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. Author manuscript; available in PMC: 2023 Aug 1.
Published in final edited form as: Nat Mater. 2022 Jul 11;21(8):939–950. doi: 10.1038/s41563-022-01293-3

Fig. 3 |. Viscoelasticity of ECM controls dendritic cell differentiation and immature monocytes expressing pSTAT3 and an endogenous inhibitor of non-canonical NF-κB, iκBζ.

Fig. 3 |

a, High-dimensional analysis of flow cytometry of cells at day 0, before encapsulation, and after 3 d in viscous or elastic gels with markers HLA-DR, CD11b, CD141, CD11c, CD1c, CD80, PDL1 and CCR2 gated on live cells (left), and gated on HLA-DR+ CD11b+ live cells (right). b, Relative fluorescence of cell populations in viscous and elastic gels identified by k-means clustering, gated on HLA-DR+ CD11b+ live cells. c, Flow cytometry of HLA-DR and CD11b with quantification of HLA-DR MFI on the right, gated on live cells. d, Dendritic cell differentiation markers, CD1c+ CD11c+, gated on CD11b+ HLA-DR+. e, MFI of CCR2+ and fractions of CD80+ and PDL1+ gated on CD11b+ HLA-DR+. f, Quantification of CD68+ cells, gated on live cells (left), and CD11c+ CD163+ (middle) and SLAMF7+ (right) cells, gated on CD11b+ HLA-DR+ cells. n = 3 biological replicates from a single donor for c–f, with P value from an unpaired t-test, two-tailed. g, Immunofluorescence imaging of pSTAT3 (Tyr705, magenta) with quantification and a P < 0.0001 provided by a Mann–Whitney non-parametric test, two-tailed, n = 89 (viscous) and n = 95 (elastic) biological replicates from a single donor. Scale bar, 10 μm. h, Flow cytometry for fraction of CD1c+ CD11c+ cells gated on CD11b+ HLA-DR+ in viscous and elastic hydrogels with and without STAT3 inhibitor C188–9 (1 μM). n = 3 biological replicates from a single donor, with P values from an ordinary one-way ANOVA with Tukey’s multiple comparisons. i, Immunofluorescence imaging of NFKBIZ (IκBζ) (magenta) with quantification and P = 0.0005 provided by a Mann–Whitney non-parametric test, two-tailed, n = 57 (viscous) and n = 22 (elastic). Scale bar, 10 μm. j, Gene expression of NFKBIZ in subsets of peripheral blood mononuclear cells (PBMCs) from the Human Blood Atlas, https://www.proteinatlas.org/humanproteome/blood. Experiments were repeated with at least three donors, unless otherwise noted. Flow staining for SLAMF7 was repeated with two donors. Data in c–i are presented as means ± s.e.m.