Journal: Nature Communications

Article Title: Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa

doi: 10.1038/s41467-018-06448-y

Figure Lengend Snippet: Characterisation of RP11 - RPE cells revealed polarity and functional defects. a Schematic of RPE differentiation timeline; b Bright-field images of iPSC-derived RPE: representative examples from at least ten independent experiments, scale bar 100 μm; c Immunostaining for basolateral markers BEST1 and Na + /K + -ATPase: representative images from three independent experiments, scale bar 50 μm; d Correct basolateral distribution of collagen IV (C-IV) and apical MERTK in unaffected control (WT3) but not RP11 RPE cells: representative images from three independent experiments, scale bar 50 μm; e , f ELISA assays for apical and basal secretion of PEDF and VEGF, respectively, in control and RP11 - RPE cells; g Trans-epithelial resistance measurements revealed a significant difference between patient and RP11 - RPE cells; h Reduced phagocytic capacity in RP11 - RPE cells. Statistical significance is calculated for MFI (mean fluorescence intensity) values. e – h Data shown as mean ± SEM, n = 3. Statistical significance of pairwise comparisons is indicated by n.s.: not significant; *** p < 0.001; **** p < 0.0001 (Student’s paired t test). b – h Data obtained from RPE at week 21 of differentiation

Article Snippet: Cells were treated with primary antibodies anti-bestrophin (Abcam, ab2182, 1:300), anti-sodium potassium ATPase (Alexa Fluor® 488 conjugate) (Abcam, ab197713, 1:50), pericentrin (Abcam, ab28144, 1:500), MERTK (Bethyl, A300-222A, 1:200), ARL13B (Proteintech, 17711-1-AP, 1:500), collagen IV (Abcam, ab6586, 1:200), PRPF31 (Abnova, PAB7154, 1:500) and SNRPB monoclonal antibody (Y12) (ThermoFisher, MA5-13449, 1:500), overnight at 4 °C, and with secondary antibodies anti-rabbit FITC (Sigma, T9887, 1:500) or anti-mouse FITC (Jackson Immuno Research, 715-095-151, 1:500) and anti-mouse Cy3 (Jackson Immuno Research, 115-165-003, 1:500) or anti-rabbit Cy3 (Jackson Immuno Research, 111-165-003, 1:500) diluted in PBS for 1 h at room temperature.

Techniques: Functional Assay, Derivative Assay, Immunostaining, Control, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: Nature Communications

Article Title: Mouse pulmonary interstitial macrophages mediate the pro-tumorigenic effects of IL-9

doi: 10.1038/s41467-022-31596-7

Figure Lengend Snippet: a – d LLC cells were directly injected to the lung. Tumor appearance ( a ) and tumor incidence ( b ) ( n = 2 experiments) were assessed after resection. Tumor weight ( c ) ( n = 8 mice for WT group, n = 10 mice for Il9r −/− group) was calculated from two independent experiments. d Lung cells from tumor bearing mice and cells from tumor foci were isolated for flow analysis ( n = 5 mice for lung Mac group, n = 6 mice for TAM group). TAM: tumor associated macrophage. e Venn graph showing the overlap genes from human TAMs and mouse IMs. f – h WT (CD45.1) and Il9r −/− mice (CD45.2) bone marrow cells were mixed in 1:1 ratio and transferred to lethally irradiated recipient mice (CD45.1 + CD45.2 + mice). After reconstitution, chimeric mice were intravenously injected with B16 tumor cells, donor lung macrophages were analyzed by flow cytometry ( n = 9 mice). i – k Il9r −/− mice were injected with B16 tumor on day 0. Fluorescent bead-labeled monocytes were transferred to recipient mice on day 10 ( i ). Tumor growth ( j ) and lung macrophages ( k ) were analyzed on day 18, dot plots were gated on MerTK + CD64 + SiglecF − live cells ( n = 4 mice per group). Data are the mean ± SEM. Unpaired two-tailed Student t -test was used for comparison in c , d and j . Two-way ANOVA with Sidak’s multiple comparisons was used for comparisons in h .

Article Snippet: The tumor cell line was starved in FBS-free media for 24 h. Total macrophage was isolated from d20 tumor bearing mice and isolated by using anti-Mertk-biotin antibody (Miltenyi Biotec).

Techniques: Injection, Isolation, Irradiation, Flow Cytometry, Labeling, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Mouse pulmonary interstitial macrophages mediate the pro-tumorigenic effects of IL-9

doi: 10.1038/s41467-022-31596-7

Figure Lengend Snippet: a Kaplan–Meier plots showing differences in survival among lung cancer patients ( n = 982 donors) by using data and online tools described in Methods for ARG1 and IL6 . b Comparison of gene expression in normal lung tissue and metastatic lung tissues by using data and online tool described in the method. The bars represent the proportions of metastatic tumor samples that show higher expression of the selected gene compared to normal samples at each of the quantile cutoff values (minimum, 1st quartile, median, 3rd quartile, maximum). c IL9R and IL6 gene expression were analyzed in cells from normal lung tissue and cells in the lung tumor ( n = 8 donors for non-tumor group and n = 9 donors for lung cancer group in left panel, n = 6 donors for non-tumor group and n = 4 donors for lung cancer group in right panel). d Serum IL-9 level, IL-6 level and arginase activity were analyzed from healthy donors and lung cancer patient samples. ( n = 5 donors for left panel, n = 7 donors for non-tumor group and n = 4 donors for lung cancer group in middle panel, n = 7 donors for non-tumor group and n = 5 donors for lung cancer group in right panel). e Dot plot showing gene expression in macrophages between normal lung tissue and lung tumor. f , g Immunohistochemistry staining of CD68 and IL-9R. Protein expression quantification was performed by using Image J software ( n = 7 donors), Scale bar = 100 µm. h – l WT mice were intravenously injected with B16 tumor cell line, 7 days after tumor inoculation, tumor bearing mice were intravenously injected with nanoparticle-siRNA complexes every 72 h. Scr/ Il9r -siRNA was conjugated with Alexa Flour 555. Nanoparticles were tagged with SIRPα peptide ( h ). i Lung tumor growth were analyzed on day 21 ( n = 6 mice for Scr-siRNA group, n = 7 mice for Il9r -siRNA group). j IL-9R expression in siRNA + (Alexa Flour 555) lung macrophages were analyzed by flow ( n = 6 mice for Scr-siRNA group, n = 7 mice for Il9r -siRNA group). k Arg1 production from siRNA + (Alexa Flour 555) macrophages were analyzed by flow ( n = 6 mice for Scr-siRNA group, n = 7 mice for Il9r -siRNA group). l siRNA + (Alexa Flour 555) lung macrophages were sorted by gating on Alexa Flour 555 + MerTK + CD64 + live cells. Gene expression was analyzed ( n = 6 mice for groups in left panel, n = 6 mice for Scr-siRNA group and n = 7 mice for Il9r -siRNA group in right panel). Data are the mean ± SEM. Unpaired two-tailed Student t -test was used for comparison.

Article Snippet: The tumor cell line was starved in FBS-free media for 24 h. Total macrophage was isolated from d20 tumor bearing mice and isolated by using anti-Mertk-biotin antibody (Miltenyi Biotec).

Techniques: Comparison, Gene Expression, Expressing, Activity Assay, Immunohistochemistry, Staining, Software, Injection, Two Tailed Test