Journal: Cell reports

Article Title: Dissecting the spermatogonial stem cell niche using spatial transcriptomics

doi: 10.1016/j.celrep.2023.112737

Figure Lengend Snippet: (A) Spatial mapping of major testicular cell types (left) and transcriptional states of spermatogonia (right) in a human testis sample. ES, elongating/elongated spermatid; RS, round spermatid; SPC, spermatocyte; SPG, spermatogonium. Scale bar, 300 μm. (B) The spatial relationship between the five SPG transcriptional subtypes as quantified by the neighborhood enrichment score in two independent human Slide-seq datasets. (C) Differentially expressed LR pairs among SPG1-neighboring cell type pairs. (D) Spatial expression patterns of selective LR pairs enriched in myoid cell-SPG1 (upper panel) and endothelial cell-SPG1 (lower panel) pairs. Scale bar, 320 μm. (E) Protein expression of VWF and EFNA1 in a human testicular sample. The white dashed line outlines the blood vessel. Yellow arrowheads denote protein co-localization. Scale bar, 8 μm. (F) Bar graph showing the number of GFRA1 + ; KIT ‒ human SPG on day 7 normalized against that from day 0. Cells were treated with recombinant human ENFA protein at 0, 3, and 6 ng/mL, respectively. Data from 4 patient samples are shown.

Article Snippet: Antibodies used for IF: mouse Anti-CYP11A1 antibody (1:200, 67264-1-Ig, Proteintech), rabbit anti-PTN antibody (1:100, 27117-1-AP, Proteintech), goat anti-PTN antibody (1:100, AF252SP, R&D Systems), mouse anti-Syndecan-4 (5G9) Alexa Fluor 546 antibody (1:100, sc-12766 AF546, Santa Cruz Biotechnology), rabbit anti-Ephrin A1 antibody (1:100, ab124911, abcam), mouse Anti-VWF antibody (1:100, 66682-1-Ig, Proteintech), mouse anti-GFRa1 Alexa Fluor 647 antibody (1:100, sc-271546 AF647, Santa Cruz Biotechnology) and Alexa Fluor 488-, 594-, and 647-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Recombinant

Journal: Cell reports

Article Title: Dissecting the spermatogonial stem cell niche using spatial transcriptomics

doi: 10.1016/j.celrep.2023.112737

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used for IF: mouse Anti-CYP11A1 antibody (1:200, 67264-1-Ig, Proteintech), rabbit anti-PTN antibody (1:100, 27117-1-AP, Proteintech), goat anti-PTN antibody (1:100, AF252SP, R&D Systems), mouse anti-Syndecan-4 (5G9) Alexa Fluor 546 antibody (1:100, sc-12766 AF546, Santa Cruz Biotechnology), rabbit anti-Ephrin A1 antibody (1:100, ab124911, abcam), mouse Anti-VWF antibody (1:100, 66682-1-Ig, Proteintech), mouse anti-GFRa1 Alexa Fluor 647 antibody (1:100, sc-271546 AF647, Santa Cruz Biotechnology) and Alexa Fluor 488-, 594-, and 647-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Recombinant, DNA Library Preparation, Software

Journal: bioRxiv

Article Title: Hedgehog-dependent and Hedgehog-independent roles for Growth Arrest Specific 1 in mammalian kidney morphogenesis

doi: 10.1101/2024.05.02.592197

Figure Lengend Snippet: RET receptors are expressed normally in Gas1 -/- metanephros. (A-H) Whole-mount in situ hybridization for Ret (A-D) and Gfra1 (E-H) in wildtype (A,C,E,G) and Gas1 -/- (B,D,F,H) metanephros (MetN). (I-P) Immunofluorescent antibody detection of RET (I-L) and GFRA1 (M-P) in coronal kidney sections from E11.5 and E12.0 wildtype (I,K,M,O) and Gas1 mutant (J,L,N,P) embryos. Asterisks denote Gas1 mutant kidneys that have not undergone ureteric bud (UB) branching morphogenesis (insets; L,P). (Q) Quantitation of Etv5 MM fluorescence intensity normalized to MM area (3 sections/kidney) in E11.5 wildtype (n=6 kidneys) and Gas1 -/- (n=5 kidneys) embryos. Scale bars (A,C,E,G) 100μm; (I) 50μm. P-values were determined by a two-tailed Student’s t-test: non-significant: p>0.05 and significant: p≤0.05.

Article Snippet: Sections were blocked (1X PBS+ 10% Donkey serum + 0.3% Triton) for 1 h at 37°C, then incubated with rabbit anti-RET (1:200, Cell Signaling, Cat#3223) and goat anti-GAS1 (1:900, R&D Systems; Cat#AF2544) or goat anti- GFRA1 (1:500, R&D Systems, Cat# AF560) overnight at 4°C.

Techniques: In Situ Hybridization, Mutagenesis, Quantitation Assay, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Hedgehog-dependent and Hedgehog-independent roles for Growth Arrest Specific 1 in mammalian kidney morphogenesis

doi: 10.1101/2024.05.02.592197

Figure Lengend Snippet: GAS1 is secreted from the MM and accumulates in the UB epithelium. (A-J) Immunofluorescent detection of DAPI (gray; A,F), GAS1 (cyan; B,G), RET (magenta; C,H), HSPG2 (yellow; D,I) in metanephros coronal sections from E12.0 wildtype (A-E) and Gas1 -/- (F-J) embryos. GAS1/RET/HSPG2 merged images are shown (E,J). Dotted lines circle the ureteric bud (UB; B,G,C,H). (K) Western blot detection of immunoprecipitated endogenous GAS1 from supernatants collected from cultured wildtype and Gas1 mutant metanephric mesenchyme (MM) cells from E18.5 kidneys (n=3 technical replicates). (L-Q) Representative images of Proximity Ligation Assay (PLA) detection of GFRA1/RET PLA+ signal (yellow; M,P) merged with CDH1 (cyan; N,Q) antibody detection in metanephros coronal sections from E11.5 wildtype (L-N) and Gas1 -/- (O-Q) embryos. DAPI labels nuclei (gray; L,O). Dotted lines mark the UB (M,P). (R) Quantitation of the total number of GFRA1/RET PLA+ puncta (normalized to CDH1+ cells; 3 sections/kidney) in E11.5 wildtype (n=11) and Gas1 -/- (n=4) metanephros. (S-X) Representative images of PLA detection of GAS1/RET PLA+ signal (yellow; T,W) merged with CDH1 (cyan; U,X) antibody detection in metanephros coronal sections from E11.5 wildtype (S-U) and Gas1 -/- (V-X) embryos. DAPI labels nuclei (gray; S,V). Dotted lines mark the UB (T,W). (Y) Quantitation of the total number of GAS1/RET PLA+ puncta (normalized to CDH1+ cells; 3 sections/kidney) in E11.5 wildtype (n=16) and Gas1 -/- (n=7) metanephros. (Z-C’) Whole-mount in situ hybridization of Wnt11 expression in wildtype (Z,A’) and Gas1 -/- (B,C’) kidney explants cultured for 4 days in the presence or absence of recombinant murine GDNF (75ng/mL). Dotted lines denote the metanephros (Z’B’) or UB (B’; inset). (D’) Quantitation of the total number of Wnt11 -expressing UB branch tips in non-treated and GDNF-treated control (wildtype or Gas1 +/- ; n=8 non-treated, n=12 GDNF-treated) and Gas1 -/- (n=7 non-treated, n=8 treated) kidney explant cultures. Scale bar (F,N) 25μm; (Z) 250μm. P-values were determined by a two-tailed Student’s t-test (R,Y) or an ordinary one-way ANOVA with Tukey’s multiple comparisons test (D’): p>0.05 and significant: p≤0.05.

Article Snippet: Sections were blocked (1X PBS+ 10% Donkey serum + 0.3% Triton) for 1 h at 37°C, then incubated with rabbit anti-RET (1:200, Cell Signaling, Cat#3223) and goat anti-GAS1 (1:900, R&D Systems; Cat#AF2544) or goat anti- GFRA1 (1:500, R&D Systems, Cat# AF560) overnight at 4°C.

Techniques: Western Blot, Immunoprecipitation, Cell Culture, Mutagenesis, Proximity Ligation Assay, Quantitation Assay, In Situ Hybridization, Expressing, Recombinant, Control, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Oocytes could rearrange immunoglobulin production to survive over adverse environmental stimuli.

doi: 10.3389/fimmu.2022.990077

Figure Lengend Snippet: FIGURE 2 IgG protein increased in response to environmental LPS (A, B) Immunofluorescence shows that IgG increased more than one-fold in response to LPS, and nuclear IgG increased more significantly. (C, D) Western blot and quantification show that IgG protein level increased more than one-fold in response to LPS (Lipopolysaccharide) supplemented in the IVM (in vitro maturation) medium. (E-H) Immunofluorescence shows that IgG intensity is very low in control granular cells (GCs), (E, F) or NIH3T3 (3T3s), (G, H) and did not increase at all on exposure to LPS stimuli. (I, J) Western blot and quantification show that p-NFKB (p-p65) significantly increased in response to LPS. (K-N). Western blot and quantification show that TLR4, IRF3, and p- IRF3 didn’t change at all in response to LPS. Actin was used as a loading control. Scale bars, 20 mm. ** indicates p < 0.01. **** indicates p < 0.0001. ns, not significant.

Article Snippet: Primary antibodies: Anti Beta Actin mAb (Cat#:S6007M, Bioworld, Dublin, OH, USA); Rabbit anti OMPC (Cat#: bs20213R, Bioss Antibodies, Beijin, China); Acetyl-Histone H3 (K27)(D5E4) XP(R) rabbit mAb (Cat#: 8173s, Cell Signaling Technology, Danvers, MA, USA); Histone H3 (Tri-Methyl K4) pAb (Cat#: BS7232, Bioworld, Dublin, OH, USA); CR2 rabbit pAb (Cat#: A8407, ABclonal, Wuhan, Hubei, China); CD46 rabbit pAb(Cat#: A1653, ABclonal, Wuhan, Hubei, China); FCGR1A pAb (Cat#: BS6238, Bioworld, Dublin, OH, USA); Phospho-Histone H3(Ser 10) Mouse McAb (Cat#: 66863-1-Ig; Proteintech, Rosemont, IL, USA); Tubulin a (G436) mAb (Cat#: BS1699M; Bioworld, Dublin, OH, USA); Anti-RELA (PhosphoSer536) rabbit polyclonal antibody (Cat#: D155006; BBI, Shanghai, China), AffiniPure Goat anti-Mouse IgG, light chain specific (Jackson ImmunoResearch Laboratory, Cat#: 115-005- 174, West Grove, PA, USA); SOD2 Rabbit Recombinant mAb (Cat#: A5377; Houston, TX, USA); GPX4 Rabbit Recombinant mAb (Cat#: A5569; Houston, TX, USA); IRF3 Rabbit Recombinant mAb (Cat#: A5373; Houston, TX, USA); TLR4 rabbit pAb (Cat#: A11226, ABclonal, Wuhan, Hubei, China); Phospho-IRF3 (Ser396) Rabbit Polyclonal Antibody (Cat#: A8407, Beyotime, Tangshan, Hebei, China).

Techniques: Western Blot, In Vitro, Control