Journal: Science Advances

Article Title: Single-cell atlas of cervical organoids uncovers epithelial immune heterogeneity and intercellular cross-talk during Chlamydia infection

doi: 10.1126/sciadv.ady1640

Figure Lengend Snippet: ( A ) Immunofluorescence images of ectocervical (top) and endocervical (bottom) organoids, uninfected (left) or infected (right) for 48 hours with Chlamydia, stained for KRT5 (green), major outer membrane protein (MOMP) (red), KRT8 (gray), and DAPI (blue). ( B ) UMAP projection of single cells from ecto- and endocervical organoids, colored by infection status: uninfected (UI), infected (Inf), and bystander (Bstd). ( C and D ) UMAP showing reclustered ectocervical squamous epithelial population from (B), colored by infection status (C) and subtype identity (D). ( E ) Proportion of UI, Bstd, and Inf cells in each ectocervical squamous subtype. ( F to G ) UMAP showing reclustered endocervical columnar epithelia from (B), colored by infection status (F) and subtype (G). ( H ) Proportion of UI, Bstd, and Inf cells in each endocervical columnar subtype. ( I ) Heatmap of differentially regulated TFs between ecto- and endocervix across infection conditions; color bar depicts the TF activity scores from high (deep pink) to low (blue). ( J ) Violin plot of gene set enrichment scores for the GO term defense response to bacterium across epithelial compartments and infection states; statistical significance assessed by Wilcoxon rank-sum test with Holm-adjusted P values ( ****P ≤ 0.0001). ( K ) The relative expression of IFN-related genes across ecto- and endocervical subclusters; dot size represents the % of cells expressing a particular gene, and the color bar indicates the intensity of scaled mean expression levels ranging from high (red) to low (blue). ( L ) Gene-weighted density UMAP projections showing expression of STAT1 , STAT2 , and IRF9 across epithelial cells in (B). ( M ) Violin plot showing ISG15 expression across ecto- and endocervical organoids in uninfected, bystander, and infected states. ( N ) IHC images showing CDH1 (green), ISG15 (red), MOMP (gray), and DAPI (blue) in ecto- and endocervical organoids, uninfected (left) or infected (right). Yellow arrows mark infected cells; arrowheads indicate ISG15 + bystander cells.

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti–acetylated tubulin–Alexa Fluor 647 (1:300, Santa Cruz Biotechnology, sc-23950-AF647), mouse anti–E-cadherin–Alexa Fluor 488 (1:50, BD Biosciences, 560061), mouse anti–E-cadherin (1:50, BD Biosciences, 610181), rabbit anti–KRT5–Alexa Fluor 488 (1:300, Abcam, ab193894), mouse anti-MUC5B (1:200, Abcam, ab77995), rabbit anti-MUC21 (1:200, ProteinAtlas, HPA052028), rabbit anti-KRT8 (1:200, Abcam, ab59400), mouse-anti-KRT6 (1:50, Abcam, ab18586), recombinant rabbit anti-PAX8 (1:200, Abcam, ab239363), goat anti– C. trachomatis major outer membrane protein (1:500, Bio-Rad, 1990-0804), rabbit anti–HLA-DQA1 antibody (EPR7300) (1:200, Abcam, ab128959), rabbit anti-ISG15 polyclonal antibody (1:200, Proteintech,15981-1-AP), and for labeling the DNA, 4′,6-diamidino-2-phenylindole (DAPI, Roche, 10236276001) were used.

Techniques: Immunofluorescence, Infection, Staining, Membrane, Activity Assay, Expressing