Journal: Nature

Article Title: Mechanics of a multilayer epithelium instruct tumour architecture and function

doi: 10.1038/s41586-020-2695-9

Figure Lengend Snippet: a, Top GO terms from E15.5 basal progenitor cell genes that are upregulated by a factor of two or more in SmoM2 versus HRasG12V embryos; n = 3 independent biological replicates. Statistical significance was determined by unpaired, two-tailed t-test and P values were corrected using the Benjamini–Hochberg method. ECM genes encoding known basement-membrane components are in blue. b, Modelling the biophysical properties of the basement membrane (BM). The membrane stretching modulus (KS), bending modulus (B) and assembly rate (1/τa) are incorporated into the effective energy term (wBM). Assembly of basement membranes is proportional to the rate constant τa, and cell growth is proportional to the rate constant τg (lj and li are the lengths corresponding to cell edge j and vertex i; B is the bending modulus; C refers to curvature; see Supplementary Notes 1, 2 for details). c, Tissue shapes simulated by varying BM stiffnesses (proportional to B) and BM assembly rates. d, AFM measurements made on the BM-exposed dermal surface of EDTA-separated skin. Force-indentation curves are generated, from which the Young’s modulus or stiffness of BM (EBM) is calculated (see Methods). e, Left, diagram showing BM locations for AFM and transmission electron microscopy (TEM). Bottom left, TEM image showing electron-dense hemidesmosomes (HD) at the epidermal–ermal interface. Right, EBM and ultrastructural measurements of oncogenic lesions. AFM: SmoM2 (P), n = 13; SmoM2 (D), n = 11; HRasG12V, n = 12. TEM: SmoM2 (P), n = 14; SmoM2 (D), n = 12; HRasG12V, n = 14. One-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. f, Effects of varying BM stiffness on tumour architecture. S values (median, n = 5 independent simulations) from in silico modelling are plotted as a black line and overlaid with genetic data from Extended Data Fig. 5g (mean ± s.d.). EBM values are indicated by red dotted lines. g, Immunofluorescence of laminin LN-β1, a component of nascent BMs, at the leading edge of SmoM2 and HRasG12V lesions, compared with LN-332, a component of mature BMs, as shown by the intensity heatmap. Arrows mark epidermal BM. Scale bars, 50 μm. g, BM assembly rates measured by incorporation of fluorescent laminin into native BMs over time (SmoM2,n = 6 explants; HRasG12V, n = 5 explants; two-tailed Mann–Whitney U-test). i, Quantifications of lesion S values following Lamb1 knockdown. SmoM2: shScr, n = 14; shLamb1, n = 14. HRasG12V: shScr, n = 13; shLamb1, n = 13. Two-tailed unpaired t-test; four embryos, two litters each. j, Comparison of experimental data and vertex model simulations for conditions in f, i. S values are plotted, and example simulation snapshots for the indicated values of B and 1/τa are shown. Qualitatively distinct shape regimes include ‘budding’, ‘folding’ and ‘pearling’. All bar graphs show means + s.d.

Article Snippet: For actomyosin manipulation studies, 50 μM of the ROCK inhibitor Y-27632 or vehicle control (dimethylsulfoxide, DMSO) was added and samples were harvested after 24 h. For assays of basement-membrane assembly rate, laminin isolated from Engelbreth–Holm–Swarm (EHS) tumours (Millipore) was labelled with the AlexaFluor647 antibody labelling kit ( {"type":"entrez-protein","attrs":{"text":"A20186","term_id":"90011","term_text":"pir||A20186"}} A20186 , ThermoFisher) according to the manufacturer’s instructions, or rhodamine-labelled laminin was purchased (LMN01-A, Cytoskeleton Inc).

Techniques: Two Tailed Test, Membrane, Generated, Transmission Assay, Electron Microscopy, In Silico, Immunofluorescence, MANN-WHITNEY, Knockdown, Comparison

Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Subcellular colocalization of 24K and miRNA biogenesis proteins. ( A ) (i) Colocalization of 24K:mRFP with CFP:AtDCL1 in nuclear aggregates. ( B ) (i) Colocalization of 24K:mRFP with AtHYL1:YFP in nuclear aggregates. ( C ) (i) Colocalization of 24K:mRFP with AtSE:YFP in nuclear aggregates. (ii) The mRFP control does not colocalize in nuclear aggregates with any of the tested proteins. ( D ) Western blot analyses of extracts from N. benthamiana leaves in colocalization assays at 3 dpa. Anti-RFP (@RFP) or anti-GFP (@GFP) monoclonal antibodies were used for 24K or biogenesis proteins, respectively. Coomassie blue stain is shown as loading control. The numbers below correspond to normalized band density. Ratios between AtDCL and AtDCL1/24K (240 kDa); AtHYL1 and AtHYL1/24K (75 kDa); AtSE and AtSE/24K (110 kDa) are shown under the lines. Scale bar: 10 µm.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: Control, Western Blot, Bioprocessing, Staining

Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Interaction between 24K protein and miRNA biogenesis proteins. ( A ) In vivo analysis of BiFC assays in epidermal cells of N. benthamiana plants. The merge panels show positive interaction between N-mCitrine:24K and C-mCitrine:AtHYL1 (i) and N-mCitrine:24K and C-mCitrine:AtSE (ii) and no interaction between N-mCitrine:24K with C-mCitrine:AtDCL1 (iii). (iv). Negative control: N-mCitrine:24K + C-mCitrine (empty vector). Chloroplasts were marked in blue in the case of the negative interactions. In all cases, Fib:mRFP was used as an expression control of infiltrated samples. Scale bar: 10 µm. ( B ) Co-IP assay between 24K:mRFP and AtHYL1. INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins. (−) corresponds to the negative control using beads without @HYL1 antibody. (+) corresponds to the immunoprecipitated samples with @HYL1 attached to the beads. The anti-RFP (@RFP) and anti-HYL1 (@HYL1) monoclonal antibodies were used in each case to develop the blots. The presence of both proteins in the IP fraction indicates a positive interaction. ( C ) Co-IP assay between the 24K:mRFP and AtSE:YFP protein. The INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins with @GFP attached to the beads. Free mRFP was used as a negative control. The anti-RFP (@RFP) and anti-GFP (@GFP) antibodies were used in each case to develop the blots. The presence of both proteins in IP fraction indicates a positive interaction.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: In Vivo, Negative Control, Plasmid Preparation, Expressing, Control, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Bioprocessing