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. 2022 Sep 12;13:984302. doi: 10.3389/fimmu.2022.984302

Figure 5.

Figure 5

In vivo Traf6 reduction results in graded decrease of miR-146a mediated B-cell response. (A) Schema of BMT for Traf6 guides. (B) TRAF6 expression via Western Blot. Left panel: validation of TRAF6 deletion in 70Z/3 Cas9 cells. Repeated in duplicate. Right panel: Sorted reconstituted recipient GFP+ mCherry+ bone marrow cells show TRAF6 deletion levels. Data represent three independent experiments. One-way ANOVA with Tukey’s multiple-comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001. (C, D) Splenic B-cells from BMT recipients are stimulated ex vivo with LPS (left) or CpG (right). (C) M146aKOCas donor cells (GFP+) with sg transduced (mCherry+) cells show NT control (red) with high cellular response (similar to native miR-146aKO stimulations in Figures 2C, D ), but with graded decreased response in cells with Traf6 deletion (black and gray). (D) M146aKOCas donor (GFP+) cells without sg RNA transduction look similar among groups (red, black, gray). All bone marrow recipient groups: n = 8 mice/group, total 24 mice/exp; repeated in two independent experiments. t test; *P < 0.05, **P < 0.01, ***P < 0.001. All data represent mean ± SEM. M146aKOCas, MiR-146aKO-Cas9-eGFP mice; BMT, Bone Marrow Transplantation. ▲: *** NT vs. A, **** NT vs. B, *** A vs. B; ●: **** NT vs. A, **** NT vs. B, *** A vs. B; ◆: ***NT vs. A, **** NT vs. B, ** A vs. B; ■ : * NT vs. A, *** NT vs. B, **** A vs. B; △: * NT vs. A; ○ : * NT vs. A, *** NT vs. B, *** A vs. B; □ : * NT vs. A.