Journal: Scientific Reports

Article Title: Neuronal imaging with ultrahigh dynamic range multiphoton microscopy

doi: 10.1038/s41598-017-06065-7

Figure Lengend Snippet: Demonstration of technique. S is the raw PMT data obtained from BPAE cells (FluoCells Prepared Slide 2, Molecular Probes) while active illumination is on (normally this signal would be sent to the microscope acquisition electronics). P is the illumination power measured by the photodiode. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{log}}_{2}X$$\end{document} log 2 X is the resultant log-encoded sample strength sent to the microscope acquisition electronics. X is the linear-encoded sample strength calculated post-hoc (see below). Note the bright edges apparent in S are caused by overshoot in the feedback at 4 μ s pixel time (see Supplementary Information for details). These are canceled after computation of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{log}}_{2}X$$\end{document} log 2 X (or X ).

Article Snippet: The fluorescence signal S obtained from the microscope PMT (Hamamatsu H7422PA-40 with Prairie Ultima pre-amp) is normally routed to the microscope acquisition electronics.

Techniques: Microscopy

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: Science Advances

Article Title: Sustainable synthesis of amino-cellulose nanofibers for biomaterial platforms

doi: 10.1126/sciadv.adx4556

Figure Lengend Snippet: Morphological characterizations: ( A ) digital photograph of 0.2% (w/v) A-CNF suspension (carboxylated cellulose:PEI molar ratio, 1: 1); ( B ) atomic force microscopy (AFM) micrograph (scale bar, 400 nm); ( C and D ) transmission electron microscopy (TEM) micrographs at different magnifications (scale bars, 0.5 μm and 200 nm); ( E ) diameter distribution histogram; ( F ) digital photograph of 0.2% (w/v) A-CNF suspension (carboxylated cellulose:PEI molar ratio, 1: 2); ( G ) AFM micrograph (scale bar, 400 nm); ( H and I ) TEM micrographs at different magnifications (scale bars, 0.5 μm and 200 nm); ( J ) diameter distribution histogram; ( K ) TEM visualization of high aspect ratio (scale bar, 0.5 μm). Physicochemical properties: comparison of ( L ) aspect ratio and ( N ) amine content of A-CNF prepared in optimal conditions with literature values. Data are detailed in table S1 ( – ). Quantitative analysis: comparison of ( M ) amine content of A-CNF, based on three independent measurements. Error bars denote SD. Stability and scalability assessments: ( O ) photographs of A-CNF suspension pre– and post–8 months of storage; ( P ) pilot production tests of 1 wt % A-CNF demonstrating scalability of the production process. LCA and TEA analyses of A-CNF: ( Q ) At an electricity price of US$0.1 per kWh, the per-ton synthesis cost of A-CNF via this method and the full process cost breakdown were calculated. These were compared with expenditures for quaternization reaction, amidation reactions, and the market price of CTS per ton. ( R ) For A-CNF and CTS production, environmental impacts of the quaternization reaction, amidation reaction, and the proposed synthesis method were analyzed across 10 categories (including global warming, human health, and terrestrial ecosystems). Data were standardized to enable consistent, objective evaluation of environmental outcomes.

Article Snippet: Before imaging, samples were coated with platinum under a 30-mA current for 40 s. Transmission electron microscopy images were captured using a JEM-2100 microscope (JEOL, Tokyo, Japan).

Techniques: Suspension, Microscopy, Transmission Assay, Electron Microscopy, Comparison

Journal: Science Advances

Article Title: Sustainable synthesis of amino-cellulose nanofibers for biomaterial platforms

doi: 10.1126/sciadv.adx4556

Figure Lengend Snippet: ( A ) Schematic and scanning electron microscopy (SEM) images show the cross-sectional views of A-CNF scaffold with varying solid contents. Insets provide digital photographs of each scaffold (scale bars, 500 μm). ( B ) Micro-CT reconstructed three-dimensional images depict the microstructures of voids within the A-CNF scaffold. ( C ) Variations in SSA and pore dimensions as a function of solid content in the scaffold. ( D ) Analysis of void structure connectivity within A-CNF and CTS scaffolds. ( E ) Comparative analysis of porosity and density metrics for A-CNF scaffold versus CTS scaffold at different solid content levels. ( F ) Graphical representation of water absorption and retention capacities for A-CNF and CTS scaffolds. ( G ) Comparison of the biostability of A-CNF and CTS scaffolds across varying solid contents, supplemented with digital photographs documenting changes over specified time periods. h, hours. ( H ) Antimicrobial properties of the scaffolds are demonstrated through bacterial inhibition of E. coli and S. aureus in the presence of the A-CNF scaffold, compared to a control group. ( I ) The influence of solid content on the compressive strength and Young’s modulus of the A-CNF scaffold. ( J ) Ashby plots comparing the compressive strength and specific strength of the A-CNF scaffold to four other documented scaffolds. ( K ) A comparative overview of Young’s modulus in the A-CNF scaffold with natural in vivo tissues, illustrating a broad range of tissue stiffness from soft tissues with sub-megapascal modulus to hard tissues like bone. The modulus values of common tissue engineering materials such as 8% acrylamide (AM) gel, tissue culture polystyrene, and titanium (used in dental and bone implants) are included for comparison. ( L ) Details on the gel casting preparation method used to mold the A-CNF scaffold into various biomimetic soft tissue forms (scale bars, 1 cm). All data were analyzed from three independent measurements. Error bars represent SD.

Article Snippet: Before imaging, samples were coated with platinum under a 30-mA current for 40 s. Transmission electron microscopy images were captured using a JEM-2100 microscope (JEOL, Tokyo, Japan).

Techniques: Electron Microscopy, Micro-CT, Comparison, Inhibition, Control, In Vivo

Journal: Science Advances

Article Title: Sustainable synthesis of amino-cellulose nanofibers for biomaterial platforms

doi: 10.1126/sciadv.adx4556

Figure Lengend Snippet: ( A ) Schematic of the A-CNF scaffold tailored for organoid culture with adjustable pore sizes to accommodate varying cell sizes. H9c2 and SK-BR-3 (single adult cells) and PHC (adult primary cells) were used to develop heart, mammary, and liver organoids. ( B ) Optical images display hemolysis test results for CTS and A-CNF scaffolds, using deionized (DI) water (positive control, high hemolysis) and PBS (negative control, no hemolysis). ( C ) Quantification of hemolysis ratios for each scaffold group (Error bars show means ± SD; n = 6 repeats). ( D ) Results of CCK-8 assays showing cell inhibition rates (box plot elements: lower/upper boundaries, Q1/Q3; whiskers, max/min; dashed line, mean; n = 6 repeats), and ( E ) cell viability analyses of H9c2, SK-BR-3, and PHC cells cultured on A-CNF scaffolds for 24 hours versus controls (error bars show means ± SD; n = 6 repeats). ( F ) Confocal and three-dimensional (3D) fluorescence microscopy images of H9c2, SK-BR-3, and PHC cells cultured on CTS and A-CNF scaffolds for 48 hours. Live cells stained green (calcein-AM), and dead cells stained red (propidium iodide) (scale bars, 200 μm). ( G ) Schematic illustrating the impact of scaffold pore size on cell behavior. Larger pores may lead to cell crowding and reduced adhesion efficiency, while smaller pores can restrict cell extension and adhesion. Appropriately sized pores enhance cell attachment and promote growth. ( H and I ) Graphs of cell adhesion and proliferation rates for H9c2, SK-BR-3, and PHC cells cultured on A-CNF scaffolds with different pore sizes, showing how scaffold configurations affect cellular responses (error bars show means ± SD; n = 6 repeats). ( J ) Digital photographs of heart (H9c2), mammary (SK-BR-3), and liver (PHC) organoid cultures (scale bars, 5 mm), demonstrating the supportive role of A-CNF scaffolds in maintaining morphology and function of diverse organoids. h, hours.

Article Snippet: Before imaging, samples were coated with platinum under a 30-mA current for 40 s. Transmission electron microscopy images were captured using a JEM-2100 microscope (JEOL, Tokyo, Japan).

Techniques: Positive Control, Negative Control, CCK-8 Assay, Inhibition, Cell Culture, Fluorescence, Microscopy, Staining, Pore Size, Cell Attachment Assay

Journal: Molecular cancer

Article Title: The circular RNA circDLG1 promotes gastric cancer progression and anti-PD-1 resistance through the regulation of CXCL12 by sponging miR-141-3p.

doi: 10.1186/s12943-021-01475-8

Figure Lengend Snippet: Fig. 4 CircDLG1 promotes tumor progression by increasing the infiltration of MDSCs. a C57BL/6 mice were injected with MFC-sh-NC or MFC-sh-circDLG1 cells, and the tumor volume and tumor metastasis are shown. The mice were sacrificed at 4 weeks after injection. Left: the tumor size was measured, and the tumor volume was calculated every 4 days (n = 10 mice per group), **P < 0.05, two-way ANOVA. Right: the lungs of the mice were removed and paraffin embedded, consecutive sections (4 μm) were made and stained with hematoxylin-eosin, and the micrometastases in the lungs were examined and counted under a dissecting microscope (n = 10 mice per group), **P < 0.05, Student’s t-test. All experiments were repeated three times. Scale bar, left: 5 mm; right: 100 μm. b BALB/c nude mice were injected with MFC-sh-NC or MFC-sh-circDLG1 cells. The mice were sacrificed at 4 weeks after injection. Left: the tumor size was measured, and the tumor volume was calculated every 4 days (n = 10 mice per group). ns, no significance, two-way ANOVA. Right: The lungs of the mice were removed and paraffin embedded. Consecutive sections (4 μm) were made and stained with hematoxylin-eosin, and the micrometastases in the lungs were examined and counted under a dissecting microscope (n = 10 mice per group). ns, no significance, Student’s t-test. All experiments were repeated three times. Scale bar, left: 5 mm; right: 100 μm. c IHC analysis was performed using subcutaneous tumors for CD4, CD8, Gr-1, and CD11b (Gr-1 and CD11b are mouse MDSC markers). HPF, high-power visual field. ns, no significance; **P < 0.05. Student’s t-test. d Flow cytometric analysis of immune cells in subcutaneous tumors. **P < 0.05, Student’s t-test; the experiment was repeated three times. e The expression of CD33 (a marker of human MDSCs) was detected by IHC in human gastric cancer tissues. Scale bar, 50 μm. f The correlation between circDLG1 expression measured by qRT–PCR and CD33+ cells. n = 30, *P < 0.05, Pearson correlation analysis

Article Snippet: The following antibodies were used: anti-mouse IFNγ (BD Biosciences), anti-mouse CD45 (BD Biosciences), anti-mouse Gr-1 (TONBO Biosciences), anti-mouse Ly6G (TONBO Biosciences), anti-mouse ly6C (BD Biosciences), anti-mouse CD11b (TONBO Biosciences), anti-mouse F4/80 (BioLegend), anti-human CD11b (TONBO Biosciences), anti-human CD33 (BD Biosciences), and anti-human HLA-DR (BD Biosciences).

Techniques: Injection, Staining, Microscopy, Expressing, Marker, Quantitative RT-PCR