Journal: bioRxiv

Article Title: GATA1-defective immune-megakaryocytes as possible drivers of idiopathic pulmonary fibrosis

doi: 10.1101/2023.06.20.542249

Figure Lengend Snippet: (A) First line: Double IF staining with anti-GATA1 (green) and CD42b (red, as a marker of MK) of lung sections from one IPF and one representative no-IPF patient, as indicated. Nuclei are counterstained with DAPI. The inserts show representative MK at high power fields. The signal captured in the individual channels are presented in Figure S1 . Second and third lines: Hematoxilin&Eosin (H&E) and reticulin staining of the corresponding consecutive sections confirming that the lung from the IPF patient has an overall abnormal architecture with fibrosis. Magnification 400x in the panels on the top and 200x in the middle and bottom panels. (B) Frequency of CD42b pos MK per area (0.488mm ) and percentage of GATA1 pos MK (over the total number of CD42b pos cells) observed in the lungs from IPF and no-IPF patients, as indicated. Data are presented as Mean(±SD) and as value in individual subjects (each dot represents a single subject). The data are analyzed by One-way ANOVA and significant p values are indicated within the panels. C) Confocal microscopy analyses for markers of immune (CD41, green/LSP1, red), HSC-supporting (CD61 green/MYLK4 red) and Plt-poised (CD42b red/ARNTL green) MK in the lung from a representative no-IPF and a IPF patients, as indicated. Signals are presented in the individual channel and merged mode. The signals in the green and red channel are presented individually and in combination. Original Magnification 400x. D-F) Frequency of total CD41pos and CD41pos/LPS1pos (immune-MK. D), CD61pos and CD61pos/MYLKpos (HSC-supporting MK, E) and total CD42b pos and CD42b pos /ARNTL pos (Plt-MK, F) MK detected in the lungs from three representative No-IPF and three IPF patients, as indicated. Data are presented as Mean(±SD) and as value in individual subjects (each dot represents a single subject). The data are analyzed by One-way ANOVA and significant p values are indicated within the panels.

Article Snippet: Another one with monoclonal rabbit anti mouse (Abcam cat. ab133506) and the last one incubated with antibodies against CD61 ((B-7) mouse anti mouse, Santacruz Biotechnology INC., sc-46655) conjugated with Alexa 488, and MYLK (polyclonal rabbit anti-mouse, Proteintech, 24309-1-AP); and the last one incubated with antibodies against CD42b (mouse monoclonal, Santacruz Biotechnology INC, sc-271171) and ARNTL (rabbit anti mouse/human NB100-2288, Bio-Techne), for 2 hours at room temperature.

Techniques: Staining, Marker, Confocal Microscopy

Journal: bioRxiv

Article Title: GATA1-defective immune-megakaryocytes as possible drivers of idiopathic pulmonary fibrosis

doi: 10.1101/2023.06.20.542249

Figure Lengend Snippet: (A) IHC with the CD42b antibody of lung sections from WT (wild-type) and Gata1 low littermates, as well as from P-sel null and P-sel null Gata1 low mice used as additional controls, as indicated. The panels present representative immature (MK) and mature Plt-forming (PfMK) MK found in the different groups. Scale bar 50µm. Magnification See Figure S2A for further detail. ( B ) Confocal microscopy analysis for CD42b (red) and GATA1 (green) of the MK in the lung from representative Gata1 low and WT littermates. Original Magnification 400x. The signal captured in the individual channels are presented in Figure S2B . The frequency of CD42b pos MK per area (0.488mm 2 ) and the percentage of GATA1 pos MK (over the total number of CD42b pos cells) observed in the lungs from multiple mice is presented as Mean(±SD) and as value in individual mice (each dot represents a single mouse). The data are analyzed by One-way ANOVA and significant p values are indicated within the panels. C) Frequency of CD42b pos cells with a DNA content 2N, 4N and >8N in lungs from Gata1 low mice and WT littermates (both 15-months old) determined by Flow cytometry, as indicated. Data are presented as Mean (±SD) and as value in individual mice (each symbol a single mouse) and were analyzed by ANOVA. Statistically significant p values are indicated within the panels. D) Confocal microscopy analyses for markers of immune (CD41, green/LSP1, red), HSC-supporting MK (CD61 green/MYLK4 red) and Plt-poised (CD42b red/ARNTL green) MK in the lung from representative WT and Gata1 low mice at 2-3 (young) and 15-months (old) of age, as indicated. Original Magnification 40x. The signal captured in the individual channels are presented in Figure S4 . E-G) Frequency of total CD41pos and CD41pos/LPS1pos (immune-MK, E), CD61pos and CD61pos/MYLKpos (HSC-supporting MK, F) and total CD42b pos and CD42b pos /ARNTL pos (Plt-MK, G) MK detected in the lungs from multiple young and old WT and Gata1 low mice. Data are presented as Mean(±SD) and as value in individual subjects (each dot represents a single mouse). The data are analyzed by One-way ANOVA and significant p values are indicated within the panels. H) Principal component analysis (PCA ) of the RNAseq data obtained with MK purified from the BM and lungs of individual 15-months old WT and Gata1 low littermates (each dot an individual mouse) as indicated. The strong separation and clustering of samples demonstrates the high reproducibility of the data from the same group. The differences between tissue of provenience (BM or lung) account for approximately 79% of the variance in the data. I) Log ratio vs mean average (MA) plot of up-and down-regulated genes in MK from the lungs of Gata1 low mice lungs with respect to the MK from WT lungs. F) Heatmaps for the level of expression of representative genes which characterize the signature of immune (blue), HSC-supporting (green) and Plt-poised (red) MK described by . Statistically significant (p<0.05) differences are indicated by asterisks. Interestingly, in spite of the fact that the lungs from Gata1 low mice are enriched for immune-MK, they have an overall defective immune-signature.

Article Snippet: Another one with monoclonal rabbit anti mouse (Abcam cat. ab133506) and the last one incubated with antibodies against CD61 ((B-7) mouse anti mouse, Santacruz Biotechnology INC., sc-46655) conjugated with Alexa 488, and MYLK (polyclonal rabbit anti-mouse, Proteintech, 24309-1-AP); and the last one incubated with antibodies against CD42b (mouse monoclonal, Santacruz Biotechnology INC, sc-271171) and ARNTL (rabbit anti mouse/human NB100-2288, Bio-Techne), for 2 hours at room temperature.

Techniques: Confocal Microscopy, Flow Cytometry, Purification, Expressing

Journal: bioRxiv

Article Title: A role for SHARPIN in platelet linear protein ubiquitination and function

doi: 10.1101/2021.01.13.426403

Figure Lengend Snippet: A – D) Washed wild-type and cpdm/cpdm platelets, +/- indomethacin (10 µM) and ARC66096 (1 µM), were stimulated for 10 min with thrombin (n = 11 mean ± s.e.) or CRP-XL (n = 11 mean ± s.e.) in the presence of PE-conjugated JON/A antibody directed against the high affinity form of integrin αIIbβ3 (A, C) and FITC-conjugated (Wug.E9) antibody (B, D) against the α-granule marker CD62P (P-selectin) before fixation and analysis. E & F) ATP release from washed wild-type and cpdm/cpdm platelets was monitored for 5 minutes in response to indicated concentrations of thrombin (n = 6 mean ± s.e.) or CRP-XL (n = 4 mean ± s.e.). Data expressed as % of ATP standard. Results were analysed by 2-way ANOVA with Bonferroni’s post-test. P values reported for genotype variable. α = 0.05. Star notation indicates individual post-test comparisons by genotype. **: p<0.01, ***: p<0.001.

Article Snippet: FITC-conjugated integrin β1 (CD29, HM beta 1-1, #MCA2298) and FITC-conjugated integrin β3 (CD61, #MCA2299) were from Bio-Rad (Oxford, UK).

Techniques: Marker

Journal: bioRxiv

Article Title: A role for SHARPIN in platelet linear protein ubiquitination and function

doi: 10.1101/2021.01.13.426403

Figure Lengend Snippet: Washed platelets were stimulated with the indicated agonists for 10 min in the presence of of PE-conjugated JON/A antibody directed against the high affinity form of integrin αIIbβ3 (A, C) and FITC-conjugated (Wug.E9) antibody (B, D) against the α-granule marker CD62P (P-selectin) before fixation and analysis. A & B) Integrin activation and α-granule secretion induced by ADP (10 µM) plus U46619 (30 µM) (n=7 mean ± s.e.). C) ATP release from washed wild-type and cpdm/cpdm platelets was monitored for 5 minutes in response to ADP (10µM)+U46619 (30µM) (n = 6 mean ± s.e.). Results in A - C were analysed by 2-way ANOVA with Bonferroni’s post-test. P values reported for genotype variable. α = 0.05. Star notation indicates individual post-test comparisons by genotype. Results in C were analysed by Wilcoxon test *: p<0.05, **: p<0.01, ***: p<0.001.

Article Snippet: FITC-conjugated integrin β1 (CD29, HM beta 1-1, #MCA2298) and FITC-conjugated integrin β3 (CD61, #MCA2299) were from Bio-Rad (Oxford, UK).

Techniques: Marker, Activation Assay