p creb p atf1  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: Transient SUMOylation inhibition in human pre-adipocytes stably imprints a transcriptional beiging fate

doi: 10.1093/nar/gkag232

Figure Lengend Snippet: Effect of transient SUMOylation inhibition in pre-adipocytes on cAMP-PKA-p38 signaling and adaptive thermogenesis in mature adipocytes. ( A ) Heatmap showing the stable upregulation of most genes involved in adaptive thermogenesis and cAMP-PKA-p38 signaling 22 days after adipogenic induction. Z -scores were calculated and plotted in GraphPad Prism. Treatments of pre-adipocytes were performed as shown in Fig. . ( B ) Western blot analysis of PKA substrates phosphorylation, assessed using a pan-PKA-target antibody, 22 days after adipogenic induction. TBP was used as a loading control. The asterisk (*) indicates substrates significantly affected by TAK-981 and/or rosiglitazone treatment. ( C ) Quantification of PKA substrate signals from panel (B). The signal for PKA substrates and TBP was quantified using Fiji software, with PKA substrate signals normalized to TBP. Error bars represent the standard deviation of four independent experiments. Student’s t -test was used to calculate P -values. ( D ) Western blot analysis of p-CREB, p-ATF1, p38, p-p38, and p-ATF2, 22 days after adipogenic induction. TBP was used as a loading control. ( E ) Quantification of p-CREB/ATF-1 signals from panel (D). Signals were normalized to TBP. Error bars represent the standard deviation of three independent experiments. Student’s t -test was used to calculate P -values. ( F ) Western blot analysis of p-CREB in the absence or presence of the PKA inhibitor RP-8-CPT-cAMPS. TBP was used as a loading control. Quantification of p-CREB, normalized to TBP, is showed in the lower panel. Error bars represent the standard deviation of two independent experiments. Quantification of p-p38 ( G ) and p-ATF2 ( H ) from experiment in panel (D). Signals were normalized to TBP for p-ATF2 and to p38 for p-p38. Error bars represent the standard deviation of three independent experiments. Student’s t -test was used to calculate P -values.

Article Snippet: The following antibodies were used: UCP1, abcam, ab209483; PKA phospho-substrates, Cell Signaling, 9624; p-CREB/p-ATF1, Cell Signaling, 9198; p38 MAPK, Cell Signaling, 9212; p-p38 MAPK, Cell Signaling, 9211; p-ATF2, Cell Signaling, 24329; SUMO2/3, abcam, ab81371; PPARG, Cell signaling, 2443; and TBP, Protein Tech, 22006-1-AP or abcam, 282715; γ-tubulin, Sigma, T5326: CEBPB, Santa Cruz, sc-7962 quantifications were performed using FiJi [ ].

Techniques: Inhibition, Western Blot, Phospho-proteomics, Control, Software, Standard Deviation

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a Human PBMCs (1 × 10⁶) were activated with anti-CD3/CD28 beads for 4 days, then treated with NECA ± TAIII for 24 h. Bulk RNA sequencing was performed, and a heatmap displays selected mRNAs and surface markers of Treg and Teff subsets. b PBMCs were stimulated with CD3/CD28 beads and treated with indicated compounds for 48 h. IFN-γ and IL-2 in supernatants were measured by ELISA. Data represent mean ± SEM of three independent experiments, each in triplicate; analyzed by ordinary one-way ANOVA with Dunnett’s test. c T cells were generated as in ( a ). IFN-γ, IL-2, and FoxP3 transcription ( c ) and FoxP3 expression during 10-day induced Treg (iTreg) differentiation ( d ) were determined by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. e – f Human CD4⁺ naive T cells were skewed to Tregs for 7 days. Suppressive function was assessed by co-culture with CFSE-labeled responder Teff cells; CFSE dilution in CD4⁺CFSE⁺ Teff cells was analyzed by flow cytometry to determine the percentage of undivided Teff cells. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test. g – h iTreg cells (4-day differentiation) were transduced with shCK or shA2AR constructs (shA2AR-2, -3), and mRNA levels of indicated genes, including FoxP3, were measured by qRT-PCR. Data are presented as mean ± SD ( n = 3 biological replicates); One-way ANOVA with Dunnett’s test. i iTreg cells were generated and the recruitment change of CREB on FoxP3a (FoxP3) promoter w/o TAIII treatment was confirmed by CUT & RUN assay. Data are presented as mean ± SD ( n = 3 biological replicates); ordinary one-way ANOVA with Dunnett’s test.

Article Snippet: For immunoblotting, the following primary antibodies were used: β-Actin (Yeason, 30101ES60, 1:5000), CREB (Cell Signaling Technology, 9197 T, 1:1000), phospho-CREB (Ser133; Cell Signaling Technology, 9198 T, 1:1000), A2AR (Santa Cruz Biotechnology, sc-32261, 1:1000), and GAPDH (Cell Signaling Technology, #2118, 1:5000).

Techniques: RNA Sequencing, Enzyme-linked Immunosorbent Assay, Generated, Expressing, Quantitative RT-PCR, Co-Culture Assay, Labeling, Flow Cytometry, Transduction, Construct

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a Photosensitive biotinylated-TAIII (TAIII-P) was generated via click chemistry and used for proximity labeling. b Human T cells isolated from PBMCs were treated with TAIII-P (0–20 μM) for 4 h. Association with A2AR was assessed by avidin-biotin pulldown and western blot (upper), and quantified using ImageJ (lower). Data are presented as mean ± SD from four independent experiments ( n = 4 biological replicates); two-way ANOVA with Dunnett’s test. c – d Competition assay in cell lysates pretreated with TAIII, followed by TAIII-P pulldown. Relative A2AR levels were quantified. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test. e 293 T cells were co-transfected with A2AR and a CREB luciferase reporter, followed by treatment with the indicated compounds. CREB transcriptional activity was measured using a luciferase assay. NECA was not included, as A2AR overexpression alone was sufficient to activate the CREB reporter. f 293 T cells transfected with A2AR were incubated with NECA (0.1 μM) and TAIII or AZD4635 for 30 min. cAMP levels were determined by LANCE assay; fluorescence ratios (615/665 nm) were proportional to cAMP. Blank wells were negative controls. Data are presented as mean ± SD from four independent experiments ( n = 4 biological replicates); ordinary one-way ANOVA with Dunnett’s test. g 293 T cells transfected with or without A2AR were treated with indicated compounds; cAMP levels were measured by LANCE assay. Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test. h Predicted mode of binding of TAIII to A2AR based upon molecular modeling (PDB ID: 4EIY). i – j CREB-Fluc reporters with wild-type or mutant A2AR residues were used to assess the contribution of three residues to TAIII binding ( i ) and dose-dependent effects ( j ). Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Dunnett’s test.

Article Snippet: For immunoblotting, the following primary antibodies were used: β-Actin (Yeason, 30101ES60, 1:5000), CREB (Cell Signaling Technology, 9197 T, 1:1000), phospho-CREB (Ser133; Cell Signaling Technology, 9198 T, 1:1000), A2AR (Santa Cruz Biotechnology, sc-32261, 1:1000), and GAPDH (Cell Signaling Technology, #2118, 1:5000).

Techniques: Generated, Labeling, Isolation, Avidin-Biotin Assay, Western Blot, Competitive Binding Assay, Transfection, Luciferase, Activity Assay, Over Expression, Incubation, Fluorescence, Binding Assay, Mutagenesis