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. 2022 Apr 22;11:e72313. doi: 10.7554/eLife.72313

Figure 3. YTHDF2 deficient plasma cells fail to accumulate in the bone marrow.

(A) Left: Representative flow cytometry analysis of cells expressing CD45.2 and CD19 in spleen of competitive chimeras twelve weeks after reconstitution. Numbers refer to the proportion of viable single cells within CD45.2 + CD19 + gate. Right: The number of cells expressing CD45.2 and CD19, n ≥ 10. (B) Representative flow cytometry analysis of NIP and IgG1 intracellular staining on spleen cells gated as CD45.2 + CD138+, CD267 + cells at day 21. (C) Representative flow cytometry analysis of CD138 and CD267 staining on bone marrow cells gated as CD45.2 + at day 21. (D) Representative flow cytometry analysis of NIP and IgG1 intracellular staining on bone marrow cells gated as CD45.2 + CD138+, CD267 +at day 21. For each condition, the numbers in the bottom right corner of left-hand panels show the number of events plotted. The numbers adjacent to the gates indicate the proportion of cells within the gate. For each condition, the right-hand plots show the number of cells per million viable cells. Symbols represent data from an individual Ythdf2fl/fl control (closed circles, n = 3) and Ythdf2fl/fl-Vav1-iCre knockout (open squares, n = 6) mouse. Statistical significance was determined by two-tailed unpaired Student’s t-test. The data are from a single immunisation experiment.

Figure 3.

Figure 3—figure supplement 1. Absence of cells that escaped Cre mediated deletion.

Figure 3—figure supplement 1.

(A) Left: Representative histograms of GFP-YTHDF2 fusion protein expression in surface stained CD45.2 positive total splenic B cells, splenic germinal centre B cells, splenic plasmablasts of competitive chimeras. Right: proportion of CD45.2 + cells within Ythdf2 CKO competitive chimeras expressing GFP-YTHDF2 +fusion protein, n = 6. (B) Left: Representative histograms of GFP-fluorescence in CD45.2 positive bone marrow plasma cells of competitive chimeras following fixation and intracellular staining for NIP and GFP. Right: proportion of CD45.2 + cells within Ythdf2 CKO competitive chimeras expressing GFP-YTHDF2 +fusion protein, n = 6. (C) Enumeration of CD138 +CD267 + NIP + IgG1 + cells that stained or lacked staining for CD45.2 in Ythdf2 CKO competitive chimeras at day 21, n = 6. (D) Representative flow cytometry analysis of CD138, CD267 staining on bone marrow cells of competitive chimeras gated as CD45.2- at day 21. (E) Representative flow cytometry analysis of NIP and IgG1 intracellular staining on bone marrow cells of competitive chimeras gated as CD45.2- CD138+, CD267 +at day 21. For each condition, the numbers in the bottom right corner of left-hand panels show the number of events plotted; the numbers adjacent to the gates indicate the proportion of cells within the gate. For each condition, the right-hand plots show the number of cells per million viable cells. Symbols represent data from an individual Ythdf2fl/fl control (closed circles, n = 3) and Ythdf2fl/fl-Vav1-iCre knockout (open squares, n = 6) mouse.