Journal: The EMBO Journal
Article Title: Immediate early splicing controls translation in activated T-cells and is mediated by hnRNPC2 phosphorylation
doi: 10.1038/s44318-025-00374-8
Figure Lengend Snippet: ( A , B ) PKCθ inhibition abolishes IES. After Jurkat cell treatment with PKCθ inhibitor for 30 min (left) or DMSO (right), cells were stimulated with PMA for the indicated times. Chromatin-associated RNA was investigated for RPL10 ( A ) and eIF5A ( B ) IES by radioactive, splicing-sensitive RT-PCR and quantified (bottom, data are presented as % IR, mean ± SD, number of biological replicates: n = 3, RPL10 ( A ): PKCθ inh. vs. DMSO (0 min): P = 0.0249, 15 min: P = 0.0019, 30 min: P = 0.0026, 90 min: P = 0.0046, 240 min: P = 0.0131, eIF5A (B): PKCθ inh. vs. DMSO (0 min): P = 0.1109, 15 min: P = 0.0081, 30 min: P = 0.0162, 90 min: P = 0.0462, 240 min: P = 0.2038, Student’s unpaired t test). ( C ) PKCθ inhibition reduces hnRNPC2 phosphorylation. After Jurkat cell treatment with PKCθ inhibitor (left) or DMSO (right), cells were stimulated with PMA for the indicated times and protein lysates were investigated for phosphorylation of hnRNPC2 by western blot (top) and quantified (bottom, data are presented as % hnRNPC2 phosphorylation of total hnRNPC2, mean ± SD, number of biological replicates: n = 3, PKCθ inh. vs. DMSO (15 min): P = 0.0004, 30 min: P = 0.0103, Student’s unpaired t test). ( D , E ) PKCθ overexpression induces IES in an hnRNPC2-dependent manner. HEK293 cells were transfected with an empty FLAG vector or an overexpression (OE) vector for PKCθ. Additionally, HEK293 cells with hnRNPC2-deletion were transfected with an overexpression vector for PKCθ. After 48 h, cells were stimulated by PMA for the indicated time points and chromatin-associated RNA was investigated by radioactive, splicing-sensitive RT-PCR for IR in RPL10 ( D ) and eIF5A ( E ). Bottom: data are presented as % IR, mean ± SD, number of biological replicates: n = 3, RPL10 (D): 0 vs. 15 min (PKCθ_OE in HEK293_WT), P = 0.0088, 0 vs. 30 min (PKCθ_OE in HEK293_WT), P = 0.0383, PKCθ_OE in HEK293_WT vs. empty vector in HEK293_WT (15 min): P = 0.0018, PKCθ_OE in HEK293_WT vs. empty vector in HEK293_WT (30 min): P = 0.0911, PKCθ_OE in HEK293_WT vs. PKCθ_OE in HEK293_ΔhnRNPC2 (15 min): P = 0.01, PKCθ_OE in HEK293_WT vs. PKCθ_OE in HEK293_ΔhnRNPC2 (30 min): P = 0.0119, eIF5A ( E ): 0 vs. 15 min (PKCθ_OE in HEK293_WT), P = 0.024, 0 vs. 30 min (PKCθ_OE in HEK293_WT), P = 0.016, PKCθ_OE in HEK293_WT vs. empty vector in HEK293_WT (15 min): P = 0.0115, PKCθ_OE in HEK293_WT vs. empty vector in HEK293_WT (30 min): P = 0.009, PKCθ_OE in HEK293_WT vs. PKCθ_OE in HEK293_ΔhnRNPC2 (15 min): P = 0.001, PKCθ_OE in HEK293_WT vs. PKCθ_OE in HEK293_ΔhnRNPC2 (30 min): P = 0.0322, Student’s unpaired t test). ( F , G ) Analysis as in ( D , E ) using HeLa cells ( RPL10 ( F ): 0 vs. 15 min (PKCθ_OE in HeLa_WT), P = 0.0005, 0 vs. 30 min (PKCθ_OE in HeLa_WT), P < 0.0001, PKCθ_OE in HeLa_WT vs. empty vector in HeLa_WT (15 min): P = 0.011, PKCθ_OE in HeLa_WT vs. empty vector in HeLa_WT (30 min): P = 0.0163, PKCθ_OE in HeLa_WT vs. PKCθ_OE in HeLa_ΔhnRNPC2 (15 min): P = 0.0018, PKCθ_OE in HeLa_WT vs. PKCθ_OE in HeLa_ΔhnRNPC2 (30 min): P < 0.0001, eIF5A ( G ): 0 vs. 15 min (PKCθ_OE in HeLa_WT), P = 0.0044, 0 vs. 30 min (PKCθ_OE in HeLa_WT), P = 0.011, PKCθ_OE in HeLa_WT vs. empty vector in HeLa_WT (15 min): P = 0.0128, PKCθ_OE in HeLa_WT vs. empty vector in HeLa_WT (30 min): P = 0.0531, PKCθ_OE in HeLa_WT vs. PKCθ_OE in HeLa_ΔhnRNPC2 (15 min): P = 0.0015, PKCθ_OE in HeLa_WT vs. PKCθ_OE in HeLa_ΔhnRNPC2 (30 min): P = 0.0063 (Student’s unpaired t test). .
Article Snippet: PKCθ inhibitor , Selleck , S6577.
Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Over Expression, Transfection, Plasmid Preparation
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