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

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: Reagents and tools table

Article Snippet: PKCθ inhibitor , Selleck , S6577.

Techniques: Recombinant, Construct, Sequencing, Reverse Transcription, SYBR Green Assay, Marker, Western Blot, Software, Electroporation, Blocking Assay, Imaging, Mass Spectrometry

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: Reagents and tools table

Article Snippet: PKCθ inhibitor , Selleck , S6577.

Techniques: Recombinant, Construct, Sequencing, Reverse Transcription, SYBR Green Assay, Marker, Western Blot, Software, Electroporation, Blocking Assay, Imaging, Mass Spectrometry

Journal: PLOS Pathogens

Article Title: Activating PKC-ε induces HIV expression with improved tolerability

doi: 10.1371/journal.ppat.1012874

Figure Lengend Snippet: Similar expression of PKC-θ in both T cells and platelets was observed, whereas PKC-η and ε were abundant in T cells but undetected in platelets. Cell lysates of isolated CD4 + T cells and platelets from healthy donors (n = 3) were tested for expression of novel PKC isoforms θ, η, and ε by western blotting utilizing PKC-θ and PKC-η/ε antibodies. Expression of GAPDH was used for normalization to enable accurate interpretation of differences in protein expression.

Article Snippet: For experiments shown in , primary antibodies against PKC-ɛ and -ɳ (LSBio, catalog C172661, 1:2000 dilution), PKC-θ (Cell Signaling Technology, catalog 13643, 1:1000 dilution), and GAPDH (Cell Signaling Technology, catalog 5174S, 1:1000 dilution) at RT for 60 minutes and secondary antibodies rabbit anti-mouse IgG-HRP (Santa Cruz Biotech, catalog sc-358914, 1:2000) and anti-rabbit IgG-HRP (Cell Signaling Technology, catalog 7074, 1:2000 dilution) at RT for 60 minutes were used.

Techniques: Expressing, Isolation, Western Blot

Journal: PLOS Pathogens

Article Title: Activating PKC-ε induces HIV expression with improved tolerability

doi: 10.1371/journal.ppat.1012874

Figure Lengend Snippet: Selectivity of PKC inhibitors towards PKC isoforms a .

Article Snippet: To study reactivation of latent cells by various PKC isoforms, 30 μg of pcDNA3.1 RFP vectors containing either PKC-ε, -η, -θ, or -δ, were transfected (GenScript’s Neon transfection system) according to the manufacturer’s instructions [ ].

Techniques:

Journal: PLOS Pathogens

Article Title: Activating PKC-ε induces HIV expression with improved tolerability

doi: 10.1371/journal.ppat.1012874

Figure Lengend Snippet: C-232A exhibits selectivity for novel PKC isoforms.

Article Snippet: To study reactivation of latent cells by various PKC isoforms, 30 μg of pcDNA3.1 RFP vectors containing either PKC-ε, -η, -θ, or -δ, were transfected (GenScript’s Neon transfection system) according to the manufacturer’s instructions [ ].

Techniques: Translocation Assay

Journal: PLOS Pathogens

Article Title: Activating PKC-ε induces HIV expression with improved tolerability

doi: 10.1371/journal.ppat.1012874

Figure Lengend Snippet: C-233 exhibits PKC-ε selectivity.

Article Snippet: To study reactivation of latent cells by various PKC isoforms, 30 μg of pcDNA3.1 RFP vectors containing either PKC-ε, -η, -θ, or -δ, were transfected (GenScript’s Neon transfection system) according to the manufacturer’s instructions [ ].

Techniques: