Journal: bioRxiv

Article Title: Neuron-dependent tuft cell expansion initiates sinonasal allergic Type 2 inflammation

doi: 10.1101/2023.07.04.547596

Figure Lengend Snippet: (A) Protocol for treatment of WT mice with either 3 i.n. doses every other day of PBS (gray), FAM with blank peptide conjugated to saporin (FAM 3X+Blank-SP, blue), [Sar ,Met(O 2 ) ]–Substance P conjugated to saporin (SSP-SAP 3X, green), or FAM with SSP-SAP (SSP-SAP 3X + FAM 3X, red) analyzed on D5. Data depicted in (C), (E), (G), (H), (K), (L), (O), and (P). (B) Protocol for treatment of WT mice with 3 i.n. doses every other day of either the NK1R inhibitor FK888 and PBS (FK888 3X + PBS 3X, gray), vehicle with FAM (Vehicle 3X + FAM 3X, blue), the NK1R inhibitor with Sar-SP (FK888 3X + Sar-SP 3X, green), or FK888 with FAM (FK888 3X + FAM 3X, red). Data depicted in (D), (F), (I), (J), (M), (N), (Q), and (R). (C-D) Sinonasal wash levels of IL-5 (left) and IL-13 (right) in (C) protocol (A), ablate or (D) protocol (B), inhibit. (E-F) Sinonasal wash levels of (E) IL-25 (left) and CysLT (right) for protocol (A, ablate) or (F) for protocol (B, inhibit). (G-J) Total number of respiratory tissue (G,I) Tuft-1 and (H,J) Tuft-2 cells in the (G-H) protocol (A), ablate or (I-J) in protocol (B), inhibit. (K-N) Total number of respiratory tissue (K, M) Ki-67+ basal cells and (L, M) ILC2s in (K-L) protocol (A), ablate or (M-N) protocol (B) inhibit. (O-R) Total number of respiratory tissue (O, Q) CD4+ TH2 cells and (P, R) eosinophils in (O-P) protocol (A), ablate or (Q-R) protocol (B), inhibit. Data are mean ± SEM, representative of 2 experiments (4-5 mice/treatment), one-way ANOVA with Tukey’s post-hoc tests. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. See also and .

Article Snippet: To inhibit NK1R, mice received 30 nmol of the antagonist FK888 (Tocris, cat #2400), which has been previously used i.n. in guinea pigs .

Techniques:

Journal: bioRxiv

Article Title: Neuron-dependent tuft cell expansion initiates sinonasal allergic Type 2 inflammation

doi: 10.1101/2023.07.04.547596

Figure Lengend Snippet: (A-K) Outcomes in ablation experiment following PBS (gray), FAM 3X + Blank-SAP (blue), SSP-SAP 3X (green), or SSP-SAP 3X + FAM 3X (red) for: (A) sneezing-like behavior evoked by capsaicin (12µM i.n.), which was counted from 10 min sound and video recording in a treatment-blind manner, numbers and/or percentages of all (B) Tuft-1 cells, (C) NK1R+ Tuft-1 cells, (D) all Tuft-2 cells, (E) NK1R+ Tuft-2 cells, (F) total basal cells, (G) Ki67+ basal cells, (H) NK1R+ basal cells, (I) ILC2, (J) CD4+ TH2 cells, and (K) eosinophils in the respiratory epithelium assessed by flow cytometry. (L-V) Outcomes of inhibitor experiment following PBS (gray), vehicle + FAM 3X (blue), FK888 3X +SAR-SP 3X (green), or FK888 3X + FAM 3X (red) for: (L) sneezing-like behavior evoked by capsaicin, numbers and/or percentages of all (M) Tuft-1 cells, (N) NK1R+ Tuft-1 cells, (O) all Tuft-2 cells, (P) NK1R+ Tuft-2 cells, (Q) total basal cells, (R) Ki67+ basal cells, (S) NK1R+ basal cells, (T) ILC2, (U) CD4+ TH2 cells, and (V) eosinophils in the respiratory epithelium assessed by flow cytometry. Data are mean ± SEM, representative of 2 experiments (4-8 mice/treatment) one-way ANOVA with Tukey’s post-hoc tests. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: To inhibit NK1R, mice received 30 nmol of the antagonist FK888 (Tocris, cat #2400), which has been previously used i.n. in guinea pigs .

Techniques: Flow Cytometry

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) H3K27me3 ChIP-qPCR in ESCs (Left) and EpiLCs (Middle) in WT and the ∆ mutant. There is no significant effect on polycomb dynamics in ESCs. In EpiLCs, the ∆ mutant retains residual H3K27me3 relative to WT. H3K27me3 ChIP-qPCR in WT and ∆ ESCs and EpiLCs at control loci are displayed in right panel. Data shown as ±s.e.m. from three biological replicates for each genotype. ( B ) Alleles generated by CRISPR/Cas9-mediated mutagenesis of Eed in WT and ∆ contexts. ( C ) Western blot confirming loss of EED protein and H3K27me3 in Eed-/- cell lines. ( D ) Western blot showing no detection of H3K27me3 loss after four days of incubation with PRC2 inhibitor. Negative control exhibited strong H3K27me3 signal. ( E ) RT-qPCR of Zdbf2 in absence of CTCF partition with EZH2 inhibitor. In ∆CTCF_PS mutants, there is a further increase of Zdbf2 de-repression when H3K27me3 is depleted. Data shown as ±s.e.m. from three biological replicates for each genotype. Statistical analyses were performed by two-tailed unpaired t-test: *p≤0.05, **≤0.01, ***p≤0.001.

Article Snippet: For EZH2 inhibition experiments, the EZH2 incubator UNC 1999 (or its negative control UNC 2400, Tocris Bioscience) was added to media at a 1 μM final concentration for four days.

Techniques: ChIP-qPCR, Mutagenesis, Control, Generated, CRISPR, Western Blot, Incubation, Negative Control, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet:

Article Snippet: For EZH2 inhibition experiments, the EZH2 incubator UNC 1999 (or its negative control UNC 2400, Tocris Bioscience) was added to media at a 1 μM final concentration for four days.

Techniques: CRISPR, Generated, Mutagenesis, Negative Control, Recombinant, Software

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet: ( A ) H3K27me3 ChIP-qPCR in ESCs (Left) and EpiLCs (Middle) in WT and the ∆ mutant. There is no significant effect on polycomb dynamics in ESCs. In EpiLCs, the ∆ mutant retains residual H3K27me3 relative to WT. H3K27me3 ChIP-qPCR in WT and ∆ ESCs and EpiLCs at control loci are displayed in right panel. Data shown as ±s.e.m. from three biological replicates for each genotype. ( B ) Alleles generated by CRISPR/Cas9-mediated mutagenesis of Eed in WT and ∆ contexts. ( C ) Western blot confirming loss of EED protein and H3K27me3 in Eed-/- cell lines. ( D ) Western blot showing no detection of H3K27me3 loss after four days of incubation with PRC2 inhibitor. Negative control exhibited strong H3K27me3 signal. ( E ) RT-qPCR of Zdbf2 in absence of CTCF partition with EZH2 inhibitor. In ∆CTCF_PS mutants, there is a further increase of Zdbf2 de-repression when H3K27me3 is depleted. Data shown as ±s.e.m. from three biological replicates for each genotype. Statistical analyses were performed by two-tailed unpaired t-test: *p≤0.05, **≤0.01, ***p≤0.001.

Article Snippet: Chemical Compound, Drug , EZH2 Inhibitor Negative Control , Tocris Bioscience , UNC 2400 , .

Techniques: ChIP-qPCR, Mutagenesis, Control, Generated, CRISPR, Western Blot, Incubation, Negative Control, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: Dynamic enhancer partitioning instructs activation of a growth-related gene during exit from naïve pluripotency

doi: 10.7554/eLife.44057

Figure Lengend Snippet:

Article Snippet: Chemical Compound, Drug , EZH2 Inhibitor Negative Control , Tocris Bioscience , UNC 2400 , .

Techniques: CRISPR, Generated, Mutagenesis, Negative Control, Recombinant, Software