Journal: Developmental Cell

Article Title: A Balance of Yki/Sd Activator and E2F1/Sd Repressor Complexes Controls Cell Survival and Affects Organ Size

doi: 10.1016/j.devcel.2017.10.033

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Article Snippet: Rabbit α-YAP , ABclonal , Cat#A1002.

Techniques: Virus, Recombinant, Purification, Mutagenesis, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: Multi-tier mechanics control stromal adaptations in swelling lymph nodes

doi: 10.1101/2021.07.28.454217

Figure Lengend Snippet: Capsule fibrosis constrains lymph nodes at late timepoints. ( a ) Overview of intravital UV-laser cut experiments of the SCS fLECs. Inguinal LNs from FRC-mGFP mice, in which SCS fLECs are sparsely labeled with a mGFP are surgically exposed. A high UV-laser cuts the cell along 20 µm in one plane after which the local recoil is imaged. Images depict stills from before (t=-1s), directly after (t=0s) and late after (t=10.2s) cutting (scale bars = 5 µm), with corresponding kymograph along the recoil axis (scale bar x-axis = 1s, y-axis = 2 µm). Scissor and line indicate location of cut and arrows the recoiling cell. Dashed lines in kymograph indicate slopes used to calculate the recoil velocity, and vertical white line the cut. ( b ) Quantification of experiments as described in a for homeostatic (D0) and inflamed (D2, D4, D8, D14) LNs. Each datapoint represents a recoil-measurement of a single fLEC. Data pooled from 3 mice and at least two experiments. Mean±SEM are depicted and connected with a line. ( c ) Representative confocal images of LN capsules from PROX1-GFP mice in homeostasis (D0) and inflammation (D4, D8 and D14), in which LECs are labeled by a cytoplasmic GFP. Mesenchymal cells are stained for PDGFR-β, and nuclei are counterstained with DAPI. Scale bars = 20 µm. ( d ) Quantification of the capsule thickness measured from the SCS as depicted in c. Each datapoint represents the average of 3 measurements per LN. Mean±SEM are connected with a line. Data pooled from 5 LNs derived from 3 mice per timepoint. ( e ) Overview of micro-pipette assay for capsule stiffness measurements. Capsules of explanted popliteal LNs are labeled by brief incubation with Alexa Fluor 647-conjugated anti-ERTR7 antibody, upon which a small diameter glass pipette is placed on the capsule and a defined negative pressure applied. The ECM ‘tongue’ entering the pipette is subsequently measured. The local effective Young’s Modulus of the capsule is derived using Laplace’s law. Scale bar = 50 µm. ( f ) Quantification of measurements as described in e for homeostatic (D0) and inflamed (D2, D4, D8 and D14) LNs. Each datapoint represents the average of 3 measurements per LN. Mean±SEM are connected with a line. Data pooled from 5 LNs derived from 3 mice per timepoint. ( g ) Quantification of the Capsule swelling resistance as measured by the Passive Tension, given by multiplying the capsule thickness and Young’s modulus from d and f , respectively. Each datapoint represents a single LN. Mean±SEM are connected with a line. ( h ) Schematic of the mechanical dynamics of the swelling LN. For statistical analysis see Supplementary Information, table 1. ns, not significant. **P<0.01, ***P<0.001.

Article Snippet: The following primary antibodies were used: α-CD3ε AF488 (17A2)(eBioscience), α-B220-Biotin (RA3-6B2) (eBioscience), α-Collagen IV-Biotin (Abcam), α-CCL21-Biotin (R&D Systems), α-PDPN-Biotin (8.1.1) (eBioscience), α- PDGFR-β (R&D Systems), α-YAP/TAZ (D24E4) (Cell Signal), α-cleaved Caspase 3-AF647 (Asp175) (Cell Signal), α-Ki67-APC (SolA15) (eBioscience), α-ICAM-1 (YN1/1.7.4) (BioXCell), α- VCAM-1 (Phe25-Glu698) (R&D Systems), α-Fibroblast Marker-AF647 (ERTR7) (Santacruz Biotech). α-PNAd (MECA-79) was derived from a concentrated hybridoma supernatant (kind gift from Christine Moussion).

Techniques: Labeling, Capsules, Staining, Derivative Assay, Transferring, Incubation