Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Heatmap of differentially expressed genes in RIVA cells containing vector or BCL10 mutants, (FC ≥ 1.5x up or down) . B Venn diagram of the number of differentially expressed genes in BCL10 mutants compared to vector. C Top 10 upregulated (adjusted p value ≤ 0.001) Jensen compartment complexes using genes upregulated with a FC ≥ 2. D The top overlapping Hallmark gene set pathways upregulated (FDR q-value ≤ 0.25) in BCL10 mutants. E Western blot of RIVA and HBL1 cells induced with doxycycline for 24 h and probed as indicated. F TNFα ELISA assay on cell supernatant from RIVA, HBL1, and U2932 cells containing vector or BCL10 mutants. G Boxplot of gene expression levels of TNFRSF13B and USP2 of untreated DLBCL cases comparing mutant versus wildtype BCL10 cases.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Gene Expression, Mutagenesis

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Gene ontology analysis of genes upregulated in BCL10 mutant RIVA cells showing top 10 of 49 significantly upregulated ontologies (adjusted p < 0.001). Ontologies that were completely overlapping in genes were combined. B GSEA “BIOCARTA_CYTOKINE_PATHWAY” for BCL10 mutants (q = 0.082 S136X, q = 0.176 for R58Q). C Venn diagram of cytokine genes upregulated in BCL10 mutants with array validations indicated. D Boxplot of gene expression levels of CCL22 and IL7 of untreated DLBCL cases comparing mutant versus wildtype BCL10 cases. E ELISA assays of IL6, IL7 and TNFβ on cells supernatant of RIVA and HBL1 cells induced for 24 (IL7 and TNFβ) or 72 h (IL6) with doxycycline, ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Mutagenesis, Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Genes that were upregulated (FC ≥ 2) in both mutants were analyzed for enrichment in regulation by transcription factors from the ENCODE project through the online transcription factor analysis tool ChIP-X Enrichment Analysis Version 3 (ChEA3). Sixteen transcription factors were significantly upregulated in BCL10 mutants (FDR ≤ 0.05). B Schematic of transcription factors identified to be upregulated in ( A ). C Boxplot of gene expression levels of BATF and IRF4 of untreated DLBCL cases comparing mutant versus wildtype BCL10 cases. D Western blot of RIVA and HBL1 cells induced with doxycycline for 24 h and probed as indicated. E Western blot of RIVA and U2932 cells induced with doxycycline for 24 h and probed as indicated. F ELISA assays in RIVA AND U2932 cell supernatant probing for CXCL10 (RIVA - S136X: p = 0.0046, R58Q: p = 0.0003, U2932- S136X: p = 0.0003, R58Q: p = 0.0028).

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Gene Expression, Mutagenesis, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Dose response viability assays of RIVA cells treated with pirtobrutinib (S136X: p = 0.033, R58Q: p = 0.019), acalabrutinib (S136X: p = 0.07, R58Q: p = 0.07), and ibrutinib (S136X: p = 0.023, R58Q: p = < 0.0001). B Epigenetic Library (TargetMol) on doxycycline induced RIVA cells. Hits are compounds that significantly inhibited cell viability and compounds that had an increase or decrease in viability two standard deviations from the mean of the difference between BCL10 S136X and vector after 72 h of 10 μM drug treatment. Targets in red highlight mutant-driven resistance, mutant-sensitive targets are in blue, and compounds that sensitized both vector and BCL10 S136X are in purple. C Schematic of hits from 4B. D Dose response viability assays of RIVA cells treated with selcidemstat (p=ns), tovorafenib (p = ns), ulixertinib (S136X: p = 0.0125, R58Q: p = 0.0103), JNKi (p = ns), duvelisib (S136X: p = 0.126, R58Q: p = 0.0264), idelalisib (S136X: p = ns, R58Q: p = 0.004), capivasertib (S136X: p = 0.0002, R58Q: p = 0.005), venetoclax (S136X: p = 0.008, R58Q: p = 0. 0.046), and AZD1208 (PIMi) (S136X: p = 0.019, R58Q: p = 0.040).

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Plasmid Preparation, Mutagenesis

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Normalized counts from gene expression data for BCL2 family genes BCL2, BCL2L1 , and BCL2A1 in the BCL10 mutants. B Western blot of proteins implicated in venetoclax resistance including BCL-xL, BFL1, and BCL2 in doxycycline induced RIVA cells. C Transcript levels of doxycycline induced RIVA cells treated with DMSO or 5uM pirtobrutinib for 24 h: BCL2 (vector: p = 0.0325, R58Q: p = 0.005, S136X: p = 0.0006), BCL2L1 (vector: p=ns, R58Q: p = 0.0484, S136X: p = 0.0007), BCL2A1 (vector: p = 0.0087, R58Q: p = 0.0121, S136X: p = 0.0005). D Mitochondrial membrane potential flow cytometry assay in RIVA vector compared to RIVA R58Q and S136X at baseline. E Time course for BCL-xL, BFL1, and BCL2 in 5 μM pirtobrutinib treated RIVA cells. F Transcript levels of VDACs and TOMM22 after 24-h treatment with pirtobrutinib in RIVA cells. G Mitochondrial membrane potential flow cytometry assay in RIVA vector compared to RIVA R58Q and S136X after treatment with DMSO, pirtobrutinib, venetoclax or the combination for 2, 6 or 20 h. H Western blot of doxycycline induced RIVA cells treated with DMSO, venetoclax, pirtobrutinib or the combination for 24 h.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Gene Expression, Western Blot, Plasmid Preparation, Membrane, Flow Cytometry

Journal: Blood Cancer Journal

Article Title: Bruton’s tyrosine kinase inhibition re-sensitizes multidrug-resistant DLBCL tumors driven by BCL10 gain-of-function mutants to venetoclax

doi: 10.1038/s41408-025-01214-y

Figure Lengend Snippet: A Upregulated transcription factor protein-protein interactions in genes upregulated by the BCL10 mutants probed through the Enrichr website ( p < 0.05). B Western blot showing phosphorylated STAT3 and total STAT3 in HBL1 and RIVA cells containing BCL10 mutants. C IL6 transcript levels of RIVA vector and mutants treated with pirtobrutinib for 24 h ( p < 0.0001). D IL6 cytokine levels in RIVA vector and mutants supernatant treated/untreated with 10uM pirtobrutinib for 24 h. E , F Western blot of doxycycline induced RIVA cells treated with DMSO, venetoclax, pirtobrutinib or the combination for 24 h. G Western Blot analysis of RIVA S136X cells treated with increasing concentrations of ruxolitinib with or without tocilizumab (IL6 inhibitor). H Western Blot analysis of RIVA S136X cells treated with increasing concentrations of ruxolitinib with or without the addition of TNFa. I IL6 transcript levels of RIVA S136X cells treated with TNFa alone or with ruxolitinib. J Schematic of TNFa / IL6/JAK/STAT signaling in BCL10 mutant cells. K Dose response viability assays of RIVA and HBL1 cells treated with pirtobrutinib (HBL1 clone1: p = 0.0027, HBL1 clone2: p = 0.0013, RIVA clone1: p = 0.0029, RIVA clone2: p < 0001) and ibrutinib (HBL1 clone1: p = 0.0401, HBL1 clone2: p = 0.0028, RIVA clone1: p = 0.3089, RIVA clone2: p < 0001), clones1 and 2 are the mutation (S136X) engineered at BCL10 gene locus. L Synergy assessments of venetoclax plus pirtobrutinib in endogenous BCL10 mutants (S136X) with reported synergy score.

Article Snippet: To more fully assess impact of BCL10 mutations on treatment resistance, we interrogated the 960-compound TargetMol Epigenetic Library.

Techniques: Protein-Protein interactions, Western Blot, Plasmid Preparation, Mutagenesis

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Schematic diagram showing domain organization and variant fragments of Doc2B and Munc13-1. ( B ) Binding of MUN to GST-Doc2B FL or its variant fragments measured by GST pull-down experiments and quantification of the binding. ( C ) ITC-based measurements of MUN binding to GST-Doc2B 13 to 37 (Mid, left) and to GST-Doc2B 13 to 80 (Mid-L, right). ( D ) ITC-based measurements of the binding affinities between GST-Doc2B FL or its variant fragments and MUN. ( E ) 2D 1 H- 15 N HSQC spectra of 13 C/ 15 N-labeled Doc2B 1 to 80 before (black) and after (red) addition of MUN. Cross-peaks of residues that are chosen for mutation to detect MUN binding are labeled along with their corresponding residue number (cyan). ( F ) Peak intensity alteration of 13 C/ 15 N-labeled Doc2B 1 to 80 protein expressed as ratio between integrated peak volumes after ( V ) and before ( V 0 ) addition of unlabeled MUN. ( G ) Binding of Doc2B FL or its variant mutations to GST-MUN measured by GST pull-down experiments and quantification of the binding. ( H ) Binding of MUN or its variant fragments to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Red asterisk shows the band of bound MUN-BC. ( I ) Binding of MUN or its variant mutations to GST-Doc2B FL measured by GST pull-down experiments and quantification of the binding. Data of GST pull-down experiments are processed by ImageJ (National Institutes of Health) and presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001; ns, not significant.

Article Snippet: For detecting Doc2B binding to EphB2 or Munc13-1 from brain samples, adult mouse brain was initially ground in buffer B supplied with 1% Triton X-100 and proteinase inhibitor cocktail (TargetMol) and then incubated with GST-Doc2B FL at 4°C overnight.

Techniques: Variant Assay, Binding Assay, Labeling, Mutagenesis, Residue

Journal: Science Advances

Article Title: Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release

doi: 10.1126/sciadv.adi7024

Figure Lengend Snippet: ( A ) Illustration of the FRET assay for detecting MUN-catalyzed SNARE complex assembly starting from the Munc18-1/Syx1 (1 to 261) complex in the presence of Syb2 (29 to 96), SN25, and MUN. FRET signal between BDPY-labeled Syb2 S61C (donor) and TMR-labeled SN25 S187C (acceptor) was monitored. ( B to D ) MUN-catalyzed SNARE complex assembly by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (B), addition of different concentrations of Doc2B Mid (C), and addition of MUN NFAA or Doc2B I20A, which disrupts Doc2B-MUN interaction (D). Decrease of donor fluorescence at 1500 s is shown in the column at the right of the chart. ( E ) Illustration of Munc13-catalyzed lipid mixing between liposomes bearing Syb2 (1 to 116) and liposomes bearing the Munc18-1/Syx1 (1 to 288) complex in the presence of SN25, Syt1 C2AB, Ca 2+ , and Munc13-1 (C 1 C 2 BMUN). Donor (NBD) fluorescence was monitored at 538 nm. ( F to H ) Munc13-catalyzed lipid mixing by addition of Doc2B Mid, Doc2B Mid-L, or Doc2B FL, respectively (F); addition of C 1 C 2 BMUN NFAA or Doc2B I20A (G). Munc13-catalyzed lipid mixing at 1000 s is shown in (H). F 1 , fluorescence intensity observed as a function time; F 0 , initial fluorescence intensity. Data are presented as means ± SEM ( n = 3). Statistical significance and P values were determined by one-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; *** P < 0.001; **** P < 0.0001.

Article Snippet: For detecting Doc2B binding to EphB2 or Munc13-1 from brain samples, adult mouse brain was initially ground in buffer B supplied with 1% Triton X-100 and proteinase inhibitor cocktail (TargetMol) and then incubated with GST-Doc2B FL at 4°C overnight.

Techniques: Labeling, Fluorescence, Liposomes

Journal: Acta pharmaceutica Sinica. B

Article Title: Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis.

doi: 10.1016/j.apsb.2021.07.018

Figure Lengend Snippet: Figure 1 Representative structures of BRD4 inhibitors.

Article Snippet: The 30 hits were then purchased from Topscience corporation and evaluated toward BRD4 using HTRF assay.

Techniques:

Journal: Acta pharmaceutica Sinica. B

Article Title: Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis.

doi: 10.1016/j.apsb.2021.07.018

Figure Lengend Snippet: Figure 2 Discovery of potent 4-phenylquinazoline derivatives as BRD4 inhibitors.

Article Snippet: The 30 hits were then purchased from Topscience corporation and evaluated toward BRD4 using HTRF assay.

Techniques:

Journal: Acta pharmaceutica Sinica. B

Article Title: Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis.

doi: 10.1016/j.apsb.2021.07.018

Figure Lengend Snippet: Figure 3 (A) Workflow of virtual screening. (B) The structure of hit 5. (C) The predicted binding modes of hit 5 (purple) with BRD4 BD2 (PDB code: 6C7Q).

Article Snippet: The 30 hits were then purchased from Topscience corporation and evaluated toward BRD4 using HTRF assay.

Techniques: Binding Assay

Journal: Acta pharmaceutica Sinica. B

Article Title: Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis.

doi: 10.1016/j.apsb.2021.07.018

Figure Lengend Snippet: Figure 6 (A) The surface binding modes of molecule C-34 (yellow) to BRD4(2) (blue) (PDB code: 6C7Q). (B) The docking model of de- rivative C-34 (yellow) binds to BRD4(2) (blue). Water molecules are shown as red spheres.

Article Snippet: The 30 hits were then purchased from Topscience corporation and evaluated toward BRD4 using HTRF assay.

Techniques: Binding Assay

Journal: Acta pharmaceutica Sinica. B

Article Title: Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis.

doi: 10.1016/j.apsb.2021.07.018

Figure Lengend Snippet: Figure 8 C-34 can effectively bind to BRD4 at the cellular level. (A) NRCFs were treated with 10 mmol/L C-34, C-34N or JQ1 for 4 h, respectively. Next, the cells were collected and heated from 43 to 61 C. The Western blot analysis of BRD4 expression level. (C) NRCFs were pretreated with 0, 1, 5, 10 mmol/L C-34, and then heated at 55 C. The Western blot analysis of BRD4 expression level. (B and D) The quantitative analysis of BRD4 expression level. Date were expressed as mean SD, n Z 3. Three individual experiments were implemented for per group. **P < 0.01, ***P < 0.001 vs. control.

Article Snippet: The 30 hits were then purchased from Topscience corporation and evaluated toward BRD4 using HTRF assay.

Techniques: Western Blot, Expressing, Control

Journal: Acta pharmaceutica Sinica. B

Article Title: Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis.

doi: 10.1016/j.apsb.2021.07.018

Figure Lengend Snippet: Figure 11 Compound C-34 can alleviate Ang II-induced activation of TGF-b1/Smad2/3 signaling pathway in NRCFs. The expression levels of a-SMA, collagen I, collagen III, TGF-b1, p-Smad2, Smad2, p-Smad3, Smad3, BRD4 and c-MYC were measured by Western blot and the quantitative analysis normalized to GAPDH. Three individual experiments were carried out for per group. Data were expressed as mean SD, n Z 3. **P < 0.01, ***P < 0.001 vs. control, ##P < 0.01, ###P < 0.001 vs. Ang II group, ʌP < 0.05, ʌʌP <0.001 vs. C-34 treatment group.

Article Snippet: The 30 hits were then purchased from Topscience corporation and evaluated toward BRD4 using HTRF assay.

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Acta pharmaceutica Sinica. B

Article Title: Discovery of novel 4-phenylquinazoline-based BRD4 inhibitors for cardiac fibrosis.

doi: 10.1016/j.apsb.2021.07.018

Figure Lengend Snippet: Figure 14 Effects of C-34 on ISO-induced pathological cardiac remodeling in vivo. (A) Representative traces of M-mode echocardiography were used to quantify ejection fraction. (B) EF%, ejection fraction. (C) FS%, fractional shortening. (D) HW/BW, ratio of heart weight to body weight. (E) HW/TL, ratio of heart weight to tibia length. (F) Masson trichrome staining from experimental mice’s heart sections. Blue staining indicates fibrosis. Scale bar Z 50 mm. (G) The quantitative results of relative fibrotic area by Image J software (n Z 5 each group). (H) The images of HE staining (n Z 5 each group). Scale bar Z 50 mm. (I) and (J) The expression levels of collagen I, BRD4, c-MYC, TGF-b1, p-Smad2 and p-Smad3 were measured by Western blot and quantitative analysis normalized to GAPDH (n Z 3 each group). The dates were represented as mean SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. ISO group.

Article Snippet: The 30 hits were then purchased from Topscience corporation and evaluated toward BRD4 using HTRF assay.

Techniques: In Vivo, Staining, Software, Expressing, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Acid sphingomyelinase recruits palmitoylated CD36 to membrane rafts and enhances lipid uptake

doi: 10.1016/j.jbc.2025.110213

Figure Lengend Snippet: OxLDL increases ASMase expression and promotes sphingomyelin (SM) catabolism. A , schematic illustration of SM catabolism. SM is hydrolyzed by ASMase to generate ceramide and phosphatidylcholine. Ceramidases convert ceramides into sphingosine and fatty acids. B , Western blot detection of ASMase expression is performed in cells treated with varying concentrations of oxLDL (50–120 μg/ml; one-way ANOVA followed by Tukey’s post hoc test, n = 3). C , RT-qPCR analysis measures ASMase mRNA levels in oxLDL (50 μg/ml) treated macrophages. D , ASMase enzymatic activity is assessed in cells treated with oxLDL at concentration of 50 μg/ml. E , OxLDL-treated cells are seeded on coverslip and incubated with SM-binding protein, lysenin-His (1 μg/ml). Membrane SM is detected using immunofluorescence staining and quantified by flow cytometry. F – H , total cellular levels of SM and ceramides, and SM/ceramide ratio are measured by LC-MS/MS in oxLDL-treated cells (50 μg/ml). I and J , lipidomics analysis of specific SM and ceramide species in cells treated with oxLDL (50 μg/ml). All comparisons were conducted with Student's t test except in ( B ) (mean ± SD, n = 3). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant.

Article Snippet: After blocking with 2% BSA/PBS, 1 μg/ml lysenin-His (TargetMol) dissolved in the blocking solution was added and incubated at 4 °C overnight.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Activity Assay, Concentration Assay, Incubation, Binding Assay, Membrane, Immunofluorescence, Staining, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: A biomimetic senotherapy replenishing MAT2A promotes wound regeneration in preclinical models

doi: 10.1038/s41467-025-65659-2

Figure Lengend Snippet: A Effect of endogenous Mat2a knockdown followed by restoration of Mat2a WT, MUT1 or SAM (500 μM) on pericyte senescence, as determined by SAHF formation (H3K9me3 staining) and SA-β-gal staining ( n = 3). B Western blot assessment of the expression levels of P21 and Lamin B1 in the indicated pericytes. C – E OCR measurement, maximal respiration analysis and cellular ATP assessment of pericytes following endogenous Mat2a knockdown with Mat2a WT, MUT1 or SAM (500 μM) restoration ( n = 3). F Scheme displaying the procedure used for identifying the specific targets of MAT2A through proteomic and IP-MS analysis. The workflow was created with BioRender.com. G Heatmap showing the change direction of differential proteins in pericytes with or without Mat2a knockdown. H Display showed the differentially regulated proteins, categorized per known or predicted function(s), literature and sequence similarity. Circle size was proportional to the number of differentially expressed proteins. I Intersection of the results from the proteomics and IP-MS analyses. J Scheme displaying the HMGCS1-mediated MVA pathway. K IP and WB analyses showing the interaction of MAT2A and HMGCS1 in 293T cells with indicated transfections. L In vitro binding analysis of MAT2A and HMGCS1 with GST pull-down assays. M Design of MAT2A and HMGCS1 truncations. N IP and WB analysis representing the interactions between Flag-tagged truncated MAT2A and His-tagged PRMT1 proteins in 293T cells. O IP and WB analysis representing the interactions between His-tagged truncated HMGCS1 and Flag-tagged MAT2A proteins in 293T cells. P Molecular docking showing the interaction between MAT2A truncation (slate) and HMGCS1 truncation (cyan). Q Docked positions of MAT2A and HMGCS1 and design of the mutations of binding sites between MAT2A and HMGCS1. R IP and WB analysis of the interactions between FLAG-tagged MAT2A mutation (MUT2) and His-tagged HMGCS1 mutation in 293T cells. Data were shown as mean ± SD. n = 3 biologically independent samples ( A , C , D , E ). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test ( A , D , E ). n.s. no significance. Source data are provided as a Source Data file.

Article Snippet: D OCR measurement in pericytes with Mat2a knockdown followed by transfection of Hmgcs1 , or CoQ (TargetMol, USA, T2796) restoration.

Techniques: Knockdown, Staining, Western Blot, Expressing, Protein-Protein interactions, Sequencing, Transfection, In Vitro, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: A biomimetic senotherapy replenishing MAT2A promotes wound regeneration in preclinical models

doi: 10.1038/s41467-025-65659-2

Figure Lengend Snippet: A HMGCS1 expression levels in pericytes with Mat2a knockdown followed by transfection of Mat2a WT or MUT2. B HMGCS1 expression levels in pericytes treated with cycloheximide (CHX, 100 μg/ml) for the indicated times (top) and relative HMGCS1 protein levels (bottom). C HMGCS1 expression levels in Mat2a -knockdown pericytes treated with or without 10 μM MG132 for 8 h. D Ubiquitination of HMGCS1 in pericytes with the indicated transfections and treatment with 10 μM MG132 for 8 h. E Identification of ubiquitination related modification factors from IP/MS data. HMGCS1 expression levels in pericytes following Otub1 knockdown with or without WT restoration. The workflow was created with BioRender.com. F Ubiquitination of HMGCS1 in 293T cells with transfection of OTUB1 or the indicated mutant and treatment with 10 μM MG132 for 8 h. G Co-localization analysis of MAT2A, OTUB1 and HMGCS1 by immunofluorescence staining in pericytes. H Binding analysis of HMGCS1 and OTUB1 following MAT2A transfection or not in 293T cells treated with 10 μM MG132 for 8 h. I Ubiquitination of HMGCS1 in 293T cells with the indicated transfection of OTUB1 and MAT2A and treatment with 10 μM MG132 for 8 h. J Binding analysis of HMGCS1 and OTUB1 following MAT2A knockdown or not in 293T cells treated with 10 μM MG132 for 8 h. K Ubiquitination of HMGCS1 in 293T cells with the indicated transfection of OTUB1 and knockdown of MAT2A and treatment with 10 μM MG132 for 8 h. L Co-localization analysis of OTUB1 and HMGCS1 following Mat2a knockdown by immunofluorescence staining in pericytes. Data were shown as mean ± SD. n = 3 biologically independent samples ( B ). Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons test ( B ). n.s. no significance. Source data are provided as a Source Data file.

Article Snippet: D OCR measurement in pericytes with Mat2a knockdown followed by transfection of Hmgcs1 , or CoQ (TargetMol, USA, T2796) restoration.

Techniques: Expressing, Knockdown, Transfection, Ubiquitin Proteomics, Modification, Protein-Protein interactions, Mutagenesis, Immunofluorescence, Staining, Binding Assay

Journal: Nature Communications

Article Title: A biomimetic senotherapy replenishing MAT2A promotes wound regeneration in preclinical models

doi: 10.1038/s41467-025-65659-2

Figure Lengend Snippet: A Cellular CoQ levels in pericytes with Mat2a knockdown. B MAT2A and HMGCS1 expression levels in pericytes with the indicated transfections. C Cellular CoQ levels in pericytes with Mat2a knockdown followed by transfection of Mat2a MUT2, Hmgcs1 , or not. D OCR measurement in pericytes with Mat2a knockdown followed by transfection of Hmgcs1 , or CoQ (TargetMol, USA, T2796) restoration. E , F ATP assessment and cell viability in pericytes with Mat2a knockdown followed by transfection of Hmgcs1 , or CoQ restoration. G SA-β-gal staining in pericytes with Mat2a knockdown followed by transfection of Hmgcs1 , or CoQ restoration. H Expression levels of P21 and Lamin B1 in pericytes subjected to the indicated treatments. I RT-qPCR analysis showing the expression levels of typical SASP components in pericytes subjected to the indicated treatments. J , K MitoDsRed+ macrophages were co-cultured with GFP+ pericytes following the indicated treatment. GFP+ MitoDsRed+ pericytes were quantified via flow cytometry ( J ), as summarized in ( K ).Data were shown as mean ± SD. n = 3 biologically independent samples ( A , C – G , I , K ). Statistical significance was determined using two-tailed unpaired t test ( A ), one-way ANOVA with Tukey’s multiple comparisons test ( C – E , G , I , K ) and two-way ANOVA with Tukey’s multiple comparisons test ( F ). n.s. no significance. Source data are provided as a Source Data file.

Article Snippet: D OCR measurement in pericytes with Mat2a knockdown followed by transfection of Hmgcs1 , or CoQ (TargetMol, USA, T2796) restoration.

Techniques: Knockdown, Expressing, Transfection, Staining, Quantitative RT-PCR, Cell Culture, Flow Cytometry, Two Tailed Test