Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization directs splenic DC progenitors towards CD8α+ DC development. (A) Intracellular β-catenin expression in naïve Ex3fl/fl and Ex3DC−/− splenic CD11c+ cells. (B) Intracellular β-catenin levels in splenic CD4+ T cells isolated from Ex3fl/fl and Ex3DC−/− mice. The data show results from an individual mouse that is representative of of at least 3 experiments with 3–5 mice per group. (C) Western blot analysis of β-catenin in bone marrow-derived DC from Ex3fl/fl and Ex3DC−/− mice following cytoplasmic (C) and nuclear (N) fractionation. Antibodies against PARP and Rab5 were used for nuclear and cytoplasmic loading controls, respectively. The data are from 1 independent trial. (D and E) Comparison pre-cDC populations from the (D) bone marrow and (E) spleen of Ex3fl/fl and Ex3DC−/− mice by flow cytometry. Numbers in representative plots represent percentages of relevant populations within the indicated gate. Bar graphs show mean percentages plus standard error (S.E.) of relevant populations. The data represent the combination of 2 independent experiments (n=10 mice per group). (F) Levels of splenic pre-CD8α+ DC, defined as CD11c+CD8α−B220−CD24+, in Ex3fl/fl and Ex3DC−/− mice by flow cytometry. The data are representative of 3 independent experiments, each involving 4–5 mice per group. **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Expressing, Isolation, Western Blot, Derivative Assay, Fractionation, Comparison, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization expands splenic CD8α+ and plasmacytoid DC populations. (A–G) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− splenocytes by flow cytometry. (A and B) Percentage and total number of CD11c+ cells in Ex3fl/fl and Ex3DC−/− spleens. (C–E) Percentage and total number of (C and D) CD8α+ DC and (C and E) CD11b+ DC in naïve Ex3fl/fl and Ex3DC−/− spleens. The data are representative of at least 3 independent experiments (n=3–5 mice per group). (F and G) Percentage and total number of B220+PDCA-1+ plasmacytoid DC in naïve Ex3fl/fl and Ex3DC−/− spleens. (H and I) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− lung tissue by flow cytometry. (H) Plots from representative mice and (I) percentage of CD103+CD11b− lung DC for multiple mice are shown. (J–L) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− intestinal lamina propria by flow cytometry. (J) Plots from representative mice and percentages of (K) CD103+CD11b− and (L) CD103+CD11b+ intestinal DC for multiple mice are shown. Dots in relevant graphs represent results from individual mice. Bar graphs display means and standard errors of individual mice. The data are representative of at least 2 independent experiments (n=3–5 mice per group). *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin signaling controls Irf8 expression. (A) Semi-quantitative PCR analysis of Nfil3, Batf3, Id2, and Irf8 mRNA in CD11c+ splenocytes magnetically purified from naïve Ex3fl/fl and Ex3DC−/− mice. mRNA levels were normalized to GAPDH. The data are representative of 2 independent experiments (n=2–3 mice per group) (B) Representative flow cytometric plots of IRF8 expression by Ex3fl/fl and Ex3DC−/− CD8α− and CD8α+ splenic DC. (C) MFI of IRF8 within Ex3fl/fl and Ex3DC−/− CD8α− and CD8α+ DC and (D) the percent of CD8α+ DC expressing IRF8 are shown. Dots represent results from individual mice. The data are the combined results of 2 experiments, and the experiment was independently performed at least 3 times (n=4–5 mice per group). (E) Representative FACS plot of IRF4 expression and IRF4 MFI in Ex3fl/fl and Ex3DC−/− CD11c+ splenocytes. The data are representative of 3 independent experiments (n=4 mice per group). (F) Chromatin immunoprecipitation of naïve Ex3DC−/− Flt3L DC cultures with control IgG or β-catenin antibody followed by quantitative PCR to determine Irf8 promoter occupancy. DNA levels were normalized to 1% input chromatin. The data are representative of 2 independent experiments. (G) Quantitative PCR analysis of Axin2 and Irf8 gene expression in BMDC following 5 hr culture with DMSO or ICG-001. Fold change is relative to DMSO control. The data are from one independent trial. (H and I) Intracellular expression of IRF8 and β-catenin following ICG-001 treatment of BMDC (H) or MutuDC1940 cells (I). The data are representative of 2 (MutuDC1940 cells) and 4 (BMDC) independent experiments with 3 replicates per treatment per experiment. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Purification, Chromatin Immunoprecipitation, Control, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization enhances IL-12 production by CD8α+ DC. (A) IL-12p40 production by naïve Ex3fl/fl and Ex3DC−/− splenocytes stimulated in vitro with LPS, STAg, or media control measured by ELISA. (B) IL-12p40 production by splenic CD11c+ DC magnetically purified from Ex3fl/fl and Ex3DC−/− mice stimulated in vitro with LPS, STAg, or media control measured by ELISA. (C) IL-12p40 production by CD8α+ and CD8α− DC DC purified from naïve Ex3fl/fl and Ex3DC−/− splenocytes following in vitro stimulation with media or STAg for 48 hr measured by ELISA. (D) IL-12p40 secretion by Ex3fl/fl splenocytes pre-treated with ICG-001 for 5 hr and then stimulated overnight with LPS or STAg measured by ELISA. (E) IL-12p40 production by splenocytes (106) from Ex3DC−/− mice cultured for 5 hr with 5 μM ICG-001 or DMSO and then stimulated with media, LPS (100 ng/ml), or STAg (25 μg/ml) overnight. (F) IL-12p40 production by MutuDC1940 cells (105) pre-treated with 20 μM ICG-001 or DMSO for 2 hr and then stimulated with media or STAg (25 μg/ml) overnight. The data are representative of at least 3 (A, F) and 2 (B–E) independent experiments, each involving 3–5 mice per group, except (C), which used pooled samples from 3 mice per experiment. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: In Vitro, Control, Enzyme-linked Immunosorbent Assay, Purification, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: Constitutive DC β-catenin signaling increases the proinflammatory cytokine response to Toxoplasma. (A) Survival of Ex3fl/fl and Ex3DC−/− mice following i.p. infection with Toxoplasma Type II strain ME49 (25 cysts) (n=4–6 mice per group). The data are representative of at least 3 experiments. (B) Quantitative PCR amplification of parasite (B1 gene) and host DNA (ASL gene) isolated from Ex3fl/fl and Ex3DC−/− spleens 9 days post-infection. Parasite load is displayed as a ratio of parasite genomes to host genomes (n=3–4 mice per group). (C) IL-12p40 production by CD11c+ DC magnetically separated from Day-6 post-infection Ex3fl/fl and Ex3DC−/− splenocytes and cultured overnight without additional stimulation (n=3 mice per group). (D) IL-12p70 production by bulk splenocytes from Day-6 post-infection Ex3fl/fl and Ex3DC−/− mice (n=3 mice per group). The data are representative of 2 independent experiments. (E) IL-12p40 and IFN-γ production by splenocytes from Day-10 post-infection Ex3fl/fl and Ex3DC−/− mice cultured for 72 hr without additional stimulation (n=3–5 mice per group). The data are representative of 3 independent experiments. (F) IL-12p40, IFN-γ, and TNF-α levels in serum collected from Day-9 post-infection Ex3fl/fl and Ex3DC−/− mice (n=3–5 mice per group). The data are representative of 2 independent experiments. The means and S.E. of individual mice are shown. *, p<0.05; **, p<0.01.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Infection, Real-time Polymerase Chain Reaction, Amplification, Isolation, Cell Culture

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A, Schematic experiment setting. B, Increase in total number of cells of known (KIF-P1 and -P2, SYT-T1, -T2, -P1, -P2) and newly identified (KIF-sc1 and -sc2) TCRs upon antigen-specific stimulation and CD137-enrichment with dominance of KIF-P1 and -P2 harboring high precursor frequency. The two newly identified KIF2C-TCRs were selected based on fold change of TCR frequency and highest absolute frequency in the stimulated sample. C, Assessment of antigen-specific IFN-γ-secretion for the two newly identified TCRs KIF-sc1 and -sc2 in comparison to the known TCR KIF-P2. Cytokine secretion was measured by IFN-γ-ELISA upon 24h of co-culture of TCR-tg T cells from one representative donor with Mel15-LCLs transgenic for the mutated KIF2C P13L minigene (mut mg) and the wildtype KIF2C minigene (wt mg) as well as pulsed for 2h at 37°C with the mutated and wildtype peptide (mut pep and wt pep). An irrelevant peptide (irr peptide), target cells (LCL only) or T cells alone (T cell only) served as negative controls. D, Frequency of KIF-sc1 and -sc2 in relation to the previously identified TCR-sequences identified by deep sequencing of the TCR-β-chain in intestinal (MInt) and lung metastases (MLung) as well as corresponding non-malignant draining lymph nodes (MInt-LN1, MInt-LN2 and MLung-LN) of patient Mel15. Non-td: non-transduced.

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Transgenic Assay, Sequencing

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A-C, Mel15 LCLs were pulsed with titrated peptide concentrations (2h, 37°C) and co- incubated with TCR-tg T cells with subsequent ELISA-based assessment of IFN-γ-secretion within 24h of co-culture (A). The cellular activation level was determined after 24h by FACS staining of the extracellular level of CD137 (B) and PD-1 (C) expression (reflected by geometric mean of all CD3 + CD8 + /TCRmu + cells). The mean for ELISA data is depicted for technical triplicates of one donor; triplicates from the same donor have been pooled prior to EC FACS- staining. E:T = 1:1 (15.000 tg T cells:15.000 tumor cells). D-G, EC FACS staining at different timepoints after co-culture setup displays temporal dynamics of T cell activation marker CD137 (D, E) and inhibitory receptor LAG-3 (F, G) for TCR-tg T cells upon co-culture with JJN3-B27 peptide-pulsed target cells. A weak (0.01 µM for peptide pulsing; D, F) versus a strong (1 µM for peptide pulsing; E, G) stimulus were compared. E:T = 1:1 (10.000 tg T cells:10.000 tumor cells). H, Annexin-V/PI-staining was employed for detection of activation induced cell death (AICD) after 20h of co-culture upon strong stimulation with 1µM mut-peptide pulsed Mel15 LCLs (early apoptotic = AnnexinV + PI - , late apoptotic = AnnexinV + PI + ). E:T = 1:1 (30.000 tg T cells:30.000 tumor cells). I, Representative FACS plot of a healthy donor of CTV-analysis for all TCRmu + cells depicted after 4 days of co-culture with 1µM mut-peptide pulsed Mel15 LCLs (colors were chosen according to Figure B-E; representative wt mg-control depicted in grey). E:T = 1:1 (30.000 tg T cells:30.000 tumor cells). For all co-cultures in D-I technical triplicates per donor were pooled prior to staining; the mean and SD for biological replicates from three different human donors are shown.

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Activation Assay, Staining, Expressing, Marker, Control

Journal: bioRxiv

Article Title: High-resolution profiling of neoantigen-specific T cell receptor activation signatures links moderate stimulation patterns to resilience and sustained tumor control

doi: 10.1101/2022.09.23.508529

Figure Lengend Snippet: A, Schematic experiment setting of xenograft neoTCR-tumor rejection experiment with newly transduced T cells. B, Tumor growth kinetics are displayed as tumor area (in cm 2 ) for mut mg-U698M-tumor- bearing NSG-mice comparing neoTCR-tg T cells to the irrelevant TCR 2.5D6 until day 20. Mean values and SDs for each group of mice display rejection dynamics (n=6). Parts of this dataset were already published before . In this renewed version, tumor rejection kinetics of KIF-sc1 and -sc2 analyzed in the same experiment are included. C, Kaplan-Meier-survival curve is displayed until day 20 for tumor-bearing mice injected with different neoTCR-tg T cells. D, schematic experiment setting of xenograft neoTCR-TIL-rechallenge experiment. E-G, Ex vivo restimulation of T cells derived from TIL products on day 21 after tumor explant (TIL-P) compared to newly transduced (NEW) TCR-tg T cells from the same human donor stained for CD137 (EC; E), IFN-γ (IC; F) and GzmB (IC; G); expression was analyzed using geometric mean of all CD3 + CD8 + /TCRmu + cells after 18h of co-culture. Mut-mg and wt-mg U698M cells were used as target cells in E:T = 1:1 (50.000 tg T cells:50.000 tumor cells). Mean and SD are shown for three experimental replicates. Statistical significance is calculated with one-way ANOVA and Tukey’s multiple comparison test (*p≤0.05, ****p≤0.0001). H, Tumor growth kinetics are displayed as tumor area (in cm 2 ) for NSG-mice continuing the experiment until day 17 after second injection of in total 5x10 6 neoTCR-tg T cells (transduction rate of 55% equalized for all groups). For the TIL- P-groups, TIL-P from two mice per TCR (highest numbers of expanded neoTCR-tg T cells) were pooled. Mean values and SEMs for each group of mice display rejection dynamics (n=5 for experimental groups, n=3 for 2.5D6; due to achievement of humane endpoint criteria one 2.5D6- control mouse was sacrificed on day 13 and excluded from this graph). Statistical significance is calculated for the tumor area on day 17 with one-way ANOVA and Tukey’s multiple comparison test (***p≤0.001, ****p≤0.0001). I , Kaplan-Meier-survival curve is displayed for tumor-bearing mice injected with different TCR-tg T cells (n=5 for experimental groups, n=4 for 2.5D6 control). Survival of mice receiving TIL-P-KIF-P2 compared to TIL-P-KIF-sc1 was significantly prolonged (p=0.0019, Mantel-Cox test).

Article Snippet: Another 24h later, reactive T cells were separated using magnetic labelling and positive selection with the CD137 MicroBead Kit (Miltenyi).

Techniques: Injection, Ex Vivo, Derivative Assay, Staining, Expressing, Co-Culture Assay, Comparison, Transduction, Control

Journal: Nature

Article Title: H3K4me3 regulates RNA polymerase II promoter-proximal pause-release

doi: 10.1038/s41586-023-05780-8

Figure Lengend Snippet: ( a ) Genomic confirmation of the APEX-modified Rpb1 loci in DPY30–mAID and RBBP5–FKBP degron cells. ( b ) Sanger sequencing of the wild type and Flag-APEX2-RPB1 knock-ins. ( c ) Western blot showing the expression of APEX-modified RPB1 in DPY30–mAID and RBBP5–FKBP degron cells. ( d ) Representative brightfield images of mES cell colonies. ( e ) RT-qPCR analysis of the expression of pluripotency and differentiation genes in the knock-in cells. Data are from three biological replicates (n = 3) and are analysed using Two-way ANOVA and represented as mean ± s.d. ( f ) Titration of biotin phenol (BP). Cells stably expressing Flag-APEX2-RPB1 were pre-incubated for 30 min with the indicated concentrations of BP, followed by the addition of 1 mM H2O2 for 1 min. Cell lysates were probed with Streptavidin-HRP. Proximity biotinylation is optimal at a BP concentration of 4 mM. ( g ) Confirmation of the APEX2 functionality by protein biotinylation in the APEX2-engineered DPY30–mAID and RBBP5–FKBP degron cells. The discrete bands, denoted with asterisks, show APEX2-independent biotinylation by native enzymes. The Connexin-APEX2 overexpressed cell was severed as positive control for the APEX2 system. ( h ) SILAC-based chromatin proteomic strategy for mapping the neighbourhood interaction networks of APEX2-tagged RNAPII. ( i ) Principal component analysis (PCA) of SILAC signal in the RNAPII-APEX2 cells with or without agonist. ( j ) Distribution of SILAC ratio of RNAPII interactions quantified in the chromatin proteomic analyses. Mean log2 SILAC ratio is shown. In total, 1,901 proteins were identified in this experiment. The RNAPII-APEX2-bait (BP+H2O2) population has a right-shifted distribution compared with the no agonist negative control population, which indicates that the log2(SILAC) ratio allows us to distinguish bona fide RNAPII interactions from non-RNAPII interactions. ( k ) GO network showing significantly (q value < 0.001) enriched terms for positive RNAPII interaction neighbourhoods from RNAPII-APEX2 experiment. The most prominent pathways are indicated. Connecting lines show interaction of protein nodes. ( l ) KEGG enrichment and GO network showing significantly ( q value < 0.001) enriched terms for positive RNAPII interaction neighbourhoods from RNAPII-APEX2 experiment. ( m ) Principal component analysis (PCA) of SILAC signal in the RNAPII-APEX2 DPY30–mAID cells with or without Auxin treatment. Time trajectory is shown by the dashed arrow. ( n ) Venn diagram indicating overlap between up or downregulated targets in the indicated samples. ( o ) Heatmap representing relative protein abundance of DPY30 and selected targets in Auxin treated DPY30–mAID cells. n = 3 independently samples. ( p ) Scatterplot analysis of proteins identified by SILAC in RNAPII-APEX2 DPY30–mAID cells following Auxin treatment for 2 and 8 h. ( q ) Gene ontology-based functional classification of 228 downregulated proteins in RNAPII-APEX2 DPY30–mAID cells following Auxin treatment for 2 and 8 h. The dot size is proportional to the number of members in an enrichment set, and colour intensity reflects the p value. Significance based on clusterProfiler analysis with Benjamini-Hochberg-adjusted P values.

Article Snippet: Approximately 4 mg lysates from SILAC heavy or light cells were mixed 1:1 and incubated with 50 μl Streptavidin magnetic beads (Pierce, 88817) at 4 °C on a rotating wheel overnight.

Techniques: Modification, Sequencing, Western Blot, Expressing, Quantitative RT-PCR, Knock-In, Titration, Stable Transfection, Incubation, Concentration Assay, Positive Control, Multiplex sample analysis, Negative Control, Quantitative Proteomics, Functional Assay

Journal: Nature

Article Title: H3K4me3 regulates RNA polymerase II promoter-proximal pause-release

doi: 10.1038/s41586-023-05780-8

Figure Lengend Snippet: ( a ) Venn diagram indicating overlap of H3K4me3 interactors and RNAPII-APEX2 dependent interactors from ChIP-MS (chromatin proteomic profiling) data. ( b ) Relative enrichments of selected targets in various ChIP preparations based on ChIP-MS. ( c ) Validation of INTS11 interaction with H3K4me3 in RBBP5–FKBP degron cells at different times after dTAG-13 addition. Biotinylated proteins within lysates were enriched using Streptavidin-coated magnetic beads and analysed by Western blot. In parallel, sample in which H 2 O 2 was omitted was prepared as negative control. ( d ) Schematic representation of the dTAG INTS11 targeting strategy for the INTS11–FKBP degron mES cells. ( e ) Western blot showing the expression of INTS11 and INTS11–FKBP–HA, using antibodies recognizing INTS11 or the HA in parental and knock-in degron cells. The arrow indicates the specific HA-tagged INTS11–FKBP–HA protein. ( f ) RT-qPCR analysis showing the expression of selected pluripotency and differentiation genes in the parental and INTS11–FKBP knock-in cells. Data are from three biological replicates (n = 3) and are analysed using Two-way ANOVA and represented as mean ± s.d. ( g ) Growth curve analysis of parental and INTS11–FKBP E14 cells treated with or without dTAG-13. ( h ) INTS11 enrichment profiles and heat maps as determined by using the HA-tag in control (0 h) and Auxin-treated (2 h) DPY30–mAID; INTS11–FKBP degron cells. Genome-wide binding averages showed enrichments at the TSS regions (TSS ± 2 kb) of protein coding genes. TSS, transcription start site. Rows were sorted by decreasing ChIP–seq occupancy in the control (0 h) cells. ( i ) Correlations between TT chem -seq replicate experiments in INTS11–FKBP degron cells treated with or without dTAG-13 for the indicated times. ( j ) Average profiles for TT chem -seq for the upstream anti-sense RNAs of each annotated protein-coding gene in INTS11 degron cells. TSS, transcription start site. ( k ) RNAPII profiles of various subclasses of annotations in INTS11–FKBP degron cells with or without dTAG-13 treatment. TSS, transcription start site. mRNA, messenger RNA. snRNA, small nuclear RNA. ncRNA, non-coding RNA. eRNA, enhancer RNA.

Article Snippet: Approximately 4 mg lysates from SILAC heavy or light cells were mixed 1:1 and incubated with 50 μl Streptavidin magnetic beads (Pierce, 88817) at 4 °C on a rotating wheel overnight.

Techniques: Biomarker Discovery, Magnetic Beads, Western Blot, Negative Control, Expressing, Knock-In, Quantitative RT-PCR, Control, Genome Wide, Binding Assay, ChIP-sequencing

Journal: Clinical and Translational Medicine

Article Title: Tumor‐derived exosomal miR‐19b‐3p facilitates M2 macrophage polarization and exosomal LINC00273 secretion to promote lung adenocarcinoma metastasis via Hippo pathway

doi: 10.1002/ctm2.478

Figure Lengend Snippet: Exosomes from miR‐19b‐3p‐induced macrophages regulates Hippo signaling pathway by inducing LATS2 ubiquitination in LUAD cells. (A) A549 and H1975 cells were cultured with the CM from THP1 (Mφ) treated with NC mimics, miR‐19b‐3p mimics, miR‐19b‐3p mimics + DMSO, or miR‐19b‐3p mimics + GW4869. miR‐19b‐3p level in LUAD cell‐derived exosomes of each group was detected using qRT‐PCR. (B) A549 and H1975 cells were treated with THP1 (Mφ)/NC mimics‐Exo or THP1 (Mφ)/miR‐19b‐3p mimics‐Exo. miR‐19b‐3p level in LUAD cell‐derived exosomes of each group was detected using qRT‐PCR. (C) A series of qRT‐PCR analyses illustrated the fold changes of exosomal miR‐19b‐3p levels from A549 and H1975 cells treated with various small‐molecule inhibitors. DMSO treatment was regarded as a control. (D) Luciferase reporter assay detected YAP signaling in A549 and H1975 cells treated with THP1 (Mφ)/NC mimics‐Exo or THP1 (Mφ)/miR‐19b‐3p mimics‐Exo using 8xGTIIC‐luciferase YAP/TAZ reporters. (E) The effects of rising dose of THP1 (Mφ)/miR‐19b‐3p mimics‐Exo on the levels of p‐YAP, YAP, LATS2 and MST1 in LUAD cells were evaluated using western blot. (F) The effects of rising dose of THP1 (Mφ)/miR‐19b‐3p mimics‐Exo on the levels of nuclear or cytoplasmic YAP in LUAD cells were evaluated using western blot. (G) The effects of rising dose of THP1 (Mφ)/miR‐19b‐3p mimics‐Exo on the levels of YAP downstream genes in LUAD cells were evaluated using western blot. (H) CHX chase assay evaluated the stability of LATS2 protein in LUAD cells cultured with the exosomes of each group. Western blot was used to detect LATS2 expression in LUAD cells of each group after CHX treatment at different time points. (I) Ubiquitination assay evaluated the ubiquitination level of LATS2 protein in LUAD cells cultured with the exosomes of each group. THP1 (Mφ), macrophages induced from THP1 cells. THP1 (Mφ)/NC mimics‐Exo or THP1 (Mφ)/miR‐19b‐3p mimics‐Exo, exosomes derived from THP1 (Mφ) transfected with NC mimics or miR‐19b‐3p mimics. Data are presented as mean ± SD of at least three independent experiments. **p < 0.01

Article Snippet: To detect LATS2‐NEDD4 interaction, cell lysates were cocultured with anti‐NEDD4 (Proteintech) or IgG (Abcam) conjugated magnetic beads, and the eluted proteins underwent western blot with antibody against LATS2 or NEDD4.

Techniques: Cell Culture, Derivative Assay, Quantitative RT-PCR, Luciferase, Reporter Assay, Western Blot, Expressing, Ubiquitin Assay, Transfection

Journal: Clinical and Translational Medicine

Article Title: Tumor‐derived exosomal miR‐19b‐3p facilitates M2 macrophage polarization and exosomal LINC00273 secretion to promote lung adenocarcinoma metastasis via Hippo pathway

doi: 10.1002/ctm2.478

Figure Lengend Snippet: Macrophage‐transferred exosomal LINC00273 regulates Hippo signaling pathway by recruiting NEDD4 in LUAD cells. (A) Twenty‐four highly expressed lncRNAs (log 2 FC > 1) in lung cancer were searched out from BBCancer database. After the transfection of NC mimics or miR‐19b‐3p mimics, exosomal lncRNA levels from THP1 (Mφ) were detected, and 7 significantly up‐regulated lncRNAs by miR‐19b‐3p mimics were selected. (B) A series of luciferase reporter assay illustrated the fold change of YAP signaling in A549 and H1975 cells were treated with THP1 (Mφ)‐derived exosomes and were subjected to the knockdown of each lncRNA selected above or sh‐NC. (C) THP1 (Mφ) cells were transfected with Cy3‐LINC00273 (red) and the exosomes derived from the transfected THP1 (Mφ) cells were added to the medium of LUAD cells for incubation. Fluorescence microscopy images illustrated the transportation of THP1 (Mφ)‐derived exosomal LINC00273 to LUAD cells. FITC Phalloidin (green) and Cy3 (red) were adopted for labeling cytoskeleton of LUAD cells and LINC00273, respectively. Scale bar: 10μm. (D) LATS2, p‐YAP and YAP levels in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273 were evaluated using western blot. (E) Nuclear and cytoplasmic YAP levels in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273 were evaluated using western blot. (F) The levels of YAP downstream genes in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273 were evaluated using western blot. (G) CHX chase assay evaluated the stability of LATS2 protein in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. (H) Ubiquitination assay evaluated the ubiquitination level of LATS2 protein in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. (I) Proteins recruited by LINC00273 in A549 cells were assessed by mass spectrometric analyses following RNA pull‐down assay. The protein band corresponding to NEDD4 was indicated in the image of SDS‐PAGE gel. (J) The pull‐down enrichment product of Bio‐LINC00273‐Sense or Antisense probe was subjected to western blot to determine the existence of NEDD4. (K) The binding capacity between NEDD4 and LINC00273 was confirmed by RIP assay in A549 cells with LINC00273 overexpression. U1 RNA co‐immunoprecipitated with SNRNP70 was adopted as the positive control. (L) The combination of immunofluorescence and FISH illustrated the overlapped distribution of NEDD4 (green) and LINC00273 (red) in cytoplasm of A549 cells with LINC00273 overexpression. Scale bar: 10μm. (M) Co‐IP assay detected the level of LATS2 or NEDD4 co‐immunoprecipitated with anti‐NEDD4 (anti‐IgG as negative control) in A549 cells transfected with pcDNA3.1 or pcDNA3.1/LINC00273. THP1 (Mφ), macrophages induced from THP1 cells. Data are presented as mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: To detect LATS2‐NEDD4 interaction, cell lysates were cocultured with anti‐NEDD4 (Proteintech) or IgG (Abcam) conjugated magnetic beads, and the eluted proteins underwent western blot with antibody against LATS2 or NEDD4.

Techniques: Transfection, Luciferase, Reporter Assay, Derivative Assay, Incubation, Fluorescence, Microscopy, Labeling, Western Blot, Ubiquitin Assay, Pull Down Assay, SDS Page, Binding Assay, Over Expression, Immunoprecipitation, Positive Control, Immunofluorescence, Co-Immunoprecipitation Assay, Negative Control