Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates lung ischemia-reperfusion injury: integrative insights from network pharmacology, molecular dynamics, and experimental validation

doi: 10.3389/fphar.2026.1747977

Figure Lengend Snippet: PTE confers anti-apoptotic and anti-inflammatory protection against LIRI in a PI3K/AKT and JNK/c-Jun dependent manner. In this section, the PI3K inhibitor (LY294002) and the JNK agonist (Anisomycin) were used to further investigate the role of these two signaling pathways in PTE-mediated protection against LIRI. PTE was administered at a high dose (60 mg/kg, i.p. in vivo ; 20 µM in vitro ) at the start of reperfusion or reoxygenation. Both LY294002 (0.3 mg/kg via tail vein injection in vivo ; 10 µM in vitro ) and Anisomycin (15 mg/kg, i.p. in vivo ; 20 μg/mL in vitro ) were given 30 min prior to modeling. (A–H) In vitro and in vivo , Western blotting analysis of pro-apoptotic proteins cleaved Caspase-3 and Bax and the anti-apoptotic protein Bcl-2. (I – P) In vitro and in vivo , concentrations of TNF-α, IL-1β, IL-6, and IL-10 in cell culture supernatants or tissue homogenates were measured by ELISA. (Q,R) Representative images of MPO immunohistochemical staining and the quantitative analysis. (S,T) Representative images of TUNEL staining and the quantitative analysis. MPO staining: scale bar = 50 μm; TUNEL staining: scale bar = 100 μm. Data are presented as the mean ± SD (n = 3 or n = 6). # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, vs. IR group or OGD/R group; ^ P < 0.05, ^^ P < 0.01, ^^^ P < 0.001, ^^^^ P < 0.0001, vs. IR + PTE or OGD/R + PTE group; n.s = non-significant. c-Casp3: cleaved Caspase-3, LY: LY294002, Ani: Anisomycin, i.p.: intraperitoneal injection.

Article Snippet: Pterostilbene (PTE, HY-N0828), LY294002 (LY, HY-10108), and Anisomycin (Ani, HY-18982) were all purchased from MedChemExpress (MCE, United States), and dissolved in dimethyl sulfoxide (DMSO, D8371, Solarbio, China) before dilution to the desired concentrations for subsequent experiments.

Techniques: Protein-Protein interactions, In Vivo, In Vitro, Injection, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, TUNEL Assay

Journal: bioRxiv

Article Title: 5-Azacytidine incorporation into mRNAs disrupts translation and induces ribosome collisions

doi: 10.64898/2026.03.30.714548

Figure Lengend Snippet: (A) Immunoblots of anisomycin-induced eIF2α-P and JNK-P in HCT116 cells pre-treated with A-92 (2 µM, GCN2 inhibitor) or Nilotinib (1 µM, ZAK inhibitor). 1 µg/mL anisomycin was used to induce disomes. (B) Immunoblots of 5-AzaC-induced eIF2α-P, p38-P, JNK-P in HCT116 cells pre-treated with A-92 (2 µM, GCN2 inhibitor) or Nilotinib (1 µM, ZAK inhibitor). Vinculin serves as loading control. These blots serve as the two other biological replicates of the blot shown in . (C) Immunoblots of 5-AzaC-induced eIF2α-P and p38-P in THP-1 cells pre-treated with A-92 (2 µM, GCN2 inhibitor) or Nilotinib (1 µM, ZAK inhibitor). (D) Immunoblots of ZAK, p38-P and JNK-P in SW620 WT and ZAK KO. Vinculin serves as loading control. These blots serve as the two other biological replicates of the blot shown in . (E) Viability assay measuring the effect of A-92 (orange) and Nilotinib (red) on 5-AzaC-induced cell death in HCT116 (left), THP-1 (middle), and SW620 (right) cells. “Mock” (gray) represents the cells that are treated with 5-AzaC as well as the carrier for A-92 and Nilotinib (DMSO). (F) Viability assay measuring the effect of Nilotinib (red) on 5-AzaC-induced cell death in SW620 WT and SW620 ZAK KO (dashed lines) cells. “Mock” (gray) represents the cells that are treated with 5-AzaC as well as the carrier for Nilotinib (DMSO). (G) Colony formation assay in SW620 WT and ZNF598 KO cells with indicated concentrations of 5-AzaC. Biological replicate of . Where applicable, error bar indicates mean ± standard deviation of 3-9 biological replicates.

Article Snippet: For GCN2 and ZAK inhibitor experiments, cells were pretreated with DMSO, 2 μM A-92 (Axon Medchem, 2720) or 1 μM Nilotinib (APExBio, A8232) for 30 minutes followed by treatment with 10 μM 5-AzaC for 4 hours or 1 μg/mL anisomycin (Selleck Chemicals, S7409) for 15 minutes before harvest.

Techniques: Western Blot, Control, Viability Assay, Colony Assay, Standard Deviation

Journal: bioRxiv

Article Title: UFMylation anchors splicing factors at the ER to reprogram nuclear splicing

doi: 10.64898/2026.03.30.715226

Figure Lengend Snippet: a , Phylogenetic distribution of UFM1 across major eukaryotic lineages. Conservation status is color-coded as conserved (blue), partial loss (light blue) or total loss (grey). The inferred evolutionary origin is indicated. b , Co-evolutionary heatmap of core UFMylation machinery and co-evolved genes across representative eukaryotes. Color intensity indicates copy number; supergroups shown on the right. c , Association plot of nuclear genes co-evolving with UFMylation components (point-biserial correlation ≥ 0.3, P < 0.05). Candidates are colored by functional category, highlighting enrichment in DNA damage, chromatin, and RNA processing pathways. d , Dot plot illustrating AlphaFold2 (AF2) Multimer interaction predictions of coevolved genes against UFMylation machinery. The Y-axis represents the average protein interaction score across the top three ranked models, calculated from domain-specific Predicted Aligned Error (PAE) values rescaled from 0 to 1. Abbreviations: P1, P2: UFSP1 and 2; E1, E2, E3: UBA5, UFC1, and UFL1; C: CDK5RAP3; D: DDRGK1. Coevolved genes with Scaled PAE ≥ 0.75 are colored according to key in . e , Experimental workflow for nuclear fractionation of 10-day-old Arabidopsis Col-0 and ufm1 seedlings treated with DMSO or anisomycin (ANS, 4 h). f , GO enrichment of proteins altered by ANS. Top four terms per condition ( ≤ 300 genes, adjusted P ≤ 0.01, fold enrichment ≥ 5) are shown. Dot size and color indicate − log 10 (FDR) and fold enrichment, respectively. g, h , Scatter plots of log 2 fold-changes (ANS versus DMSO) in ufm1 (x-axis) versus Col-0 (y-axis). Proteins significantly altered in Col-0 (green, g ) or ufm1 (blue, h ) are highlighted, dot size reflects − log 10 (FDR). i , Immunoblotting analysis of RSZ22 abundance in total (whole cell lysates) and nuclear fractions from 10-day-old RSZ22 -3 × FLAG seedlings (Col-0 and ufm1 backgrounds) treated with DMSO or ANS (4 h). H3 and UGPase are nuclear and cytosolic markers. Mono-(1) and di-(2) UFMylated RPL26 are indicated. See (Fig. S1m) and source data for replicates. j , Quantification of RSZ22 nuclear abundance normalized to H3. Dots represent independent biological replicates; bars indicate mean ± standard error of the mean (s.e.m.). Statistical significance: Wilcoxon rank sum test, **** P < 0.0001; ns , not significant. Fig. S1 on page 27

Article Snippet: For chemical treatments, 7– to 10-day-old Arabidopsis seedlings grown in liquid 1/2 MS medium were treated with 100 μ M anisomycin (ANS, Santa Cruz Biotechnology) or an equal volume of DMSO (control) for 4 h under continuous light with shaking at 90 rpm.

Techniques: Functional Assay, Fractionation, Western Blot

Journal: bioRxiv

Article Title: UFMylation anchors splicing factors at the ER to reprogram nuclear splicing

doi: 10.64898/2026.03.30.715226

Figure Lengend Snippet: a–c , Quantification of alternative splicing changes induced by anisomycin (ANS)-mediated ribosome stalling in nuclear RNA from Arabidopsis thaliana seedlings ( a ) and human RKO cells ( b ), and total RNA from mouse neurons ( c ). Significant events were identified in wild-type (WT) samples using rMATS (FDR < 0.05) with an absolute change in percent spliced-in ( | ΔPSI | > 0.1), for all event types except cassette exons ( | ΔPSI | > 0.2). Events detected in WT but absent in UFM1 -deficient systems (human UFM1 knockout, mouse Ufm1 knockout, and Arabidopsis ufm1 knockout) were classified as UFM1-dependent (light shading); events in both WT and UFM1 -deficient backgrounds were classified as UFM1-independent (dark shading). Event type: A3, alternative 3 ′ splice site; A5, alternative 5 ′ splice site; MXE, mutually exclusive exons; IR, retained intron; CE, cassette exon. d–f , Scatter plots comparing percent spliced-in (PSI) value under control and ANS conditions for UFM1 dependent intron retention events. WT events are shown in yellow, and events in UFM1 -deficient systems are shown in dark yellow with black outlines; dot size reflects the False Discovery Rate (FDR). Mean Absolute Error (MAE) values summarize global splicing shifts per genotype. g, h , Genomic distribution of UFM1-dependent ( g ) and independent ( h ) retained introns across 5 ′ untranslated regions (UTRs), coding sequences (CDS), and 3 ′ UTRs. Odds ratios calculated relative to constitutive introns. Ribosome stalling-dependent intron removal is significantly enriched in 3 ′ untranslated regions (3 ′ UTRs) (Fisher’s exact test; * P < 0.05, ** P < 0.001, *** P < 0.0001). i , Length distribution of 3 ′ UTRs colored by species and shaded by type: all 3 ′ UTRs with introns (dark shade), 3 ′ UTRs with UFM1-independent IRs (medium shade), and 3 ′ UTRs with UFM1-dependent IRs (light shade). The 3 ′ UTRs undergoing stalling-induced intron retention are significantly longer than all 3 ′ UTRs with constitutive introns in the genome (Wilcoxon rank-sum test; * P < 0.05, ** P < 0.001, *** P < 0.0001). j, k , Metagene profiles of splice junction coverage ( j ) and stop codon density ( k ) relative to intron position. Compared with constitutive introns (grey), which are efficiently spliced and typically reside downstream of stop codons (canonical 3 ′ UTR introns), UFM1-dependent (green) and independent (orange) introns retain junction coverage and are enriched for premature termination codons near the 3 ′ splice site. Persistence junction signal downstream of the 55-nucleotide nonsense-mediated decay (NMD) boundary (yellow line) indicates these introns are potentially sensitive to NMD. Kb: Kilobases; B: Bases. l , Splice site strength quantified by maximum entropy (MaxEnt) scores. Both UFM1-dependent and independent introns display weaker 5 ′ and 3 ′ splice sites compared with constitutive introns (Wilcoxon rank-sum test; *** P < 0.0001), with UFM1-dependent introns exhibiting significantly weaker 5 ′ splice sites. m , Sequence logos of nucleotide composition surrounding the 5 ′ and 3 ′ splice sites. UFM1-dependent introns characterized by a conserved 5 ′ exonic CAG motif and C-rich sequences (see (Fig. S7e–g) for nucleotide probability). n , Comparison of RNA-binding protein (RBP) motif enrichment in UFM1-dependent versus UFM1-independent introns relative to constitutive introns from human. The diagonal indicates equal enrichment ( y = x ). Dashed curves denote hyperbolic significance thresholds ( y = x + c + k/x , where c = 1.0 and k = 1.5). Dot size represents the absolute difference in − log 10 (E-value) between UFM1-dependent and UFM1-independent enrichments. UFM1-dependent introns are preferentially enriched for CAG and C-rich motif-binding RBPs (e.g., SRSF3, YTHDC1, SRSF5); UFM1-independent introns enriched for G-rich binding factors (e.g., ILF2, TAF15). o, p , Subcellular localization ( o ) and signal peptide ( p ) analyses of transcripts containing stalling-induced introns. Colored according to key in . Both UFM1-dependent and independent targets are depleted for secretory pathway annotations and signal peptides (Fisher’s exact test; * P < 0.05, *** P < 0.0001). q , GO enrichment analysis of UFM1-dependent transcripts shows overrepresentations of biological processes related to membrane lipid metabolism and endomembrane-associated processes rather than protein secretion. Fig. S6 and S7 on page 33 and 35

Article Snippet: For chemical treatments, 7– to 10-day-old Arabidopsis seedlings grown in liquid 1/2 MS medium were treated with 100 μ M anisomycin (ANS, Santa Cruz Biotechnology) or an equal volume of DMSO (control) for 4 h under continuous light with shaking at 90 rpm.

Techniques: Alternative Splicing, Knock-Out, Control, Sequencing, Comparison, RNA Binding Assay, Binding Assay, Membrane