organoid size at day 10. Gorilla (G1,G2) measurements were combined and included for comparison. Day 5 mean neuroepithelial bud perimeter: Hum iZEB2 − Dox = 272 μm, Hum iZEB2 + Dox = 237 μm, and gorilla (G1,G2) = 232 μm. ∗∗∗∗ p < 0.0001, Mann-Whitney U, two-tailed, n (− Dox) = 142 neuroepithelial buds from 41 organoids from 3 independent batches; n (+Dox) = 195 neuroepithelial buds from 38 organoids from 3 independent batches; n (G1,G2) = 555 neuroepithelial buds from 114 organoids from 16 independent batches. Day 10 mean neuroepithelial bud perimeter: Hum iZEB2 − Dox = 300 μm, Hum iZEB2 + Dox = 198 μm, and gorilla (G1,G2) = 227 μm. Day 10 mean organoid area: Hum iZEB2 − Dox = 201,434 μm 2 , Hum iZEB2 + Dox = 132,325 μm 2 , and gorilla (G1,G2) = 93,447 μm 2 . ∗∗∗∗ p < 0.0001, Mann-Whitney U, two-tailed, n (− Dox day 10) = 15 organoids and 106 neuroepithelial buds from 3 independent batches, n (+ Dox day 10) = 15 organoids and 149 neuroepithelial buds from 3 independent batches, error bars are SD. (I) Representative immunofluorescence images showing the effect of BMP4 on the morphology of neural progenitor cells revealed by sparse viral labeling with GFP on day 5 untreated (− BMP4) and treated (+ BMP4) gorilla (G1) organoids with staining for GFP, SOX2, and DAPI. Arrows indicate the apical process. Scale bar, 40 μm. (J) Immunofluorescent staining for ZO1 showing apical surface areas of individual progenitor cells from BMP4-treated (+ BMP4) and untreated (− BMP4) gorilla (G1) organoids at day 5. Perimeters of some individual progenitor cells are delineated in white. Scale bar, 10 μm. (K) Quantification of individual delineated ZO1 cell perimeters as shown in (J). Mean apical surface area/cell: gorilla − BMP4 = 2.72 μm 2 and gorilla + BMP4 = 4.13 μm 2 . ∗∗∗∗ p < 0.0001, Mann-Whitney U, two-tailed, n (− BMP4) = 301 cells from 8 organoids from 2 independent batches, n (+ BMP4) = 326 cells from 8 organoids from 2 independent batches, box and whisker plots are median with min-max values, data points represent individual cells. (L) Immunofluorescence images of human (H9) and gorilla (G1) day 5 organoids untreated (− LPA) and treated (+ LPA) with staining for OCLN, ZO1, and DAPI. LPA treatment in gorilla results in increased OCLN distribution along the apicobasal axis of cells (arrowheads) and expanded apical surfaces of cells (ZO1, bottom panel). Scale bar, 40 μm (upper panels), 10 μm (bottom panels). (M) Quantification of individual delineated ZO1 cell perimeters as shown in (L). Mean apical surface area/cell: human − LPA = 5.36 μm 2 , human + LPA = 5.25 μm 2 , gorilla − LPA = 2.44 μm 2 , and gorilla + LPA = 4.73 μm 2 . ∗ p < 0.05 ∗∗∗∗ p < 0.0001, Kruskal-Wallis and post hoc Dunn’s multiple comparisons test, n (human − LPA) = 146 cells from 3 organoids of 1 batch, n (human + LPA) = 200 cells from 3 organoids of 1 batch, n (gorilla − LPA) = 375 cells from 7 organoids and 2 independent batches, n (gorilla + LPA) = 457 cells from 10 organoids and 2 independent batches, box and whisker plots show median with min-max values, data points represent individual cells. (N) Schematic summarizing the morphological changes that occur in neural progenitor cells as they transition from NE to tNE cells (purple background). ZEB2 is highlighted as a driver, which acts through BMP-responsive SMADs to downregulate epithelial features, notably tight-junction proteins (TJs, green), and involves apical constriction through rearrangements in the actin cytoskeleton (actin, magenta). See also Figure S7 .

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Staining, Expressing, Immunofluorescence, Labeling, MANN-WHITNEY, Two Tailed Test, Whisker Assay

Journal: Cell

Article Title: An early cell shape transition drives evolutionary expansion of the human forebrain

doi: 10.1016/j.cell.2021.02.050

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF), western blotting (WB) and WB blocking conditions were as follows: mouse anti-β-actin (Abcam, 8226, WB 1:2000 in BSA), mouse anti-ZEB2 (Origene, TA802113, IF 1:150, WB 1:2000 in milk), sheep anti-TBR2 (R&D Systems, AF6166, IF 1:200), mouse anti-CDH2 (BD Biosciences, 610920, IF 1:500, WB 1:1000 in milk), mouse anti-CDH1 (BD Biosciences, 610181, IF 1:500, 1:1000 in milk), rabbit anti-OCLN (Abcam, ab31721, IF 1:200, WB 1:1000 in milk), rabbit anti-EMX1 (ATLAS Antibodies, HPA006421, IF 1:100), rabbit anti-EMX1 (Origene, TA325087, WB 1:1000 in BSA), rabbit anti-BLBP (Abcam, ab32423, IF 1:200), rabbit anti-GLAST (Abcam, ab416, IF 1:200), goat anti-DCX (N-19) (Santa Cruz, sc-8067, IF 1:300), rat anti-CTIP2 (Abcam, ab18465, IF 1:200), mouse anti-HuC/D (Life Technologies, A21271, IF 1:200), mouse anti-ZO1 (BD Biosciences, 610966, IF 1:300), chicken anti-GFP (Thermo Fisher, A10262, IF 1:500), rabbit anti-GFP (Abcam, ab290, WB 1:1000 in milk), rabbit anti-EpCAM (Abcam, ab71916, IF 1:300, WB 1:1000 in milk), mouse anti-Vimentin (V9) (Santa Cruz, sc-6260, IF 1:200, WB 1:1000 in BSA) rabbit anti-PAX6 (Abcam, ab195045, IF 1:200), rabbit anti-SOX2 (Abcam, ab97959, IF 1:200), rabbit anti-SHROOM3 (ATLAS Antibodies, HPA047784, IF 1:200, WB 1:1000), mouse anti-GAPDH (Abcam, ab8245, WB 1:2000 in milk), mouse anti-TUJ1 (Biolegend, 801213, IF 1:500).

Techniques: Fluorescence, Recombinant, Knock-Out, Protease Inhibitor, Multiplex Assay, Software

Journal: Cancer Science

Article Title: Immunostimulatory oncolytic activity of coxsackievirus A11 in human malignant pleural mesothelioma

doi: 10.1111/cas.15645

Figure Lengend Snippet: ICD ‐related DAMP induction by CVA11 infection in human MPM cell lines. (A) Flow cytometric analysis of calreticulin expression at the surface of human MPM cells at 10 h after infection with CVA11 (MOI = 10). MFI, mean fluorescence intensity. (B–D) The indicated human MPM cell lines were infected with CVA11 or mock for 24 h, after which the concentrations of HMGB1 (B), annexin A1 (C), and HSP70 (D) in the supernatant were measured with ELISAs. Data are means + SD from three independent experiments. (E) Flow cytometric analysis of PD‐L1 and PD‐L2 expression at the surface of human MPM cells infected (or not) with CVA11 (MOI = 10) for 18 h. Histograms represent the measured fluorescence of cells incubated with isotype control (blue) or specific (red) antibodies, and data are representative of three independent experiments (A, E).

Article Snippet: The concentrations of HMGB1, HSP70, annexin A1, and IL‐1β in culture supernatants were measured using an HMGB1 ELISA Kit Exp (Shino‐Test), an HSP70 ELISA Kit (Proteintech), a Human Annexin A1 ELISA Kit (Abcam), and a Human IL‐1β ELISA Kit (Abcam), respectively.

Techniques: Infection, Expressing, Fluorescence, Incubation, Control