Fig 2: ERK1/2 phosphorylation is decreased with DOX-induced miR-15a/16.A, Western blot analysis of p-ERK1(Thr202) and p-ERK2(Tyr204) along with total ERK1 and ERK2 in unstimulated and 18 h TCR-stimulated T cells treated with PBS or DOX, with β-actin serving as a loading control. B, densitometry was performed on biological repeats shown in Fig. S6. C, lysates from 18 h TCR-stimulated T cells were used to conduct an ERK signaling phospho antibody array to compare DOX- versus PBS-treated cells for levels of phosphorylated amino acid residues on different signaling proteins. Darker rows indicate increased signals with DOX treatment, and lighter rows indicate decreased signals. Mean of each p-residue signal (N = 6) was normalized to mean of total residue signal (N = 6). DOX, doxycycline; ERK1/2, extracellular signal–regulated protein kinase 1/2; TCR, T-cell receptor.

Fig 3: The 3′-UTR target sequence in MEK1 is recognized by miR-15a/16.A, a gene encoding MEK1 was synthesized containing the predicted target nucleotide sequence (TGCTGCTA) or mutated sequence (AAAAAAAA) and ligated into a FLAG-tagged expression plasmid. B, Western blot was used to analyze expression in HEK293 cells of FLAG-MEK1 from each plasmid cotreated with miR-15a/16 mimics or nontargeting control mimics, with GAPDH as a loading control and biological replicates used for densitometry shown in Fig. S3. Means of replicates (N = 3) were compared using a Student’s t test and expressed as mean ± SD with ∗p < 0.05. HEK293, human embryonic kidney 293 cell line; MEK1, mitogen-activated protein kinase kinase 1.

Fig 4: Inducing higher levels of miR-15a/16 during T-cell activation decreases proliferation.A, experimental design for mice containing a DOX-inducible miR-15a/16 transgene. B, CD3+ T cell purified from transgenic mice treated either with DOX or PBS (control) was not stimulated or TCR stimulated for 18 h followed by real-time PCR analyses of miR-15a and miR-16 levels relative to U6 miR (housekeeping). C and D, CFSE-loaded transgenic T cells were treated with DOX or PBS and activated for 72 h followed by flow cytometric analysis. Because levels of CFSE fluorescence decreases as cells divide, this allowed calculation of division index and proliferation index as described in the Experimental procedures section. Means of replicates (N = 4/mouse) were compared using a Student’s t test and expressed as mean ± SD with ∗p < 0.05. CFSE, carboxyfluorescein succinimidyl ester; DOX, doxycycline.

Fig 5: Elk1 phosphorylation is decreased with DOX-induced miR-15a/16.A, Western blot analysis of p-Elk1(Ser383) and total Elk1 in unstimulated and 18 h TCR-stimulated T cells treated with PBS or DOX, with β-actin serving as a loading control. B, densitometry was performed on biological repeats of Western blots shown in Fig. S6. Means of replicates (N = 3) were compared using a Student’s t test and expressed as mean ± SD with ∗p < 0.05. DOX, doxycycline; TCR, T-cell receptor.