Article Figures & Data
Figures
- FIG 1
Generation of the conditional ERK3 knockout mouse and deletion of ERK3 mRNA and protein. (A) Targeting strategy. (B to D) ES cell screening by PCR (B and C) and Southern hybridization (D). (B and C) PCR using primer combination P1/P2 and P3/P4 (shown in panel A) to detect Erk3ex3loxneo allele. (D) Southern blot analysis using probe (shown in panel A) and ScaI-digested DNA. (E) Detection of Flp- and Cre-mediated recombination by PCR using primer combinations (P5 and P6), leading to fragments as indicated in panel A. (F) ERK3 mRNA was amplified by PCR from total RNA of BMDMs using primers for exons 2 and 4. The ERK3 KO displays a single band weaker and smaller than that of the WT, indicating loss of exon 3. (G) Western blot analysis of total protein of BMDMs by an N-terminal-ERK3 antibody (53277; Abcam) and an MK5 antibody. Equal loading is demonstrated by GAPDH detection.
- FIG 2
Characterization of the viable ERK3 KO mice. (A) Mendelian ratio of the offspring of heterozygous (HE) Erk3Δex3 mice. Fifteen litters of six crosses (total n = 105) were analyzed. No statistically significant deviation from Mendelian ratio was detected (χ2 = 4.04 < 5.99, P < 0.05). (B) Slight and transient growth retardation of male but not female ERK3 KO mice (4 to 6 mice per group; **, P < 0.05; ***, P < 0.01). (C) Histopathology of the lungs from 35-day-old WT (a), ERK3+/− (b), and ERK3 KO (c) mice revealed no significant pathological alterations. Hematoxylin and eosin staining was used. Bars, 50 µm.
- FIG 3
ERK3 is largely dispensable for intrathymic T-cell development. (A) Representative flow cytometric analysis of thymi from WT and Erk3−/− mice stained with antibodies against CD4 and CD8α. Numbers in quadrants represent frequencies. Total cellularity of thymi from WT, ERK3+/−, and ERK3−/− mice. Statistical analysis of flow cytometric results from WT, ERK3+/−, and ERK3−/− mice. Cells were defined as DN (CD4− CD8α−), DP (CD4+ CD8α+), SP4 (CD4+ CD8α−), SP8 (CD4− CD8α+), and γδ T cells (CD4− CD8α− TCRγδ+). (B) Representative flow cytometric analysis of thymi from WT and ERK3−/− mice stained with antibodies against CD4, CD8α, TCRβ, and CD5. Numbers adjacent to gates represent frequencies. For statistical analysis of flow cytometric results, cells were defined as DP1 (TCRβlo CD5lo), DP2 (TCRβint CD5hi), and DP3 (TCRβhi CD5int) thymocytes. (C) Representative flow cytometric analysis of thymi from WT and ERK3−/− mice stained with antibodies against CD4, CD8α, CCR7, and CD69. Numbers in quadrants represent frequencies. For statistical analysis of flow cytometric results, cells were defined as described for panel A and as CD69+ CCR7−, CD69+ CCR7+, and CD69− CCR7+ thymocytes. (A to C) Pooled data of two independent experiments (n = 5 for WT, n = 6 for ERK3+/−, and n = 6 for ERK3−/−). Statistically significant differences with P values of <0.05 are indicated; P values of >0.05 are not shown.
- FIG 4
ERK3 is largely dispensable for T-cell homeostasis and proliferation. (A) Treg cell numbers in thymus and spleen. Dot plots show representative flow cytometric analysis of thymi and spleen from WT and ERK3−/− mice stained with antibodies against CD4, TCRβ, Foxp3, and CD25. Numbers adjacent to gates represent frequencies. Graphs display statistical analysis of flow cytometric results from WT, ERK3+/−, and ERK3−/− mice. (B) Representative flow cytometric analysis of spleen from WT and ERK3−/− mice stained with antibodies against CD19, TCRβ, CD4, and CD8. Numbers in quadrants represent frequencies. Total cellularity of spleen from WT, ERK3+/−, and ERK3−/− mice. For statistical analysis of flow cytometric results from WT, ERK3+/−, and ERK3−/− mice, cells were defined as T cells (TCRβ+), B cells (CD19+), SP4 (TCRβ+ CD4+), and SP8 (TCRβ+ CD8+). (C) Representative flow cytometric analysis of SP4 and SP8 splenic T cells from WT and ERK3−/− mice stained with antibodies against CD4, CD8, CD44, and CD62L to identify naive (CD44− CD62L+), central memory (CD44+ CD62L+), and effector memory (CD44+ CD62L−) T-cell subsets. Pooled data from two independent experiments were used for panels A to C (n = 5 for WT, n = 6 for ERK3+/−, and n = 6 for ERK3−/−). Statistically significant differences with P values of <0.05 are indicated; P values of >0.05 are not shown. (D) Flow cytometric analysis of CD4 (upper) and CD8 (lower). T-cell proliferation from WT and ERK3–/– mice after 3 and 4 days of culture. Shown are representative contour plots of experiments with n = 2 mice per genotype.
