Impaired accumulation of NP-specific ASCs in the BM of Zfp36l1^fl/fl CD79a^Cre/+ mice results from a delayed egress from the spleen. (A) Quantification of a transwell migration assay of CD138^highB220^low cells differentiated in vitro as described in Materials and methods. The number of cells migrated toward three different concentrations of S1P in the lower chamber was normalized to the number of identically gated cells in the input sample. The B cell cultures used in the transwell assay routinely contained 10–20% of CD138^highB220^low cells (two-way ANOVA was performed on the data for 10 nM and 50 nM S1P; n = 4 and n = 6; data pooled from two or three independent experiments; each data point is a mean of technical duplicate measurements of independent biological samples, lines represent means, and the P value shown is for the main genotype effect for migration toward 10 nM and 50 nM S1P). (B) Quantification of a transwell migration assay performed as in A but toward CXCL12 (Student’s t test; n = 6; data pooled from three independent experiments; each data point is a mean of technical duplicate measurements of independent biological samples, and lines represent means). (C) Quantification of NP-specific ASCs (gated as eFluor780⁻TCRβ⁻CD138⁺Irf4⁺NIP⁺IgG1⁺ cells; full gating is depicted in Fig. S3 A) in blood of Zfp36l1^fl/fl CD79a^+/+ (closed circles) and Zfp36l1^fl/fl CD79a^Cre/+ (open squares) or Zfp36l1^fl/fl Cd23-Cre⁺ (open squares) mice at day 3 after reimmunization with NP-KLH. For each Cre-expressing line, data are pooled from two independent experiments. For the plots in panels C, D, F, and G, each symbol represents data from an individual mouse, and lines represent means (n ≥ 4; two-way ANOVA; the P value shown is for the main factor “presence of Cre”). (D) Frequency of CD138⁺NIP⁺IgG1⁺ cells in the spleen of Zfp36l1^fl/fl CD79a^+/+ (closed circles) and Zfp36l1^fl/fl CD79a^Cre/+ (open squares) mice at days 3, 4, and 5 following reimmunization with NP-KLH expressed as numbers of CD138⁺NIP⁺IgG1⁺ cells normalized to the numbers of all eFluor780⁻ cells (n ≥ 3; two-way ANOVA with Sidak’s correction for multiple testing; the P values shown are for the genotype factor for separate days). (E) Representative flow cytometry plots gated as in Fig. 1 D on CD138⁺NIP⁺IgG1⁺ cells from Zfp36l1^fl/fl CD79a^Cre/+ mice 3 d (left plot) and 5 d (right plot) after reimmunization with NP-KLH and used to identify aCasp3⁺ cells. (F and G) Plots showing proportions of aCasp3⁺ cells in the population of CD138⁺NIP⁺IgG1⁺ cells identified as in Fig. 1 D, Fig. 3 E, and Fig. S3 B in the spleen (F) and BM (G) for Zfp36l1^fl/fl CD79a^+/+ (closed circles) and Zfp36l1^fl/fl CD79a^Cre/+ (open squares) mice taken at days 3, 4, 5, and 6 following reimmunization with NP-KLH. Data are representative of at least two independent experiments (n ≥ 3; two-way ANOVA with Sidak’s correction for multiple testing; the P values shown are for the genotype factor for separate days [F] or the P value shown is for the main genotype effect [G]). For all flow cytometry plots, the numbers next to the enclosed areas (gates) represent percentages of the cells falling into the gate, and the number in the bottom right corner shows the number of cells in the plot.