Fig 2: cDC2 interact with laminin α5 of RFs via integrin α6β1(A–F) (A) Gatings employed to analyze CD11c+CD11b+ cDC2s (B) bound to laminin 511 and specific domains thereof, or fibronectin (FN) control, in the presence of function blocking integrin α6β1 antibody (GoH3); data are means ± SD of 3 experiments with triplicates in each experiment and analyzed by Student’s t test; ∗∗p <0.01. Lymph nodes from WT hosts carrying Itga6−/− or WT bone marrow were isolated 1 week after immunization and analyzed by flow cytometry for (C) antigen specific IgG1 and IgM levels, (D) CD19+Fas+GL-7+ GC B-cells, (E) CD11b+CD11c+ cDC2, (F) CD4+CXCR5+PD-1+ Tfh-cells; bar graphs show means ± SD from 3 WT and 3 Itga6−/− chimerics; data were analyzed by Student’s t test; ∗p<0.05, ∗∗p<0.01; ∗∗∗p<0.001.(G) Immunofluorescence staining at 48h after immunization for CD11b+CD11c+ cDC2 and pan-laminin to mark RFs in WT and Itga6−/− chimeras; scale bars are 150 μm.(H–J) (H) CD11b+CD11c+ double-positive cDC2s were pseudo-colored to illustrate their localization relative to the pan-laminin stained follicle borders and RFs, scale bars are 100 μm in WT and 50 μm in Itga6−/− chimeras; distances of individual cells from the follicle border were employed for quantifications in (I) and (J); data in I and J are means ± SEM from 5 sections each from 4 WT and 4 Itga6−/− lymph nodes. Data were analyzed using a Mann Whitney U test; ∗∗∗∗p <0.0001.See also Figures S4 and S5.

Fig 3: MCF10A and MCF12A cells derived from the breast tissue of patients with fibrocystic changes differ in HSF1 and EMT marker levels and morphology. (a) The expression of HSF1 and (b) EMT markers was assessed by Western blot. ACTB and HSPA8 were used as loading controls. The graph shows the results of densitometric analyses (n = 3). * p < 0.05 (significance of differences). (c) Morphology of cells in 2D culture: phase-contrast images of subconfluent (upper panel) and confluent cells (bottom panel). An example cluster of cells is marked with an ellipse; the arrow points to a round cell, and the arrowhead points to a spindle cell. Scale bar 200 µm. (d) Morphology of cells cultured in Matrigel: phase-contrast images (upper panel; scale bar 100 µm) and confocal images of spheroids stained for ITGA6, F-actin and DNA (bottom panel; scale bar 20 µm). Original western blots are presented in File S1.

Fig 4: HSF1 levels affect the expression of EMT markers and phenotype of MCF10A and MCF12A cells. (a) Western blot analysis of HSF1 and EMT markers in cells with overexpression (MCF10A) or knockdown (MCF12A) of HSF1. WT, unmodified cells; pLVX or shCTRL, cells transduced with lentiviruses containing the empty vector or control shRNA; HSF1-pLVX or sh1.HSF1, cells transduced with lentiviruses containing the HSF1 cDNA or shRNA targeting HSF1. ACTB and HSPA8 were used as loading controls. Graphs show the results of densitometric analyses (n = 3). (b) Proliferation rates assessed through crystal violet staining in MCF10A cell variants (n = 5). (c) Morphology of MCF10A cell variants in 2D culture (subconfluent; upper panel) and Matrigel (middle and bottom panels). Phase-contrast images (upper and middle panels; scale bar 100 µm) and confocal images of spheroids stained for ITGA6 and DNA (bottom panel). (d) The volume of spheroids formed in Matrigel by MCF10A cell variants. Boxplots represent the median, upper and lower quartiles, maximum and minimum. (e,f) Confocal images of spheroids grown in Matrigel for 14 or 21 days and stained for CDH1 and DNA (scale bar 20 µm and 50 µm, respectively). (g) Migratory capacity of MCF10A cell variants analyzed with Boyden chamber assay (n = 3). (h) Time of cell detachment from the plastic surface by trypsinization. Boxplots represent the median, upper and lower quartiles, maximum and minimum. *** p < 0.001, ** p < 0.01, * p < 0.05 (significance of differences). Original western blots are presented in File S1.

Fig 5: Mesenchymal and stem cell-like characteristics of MCF10A and MCF12A cell variants with altered HSF1 expression. (a) Subpopulations defined by expression of EPCAM and ITGA6 or (b) CD24 and CD44 using flow cytometry. The percentages of subpopulations shown in the bar graphs are the mean ± SEM of three independent experiments. Ovals point out cell populations, which are enriched when HSF1 expression levels are altered. * p < 0.05 (significance of differences).