Figure 4.
Functional and developmental alterations caused by miR-15a/16-1 KO in mature B cells. (A) pERK cell staining intensity per GC in spleens of WT (n = 15) and KO (n = 17) mice immunized with SRBCs identified by IHC. Graphs depict the mean plus or minus standard deviation (SD) (left). Measurements were made with HALO image analysis software. P value was calculated using the unpaired Student t test. Representative pictures are shown on the right (IHC, brown; counterstain, blue); GCs are highlighted by dotted lines. Scale bar, 50 µm. (B) Percentage of apoptotic cells per GC in spleens of WT (n = 41) and KO (n = 40) mice immunized with SRBC identified by cleaved caspase-3 IHC. Graphs depict the mean plus or minus SD (left). Measurements were made with inForm cell analysis software. P value was calculated using the unpaired Student t test. Representative pictures are shown on the right (IHC, brown; counterstain, blue); GCs are highlighted by dotted lines. Scale bar, 50 µm. (C) Area of GCs in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBCs identified by Ki-67 IHC. Graphs depict areas in paired littermate samples analyzed in parallel (left). Measurements were made with HALO Image Analysis software. P value was calculated using the ratio paired Student t test. Representative pictures are shown on the right (IHC, brown; counterstain, blue); GCs are highlighted by dotted lines. Scale bar, 300 µm. (D) Percentage of GC B cells in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBCs determined using flow cytometry. Graphs depict percentages in paired littermate samples analyzed in parallel (left). P value was calculated using the ratio paired Student t test. Representative dot plots are shown on the right. (E) GSEA analysis of signatures upregulated in DZ compared with LZ GC B cells from GSE38696 (top) and PCs compared with GC B cells from GSE11961 (bottom) in proteomes of GC B cells from KO vs WT mice. (F) Percentage of LZ and DZ GC B cells in spleens of WT (n = 3) and KO (n = 3) mice immunized with SRBCs determined using flow cytometry. Graphs depict percentages in paired littermate samples analyzed in parallel (left). P values were calculated using the ratio paired Student t test. Representative density plots are shown on the right. (G) Percentage of PCs in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBCs identified by CD138 IHC. Graphs depict percentages in paired littermate samples analyzed in parallel (left). Measurements were made with HALO image analysis software. P value was calculated using the ratio paired Student t test. Representative pictures are shown on the right (IHC, brown; counterstain, blue). Scale bar, 250 µm. (H) Percentage of PCs in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBC determined using flow cytometry. Graphs depict percentages in paired littermate samples analyzed in parallel (left). P value was calculated using the ratio paired Student t test. Representative dot plots are shown on the right. (I) Percentage of PCs per GC in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBCs identified by CD138 IHC. Graphs depict the mean plus or minus SD (left). Measurements were made with HALO image analysis software. P value was calculated using the unpaired Student t test Representative pictures are shown on the right (IHC, brown; counterstain, blue); GCs are highlighted by dotted lines. Scale bar, 100 µm. ES, enrichment score; OD, optical density; pERK, phosphorylated ERK.

Functional and developmental alterations caused by miR-15a/16-1 KO in mature B cells. (A) pERK cell staining intensity per GC in spleens of WT (n = 15) and KO (n = 17) mice immunized with SRBCs identified by IHC. Graphs depict the mean plus or minus standard deviation (SD) (left). Measurements were made with HALO image analysis software. P value was calculated using the unpaired Student t test. Representative pictures are shown on the right (IHC, brown; counterstain, blue); GCs are highlighted by dotted lines. Scale bar, 50 µm. (B) Percentage of apoptotic cells per GC in spleens of WT (n = 41) and KO (n = 40) mice immunized with SRBC identified by cleaved caspase-3 IHC. Graphs depict the mean plus or minus SD (left). Measurements were made with inForm cell analysis software. P value was calculated using the unpaired Student t test. Representative pictures are shown on the right (IHC, brown; counterstain, blue); GCs are highlighted by dotted lines. Scale bar, 50 µm. (C) Area of GCs in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBCs identified by Ki-67 IHC. Graphs depict areas in paired littermate samples analyzed in parallel (left). Measurements were made with HALO Image Analysis software. P value was calculated using the ratio paired Student t test. Representative pictures are shown on the right (IHC, brown; counterstain, blue); GCs are highlighted by dotted lines. Scale bar, 300 µm. (D) Percentage of GC B cells in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBCs determined using flow cytometry. Graphs depict percentages in paired littermate samples analyzed in parallel (left). P value was calculated using the ratio paired Student t test. Representative dot plots are shown on the right. (E) GSEA analysis of signatures upregulated in DZ compared with LZ GC B cells from GSE38696 (top) and PCs compared with GC B cells from GSE11961 (bottom) in proteomes of GC B cells from KO vs WT mice. (F) Percentage of LZ and DZ GC B cells in spleens of WT (n = 3) and KO (n = 3) mice immunized with SRBCs determined using flow cytometry. Graphs depict percentages in paired littermate samples analyzed in parallel (left). P values were calculated using the ratio paired Student t test. Representative density plots are shown on the right. (G) Percentage of PCs in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBCs identified by CD138 IHC. Graphs depict percentages in paired littermate samples analyzed in parallel (left). Measurements were made with HALO image analysis software. P value was calculated using the ratio paired Student t test. Representative pictures are shown on the right (IHC, brown; counterstain, blue). Scale bar, 250 µm. (H) Percentage of PCs in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBC determined using flow cytometry. Graphs depict percentages in paired littermate samples analyzed in parallel (left). P value was calculated using the ratio paired Student t test. Representative dot plots are shown on the right. (I) Percentage of PCs per GC in spleens of WT (n = 4) and KO (n = 4) mice immunized with SRBCs identified by CD138 IHC. Graphs depict the mean plus or minus SD (left). Measurements were made with HALO image analysis software. P value was calculated using the unpaired Student t test Representative pictures are shown on the right (IHC, brown; counterstain, blue); GCs are highlighted by dotted lines. Scale bar, 100 µm. ES, enrichment score; OD, optical density; pERK, phosphorylated ERK.

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