Journal: Cell Death & Disease

Article Title: USP12 promotes breast cancer angiogenesis by maintaining midkine stability

doi: 10.1038/s41419-021-04102-y

Figure Lengend Snippet: A The relative expression of VEGF-A and VEGF-C in the supernatant of MDA-MB-231 cells overexpressing USP12 was detected by ELISA. B The relative expression of VEGF-A and VEGF-C in the supernatant of MDA-MB-231 cells with USP12 knockdown was detected by ELISA. C , D Migration of HUVECs treated with supernatant from MDA-MB-231 cells with USP12 overexpression ( C ) and USP12 knockdown ( D ). Representative images are shown in the left panel. The quantitative results are shown in the right panel. E , F . Tube formation assay of HUVECs treated with supernatant from MDA-MB-231 cells with USP12 overexpression ( E ) and USP12 knockdown ( F ). Representative images are shown in the left panel. The quantitative results are shown in the right panel. The experiments were repeated three times. ** p < 0.01, *** p < 0.001; mean ± SD.

Article Snippet: Matrigel matrix (356234, Corning), the human VEGF-A ELISA kit (EKO588-96, Boster) and the human VEGF-C ELISA KIT (EKO539-96, Boster) were also used.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Migration, Over Expression, Tube Formation Assay

Journal: Frontiers in Neurology

Article Title: Angiogenin in the Neurogenic Subventricular Zone After Stroke

doi: 10.3389/fneur.2021.662235

Figure Lengend Snippet: Angiogenin expression in the SVZ niche: (A) schematic figure of the SVZ dissection for NSCs isolation. (B) Representative image of Western blot proving the presence of angiogenin in SVZ niche of different naïve mice and in primary NSCs in culture derived from the adult SVZ (see online for the full-length Western blot film); (C) Neurosphere immunocytochemistry phenotyping showing expected nestin and DCX markers. ANG, angiogenin; C–, negative control (NSC culture media); DCX, doublecortin; NSCs, neural stem cells; SVZ, subventricular zone.

Article Snippet: Finally, Mouse Angiogenin SimpleStep ELISA® Kit (ab208349, Abcam, Cambridge, UK) was used following manufacturer's instructions (sample dilution 1/5, and coefficient of variation of replicates <25%).

Techniques: Expressing, Dissection, Isolation, Western Blot, Derivative Assay, Immunocytochemistry, Negative Control

Journal: Frontiers in Neurology

Article Title: Angiogenin in the Neurogenic Subventricular Zone After Stroke

doi: 10.3389/fneur.2021.662235

Figure Lengend Snippet: SVZ-derived NSCs responses to angiogenin: (A) timeline of the experimental procedure of neurosphere cultures and representative images; (B,C) box plots showing quantification of the NSC-forming neurospheres after angiogenin stimulation (100 or 200 ng/ml) at different time points, the inhibition with neomycin, and the largest neurosphere diameters obtained with angiogenin treatment; n = 4–9; ** p < 0.001 and *** p < 0.001 vs. control. Box plots represent median (IQR) of the percentage vs. control values of each independent experiment. ANG/A, angiogenin; NSCs, neural stem cells; NMC, neomycin; NF, neurospheres.

Article Snippet: Finally, Mouse Angiogenin SimpleStep ELISA® Kit (ab208349, Abcam, Cambridge, UK) was used following manufacturer's instructions (sample dilution 1/5, and coefficient of variation of replicates <25%).

Techniques: Derivative Assay, Inhibition

Journal: Frontiers in Neurology

Article Title: Angiogenin in the Neurogenic Subventricular Zone After Stroke

doi: 10.3389/fneur.2021.662235

Figure Lengend Snippet: SH-SY5Y neurite differentiation: (A) timeline of the experimental procedure. (B) Box plots and representative images showing axonal/neurite outgrowth in the presence of Retinoic Acid (RA) as expected, but not with angiogenin stimulation; n = 5–6. The insert in RA shows a micrograph representative of the WimNeuron analysis. Box plots represent median (IQR) of the percentage vs. control values of each independent experiment, *** p < 0.001 vs. control.

Article Snippet: Finally, Mouse Angiogenin SimpleStep ELISA® Kit (ab208349, Abcam, Cambridge, UK) was used following manufacturer's instructions (sample dilution 1/5, and coefficient of variation of replicates <25%).

Techniques:

Journal: Frontiers in Neurology

Article Title: Angiogenin in the Neurogenic Subventricular Zone After Stroke

doi: 10.3389/fneur.2021.662235

Figure Lengend Snippet: Post-stroke SVZ neurogenesis and angiogenin expression: (A) timeline schemes describing the pre-clinical stroke and treadmill rehabilitation protocols. (B) Bar graph representing angiogenin after 3 days of treadmill rehabilitation or no rehabilitation in the SVZ area, measured by ELISA ( n = 6/group); (C) bar graph representing DCX+ quantification in the immunohistochemistry study of the SVZ ( n = 5/group) and representative immunostains of the ipsilateral hemisphere identifying the neuroblasts (DCX+). (D) Representative immunostains of the SVZ after stroke and treadmill exercise showing the distribution of nestin+ and DCX+ NSCs in the SVZ (left panel); notice that only neuroblasts (DCX+) had the most angiogenin expression, which is specifically shown in the insert magnifications and in white arrows. Bar graphs represent mean ± SEM, * p < 0.05 and ** p < 0.01 as indicated by the horizontal lines. ANG, angiogenin; CL, contralateral; DAPI, 4′,6-diamidino-2-phenylindole; DCX, doublecortin; IP, ipsilateral; RHB, rehabilitation; SVZ, subventricular zone. The white * indicates the position of the lateral ventricle with contiguous dorsolateral SVZ.

Article Snippet: Finally, Mouse Angiogenin SimpleStep ELISA® Kit (ab208349, Abcam, Cambridge, UK) was used following manufacturer's instructions (sample dilution 1/5, and coefficient of variation of replicates <25%).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: Identification of dynamic undifferentiated cell states within the male germline

doi: 10.1038/s41467-018-04827-z

Figure Lengend Snippet: Characterization of PDX1+ spermatogonia. a Representative whole-mount IF of WT (top 3 rows, n = 3 mice), Oct4-GFP and Pdx1 GFP adult tubules ( n = 2 mice per genotype). Antibodies to PDX1 and GFRα1 are goat polyclonals and distinguished by nuclear vs . cell surface staining respectively. Scale bars, 50 μm. b Mean percentage of GFRα1+ cells/chains PDX1+ ± s.e.m. from WT analysis in a ( n = 4 mice, >250 cells/chains per sample). c Representative whole-mount IF of adult Plzf-mC/CreER; Z/EG tubules D3 post-TAM ( n = 2 mice). Arrowheads: PDX1+ A s . Inset shows detail of indicated area. Scale bar, 50 μm. d Representative IF of adult WT testis sections ( n = 4 mice). Insets: details of selected areas. EOMES+ GFRα1+ (arrowheads) and EOMES− GFRα1+ cells (bracket) are shown. Tubule stage is indicated. Scale bar, 50 μm. e Representative IF of adult Oct4-GFP testis section ( n = 2 mice). Insets: immunostaining within indicated area. EOMES+ GFP− (arrowheads) and EOMES− GFP+ (bracket) spermatogonia are indicated. Asterisks: autofluorescent interstitium. Scale bar, 50 μm. f Representative flow cytometry of fixed and permeabilized testis cells from Oct4-GFP adults ( n = 2). PLZF+ cells shown. g Representative IF of adult WT testis sections ( n = 4 mice). Insets: immunostaining within indicated area. PDX1+ EOMES+ spermatogonia are indicated (arrowheads). Tubule stage is shown. Asterisk: autofluorescent interstitium. Scale bar, 50 μm. h Representative flow cytometry of fixed and permeabilized WT adult testis cells ( n = 4). Mean numbers of EOMES+ and PDX1+ cells in PDX1+ and EOMES+ gates respectively are shown ± s.e.m. i Representative flow cytometry for KI67 in indicated PLZF+ populations of fixed and permeabilized adult WT testis cells ( n = 4 mice). j Quantification of flow cytometry from i . Percentage of indicated PLZF+ fractions KI67+ . Horizontal bars: mean values ( n = 4 mice). k Representative IF of adult WT testis sections demonstrating PDX1+ A undiff localisation (arrowheads) within tubules ( n = 4 mice). Asterisks: autofluorescent interstitium. Tubule stages are shown. Scale bar, 50 μm. l Quantification of spermatogonial localisation from k . Mean values ± s.e.m. are shown ( n = 4 mice, 56–95 tubule sections per mouse). Significance vs. tubule-tubule localisation is indicated. m Representative IF of Id4 IRES-GFP adult testis sections ( n = 4 mice). Insets show immunostaining within indicated area. Arrowheads: PDX1+ EOMES+ GFP+ spermatogonia. Asterisk: PDX1 low EOMES+ GFP+ cell. Tubule stage is shown. Scale bar, 50 μm. n Representative flow cytometry of indicated PLZF+ populations of fixed and permeabilized adult Id4 IRES-GFP testis cells ( n = 4 mice). Mean numbers of PDX1+ and EOMES+ A undiff expressing GFP ± s.e.m. o Flow cytometry of Id4 IRES-GFP testis cells as in n . PLZF+ fractions are shown. Mean numbers of PLZF+ GFP+ cells positive for EOMES and PDX1 are indicated ± s.e.m. ( n = 4 mice). Significance was calculated by two-tailed Student’s t -test (*** P < 0.001, **** P < 0.0001)

Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R&D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R&D Systems, 1:1000).

Techniques: Staining, Immunostaining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of dynamic undifferentiated cell states within the male germline

doi: 10.1038/s41467-018-04827-z

Figure Lengend Snippet: Stem cell potential and molecular characteristics of PDX1+ A undiff . a Isolation of PDX1+ and PDX1− A undiff from Plzf-mC/CreER; Pdx1 GFP/+ adults by flow cytometry. A undiff are mCherry+ CD9+ c-KIT−. Gates for GFP+ and GFP− cells were set according to Plzf-mC/CreER control (left profile). Percentage of cells in GFP+ gate from representative sample is shown ( n = 7). b Pdx1-GFP+ and GFP− adult A undiff fractions were transplanted into recipient testis and analysed 8 weeks later by whole-mount IF. Images show GFP and mCherry expression in representative donor colonies. Panels on right show higher magnification details of indicated areas and grayscale panels show individual immunostaining. Scale bar, 100 μm. c Colony-forming efficiency of Pdx1-GFP+ and GFP− A undiff fractions in transplantation assays from b . Data is presented as mean number of colonies per 10 5 donor cells ± s.e.m. ( n = 15 recipient testes for Pdx1-GFP− cells and n = 13 for Pdx1-GFP+ cells). Donor cells were pooled from a total of 7 Plzf-mC/CreER; Pdx1 GFP/+ adults. d Mean length ± s.e.m. of donor colonies was measured from tiled microscope images from experiment of c . e Mean number of GFP+ cells/donor colony ± s.e.m. were calculated for a set of whole-mount samples from transplant assay of c ( n = 58 colonies from Pdx1-GFP− cells and n = 63 from Pdx1-GFP+). f Heatmap illustrates expression of indicated genes from RNA-Seq analysis of Pdx1-GFP+ and GFP− A undiff fractions isolated as in a ( n = 4 mice). Differentially expressed genes (DEG) are in bold. Cut-off for DEG is false discovery rate (FDR) < 0.05 and absolute fold change ≥1.5. g Heatmap showing differentially expressed MAPK pathway genes from KEGG analysis of RNA-Seq data from f . h Quantitative RT-PCR analysis of indicated genes in mCherry+ CD9+ c-KIT− A undiff isolated from Plzf-mC/CreER; Pdx1 GFP/+ and Plzf-mC/CreER; Pdx1 +/+ control adults. Mean values ± s.e.m. are shown ( n = 6 mice per genotype). i Representative IF of testis sections from aged (8 months) mice of the indicated genotypes ( n = 3 mice). VASA and PLZF staining identifies germ cell and spermatogonial populations respectively. Scale bar, 50 μm. Significance was calculated by two-tailed Student’s t -test (** P < 0.01, not significant (ns) P > 0.05)

Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R&D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R&D Systems, 1:1000).

Techniques: Isolation, Flow Cytometry, Control, Expressing, Immunostaining, Transplantation Assay, Microscopy, RNA Sequencing, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of dynamic undifferentiated cell states within the male germline

doi: 10.1038/s41467-018-04827-z

Figure Lengend Snippet: PDX1+ and EOMES+ spermatogonia during development and regeneration. a Representative whole-mount IF of tubules from WT mice of indicated ages (PND; postnatal day) ( n = 2 mice per age). Scale bars, 50 μm. b Representative IF of testis sections from WT mice of indicated ages ( n = 3 mice per timepoint). Insets show details of indicated areas. Arrowheads: EOMES+ PDX1+ cells. Asterisks: EOMES+ PDX1− cells. Scale bars, 50 μm. c , d Flow cytometry of fixed and permeabilized testis cells from WT mice of indicated ages. Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1 and EOMES are shown ± s.e.m. ( n = 3–5 mice/time point). e WT adults were treated with busulfan (10 mg/kg) and tubules analysed by whole-mount IF 14 days later ( n = 2 mice). Top panels: regenerative areas with GFRα1+ A al . Bottom: non-regenerative areas lacking GFRα1+ A al . Insets show details of indicated regions. Scale bar, 50 μm. f Regeneration assay of e 4 weeks post busulfan (2 areas shown). Arrowheads: PDX1+ A undiff . Scale bar, 50 μm. g Representative flow cytometry of fixed and permeabilized testis cells from WT adults untreated or busulfan treated as in e ( n = 4 busulfan-treated mice and n = 5 untreated). PLZF+ c-KIT− A undiff are shown. Percentage of A undiff KI67+ and EOMES+ are indicated. h Mean percentages of PLZF+ c-KIT− A undiff expressing PDX1, EOMES and KI67 from g are shown ± s.e.m. i Representative flow cytometry of PDX1 in indicated populations from g ( n = 4 mice per condition). Percentages of cells PDX1+ are indicated. j PDX1 levels (median fluorescent intensity) in EOMES+ PLZF+ cells from i . Mean values ± s.e.m. shown. k Quantitative RT-PCR of A undiff from Plzf-mC/CreER mice of indicated ages or 10 days post busulfan as in e . Expression levels are corrected to β-actin and normalized to an adult sample. Mean values ± s.e.m. are indicated ( n = 4 PND10 and busulfan-treated, n = 5 PND20, n = 6 adults). Selected significance values are shown. l Model of A undiff functional states. Self-renewing (curved arrows) GFRα1+ A undiff adopt different states identified by variable expression of Pdx1 , Eomes and Lhx1 . A fraction of GFRα1+ A undiff lacks PDX1, EOMES and LHX1 (grey in panel) and may represent a transitional state destined to become differentiation-primed. Niche signals differentially support self-renewing states. Given dynamic niche properties, different states predominate in development and homeostatic plus regenerative testis. The state marked by PDX1, EOMES and LHX1 is specific to homeostatic testis and potentially optimized for life-long germline maintenance. Pdx1 is downregulated and Eomes plus Lhx1 upregulated in states suited for short-term expansion during development and regeneration. Oct4-GFP+ differentiation-primed A undiff convert back to a self-renewing state under optimised culture conditions or by transplantation into recipient testis with vacant niches. Significance was calculated by two-tailed Student’s t -test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Primary antibodies were as follows: Goat anti-GFRα1 (AF560, 1:250), anti-c-KIT (AF1356, 1:250), anti-mouse/rat PDX1 (AF2517, 1:250), anti-PLZF (AF2944, 1:500), anti-LIN28A (AF3757, 1:500) and anti-SOX3 (AF2569, 1:250) (R&D Systems), rabbit anti-mCherry (ab167453, 1:2000), anti-OCT4 (ab19857, 1:500), anti-SALL4 (ab29112, 1:2000) (Abcam), chicken anti-GFP (ab13970, 1:5000) and rat anti-germ cell-specific antigen (TRA98, 1:500) (Abcam), rat monoclonal anti-mCherry clone 16D7 (Thermo Fisher Scientific, 1:2000), rabbit monoclonal anti-Cyclin D1 clone SP4 (1:250) and mouse monoclonal anti-DNMT3A clone 64B1446 (1:200) (Novus Biologicals), rat anti-KI67 clone SolA15 (eBioscience, 1:250), rabbit monoclonal anti-c-KIT clone D13A2 (1:400) and anti-RARγ clone D3A4 (1:500) (Cell Signaling Technology) and rabbit monoclonal anti-mouse EOMES clone 1219A (R&D Systems, 1:1000).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR, Functional Assay, Transplantation Assay, Two Tailed Test