Journal: iScience

Article Title: Sulfonylurea receptor 1 deficiency alleviates acute lung injury by activating PARP1 in epithelial cells

doi: 10.1016/j.isci.2026.117627

Figure Lengend Snippet: Glibenclamide disrupted SUR1-PARP1 interaction to restore DNA repair and attenuate apoptosis and inflammation in HBE cells (A) Schematic diagram of HBE cells with different treatments, followed by RNA sequencing analysis. Groups: control, 20 ng/mL; TNF-α, 100 μmol/L; glibenclamide plus TNF-α. n = 2 per group. (B) Venn diagram of differentially expressed genes. (C) GO and KEGG enrichment analyses of 280 genes in the intersection shown in (B). (D) RT-qPCR analysis of HBE cells with different treatments as indicated. (E) Co-immunoprecipitation assay in HBE cells transfected with FLAG-SUR1 plasmids and treated with 20 ng/mL TNF-α or 100 μmol/L glibenclamide as indicated. L.E., long exposure. (F) HBE cells were treated as in (E), then subjected to cytoplasmic and nuclear protein fractionation followed by western blotting. Cyto, cytoplasm; Nu, nucleus. (G) Western blot analysis of HBE cells pretreated with 100 μmol/L glibenclamide for 30 min followed by TNF-α or LPS co-treatment for 1 h. (H–J) ROS production, caspase-3 activity, and apoptosis in HBE cells under indicated treatments. (K–M) HBE cells were pretreated with 100 μmol/L glibenclamide, 5 μmol/L olaparib, or their combination for 24 h, followed by 20 ng/mL TNF-α stimulation, and subjected to western blotting, caspase-3 activity assay, and apoptosis analysis by flow cytometry. Gli, glibenclamide. Data are presented as mean ± SD ( n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. GO and KEGG enrichment analyses in (C) were performed using a hypergeometric test with false discovery rate (FDR) correction. Data in (D) and (G–M) were analyzed using one-way ANOVA followed by Tukey’s multiple-comparisons test. Representative images are shown.

Article Snippet: Caspase 3 Activity Assay Kit , Beyotime Biotechnology , Cat# C1115.

Techniques: RNA Sequencing, Analysis, Control, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Transfection, Fractionation, Western Blot, Activity Assay, Caspase-3 Activity Assay, Flow Cytometry

Journal: iScience

Article Title: Sulfonylurea receptor 1 deficiency alleviates acute lung injury by activating PARP1 in epithelial cells

doi: 10.1016/j.isci.2026.117627

Figure Lengend Snippet:

Article Snippet: Caspase 3 Activity Assay Kit , Beyotime Biotechnology , Cat# C1115.

Techniques: Cell-Signaling, Recombinant, Transfection, Caspase-3 Activity Assay, Protein Extraction, Knock-Out, Sequencing, Control, Plasmid Preparation, Software, Analysis

Journal: Advanced Science

Article Title: Fc‐Dependent Targeting of the GAS6–MERTK Axis With Monoclonal and Bispecific Antibodies Overcomes Drug Resistance Across EGFR Mutations in Exons 19, 20, and 21

doi: 10.1002/advs.78290

Figure Lengend Snippet: Design and in vitro activities of the anti‐MERTK/EGFR bispecific antibody (Bis‐EM). (A) Schematic representation of the bispecific antibody, Bis‐EM, and the parental antibodies, anti‐MERTK mAb745 and cetuximab. The EGFR‐binding arm was formatted as a CrossMab (CH1–CL swap) to ensure correct heavy–light chain pairing. Additionally, heterodimerization of the heavy chains was achieved through inserting the following mutations in the CH3 domain: Y349C, T366S, L368A, and Y407V in the EGFR arm, as well as two mutations, S354C and T366W, in the MERTK arm. V denotes an antibody variable domain, and C marks a constant antibody domain. Likewise, H denotes a heavy antibody chain, and L denotes the light chain. The table lists parameters obtained using Surface Plasmon Resonance (SPR), which determined binding affinities (dissociation constant, Kd) of the parental antibodies, as well as the Kd of Bis‐EM, toward recombinant soluble forms of human MERTK and human EGFR. CTX, cetuximab. This scheme was made using BioRender. (B, C) PC9 cells (5 × 10 4 ) were seeded on coverslips. The next day, cells were treated for various time intervals with the following antibodies: CTX (cetuximab; 10 µg/mL), mAb745 (10 µg/mL), and Bis‐EM (20 µg/mL). Next, cells were fixed (with 4% PFA), permeabilized, and stained with anti‐MERTK and anti‐EGFR antibodies, which were followed by fluorescently tagged secondary antibodies. DAPI was used to visualize nuclei. Images were taken using a spinning disk confocal microscope, and a series of Z‐stacks were taken with a Z‐step of 1 µm. To quantify the intracellular levels of the indicated receptors, the middle section of each cell was selected from the Z‐stack. For each channel, we calculated the average signal in the cytosol (see histograms in C). The obtained signal intensity was used as a threshold value to remove background cytosolic signals. The spots were counted using the function “analyze particles” of ImageJ. The signal corresponding to receptor internalization at t = 0 was used for normalization. * , p < 0.05; ** , p < 0.01; *** , p < 0.001. Scale bar, 15 µm. All experiments were repeated at least twice. (D, E) PC9 cells (3 × 10 3 cells/well) were seeded in 96‐well ultra‐low‐attachment plates and cultured overnight to generate spheroids. Spheroids were treated with the indicated mAbs (10 µg/mL) or with the bispecific antibody Bis‐EM (20 µg/mL) and cocultured with human PBMC at an effector‐to‐target ratio of 50:1. Plates were imaged continuously using the IncuCyte system. Caspase‐3/7 activation was quantified by monitoring fluorescence intensity over 5 days (D). Statistical comparisons between treatment groups were based on fluorescence values measured on day 3 (E). **** , P < 0.0001.

Article Snippet: Cleaved Caspase‐3 , 9661S , Cell Signaling Technology (CST) , AB_2341188.

Techniques: In Vitro, Binding Assay, SPR Assay, Recombinant, Staining, Spinning Disk, Confocal, Microscopy, Cell Culture, Spheroids, Activation Assay, Fluorescence

Journal: Advanced Science

Article Title: Fc‐Dependent Targeting of the GAS6–MERTK Axis With Monoclonal and Bispecific Antibodies Overcomes Drug Resistance Across EGFR Mutations in Exons 19, 20, and 21

doi: 10.1002/advs.78290

Figure Lengend Snippet: Simultaneous inhibition of MERTK and EGFR suppresses growth of NSCLC cells and associates with both inhibition of signaling and activation of apoptosis. (A) PC9 (EGFR exon 19 deletion) and H1975 cells (EGFR‐L858R in exon 21 and the “gatekeeper” mutation T790M) were seeded at low density in 6‐well plates (1000 cells/well). The next day, cells were treated for 9 days with anti‐EGFR (cetuximab), anti‐MERTK mAb (mAb745; each at 10 µg/mL), Bis‐EM (20 µg/mL), osimertinib (10 n m ), or MRX‐2843 (300 n m ). The drugs were replenished once every 72 h. Next, cells were fixed and stained with crystal violet. Images were captured using a bright‐field microscope. Colonies were quantified (CFU, colony formation units) using ImageJ. The experiment was repeated thrice. (B) PC9 and H1975 cells were seeded in 6‐well plates (5 × 10 5 cells per well). Both assays were performed in 6‐well plates. The next day, cells were treated for 9 days as in A, except that osimertinib was used at 300 n m and MRX‐2843 at 1000 n m . Media were replenished once every 3 days. Next, they were fixed and stained with crystal violet. Images of surviving cells were captured using a bright‐field microscope. Drug tolerant persister (DTP) cells were quantified using ImageJ. (C) PC9 cells (1 × 10 3 ) embedded in 5% BME were seeded in BME pre‐coated 96‐well plates. The wells contained vehicle control (CTR), mAbs (10 µg/mL), osimertinib (10 n m ), MRX‐2843 (300 n m ), Bis‐EM (20 µg/mL), and the indicated drug combinations. All experiments were stopped after 7 days. Images of the formed spheroids were captured using an OpTech IB4 microscope (bright field; 20x magnification). Percentages of the substrate covered areas were assessed using ImageJ. Significance was calculated using one‐way ANOVA, Dunnett's and Tukey's multiple comparison tests ( * , p < 0.05; ** , p < 0.01). The histograms represent the average of two independent experiments performed in duplicates. Scale bar, 50 µm. (D, E) H1975 cells were grown to confluence in 10‐cm dishes and treated for 48 h with the indicated TKIs and antibodies, either alone or in combination, at the concentrations indicated in A. Cell lysates were analyzed using immunoblotting and the indicated antibodies. Tubulin and vinculin served as loading controls. The left panel shows downstream signaling, and the right panel shows apoptosis‐related signaling. Representative blots from one of two independent experiments are shown. (F) PC9 cells expressing the Fucci reporter were treated for 1–3 days with the indicated agents or their combinations at the following concentrations: osimertinib (10 n m ), MRX‐2843 (300 n m ), cetuximab (10 µg/mL), mAb745 (10 µg/mL), and Bis‐EM (20 µg/mL). Images were collected daily from at least nine distinct fields of view. Nuclei were detected based on nuclear Fucci fluorescence, and valid nuclei were selected using circularity thresholds to remove fluorescent debris. Green and red signal intensities were measured to assign cells to S phase, G1 phase, or G2/M. Image acquisition was performed on a CV8000 system, followed by processing that made use of Fiji, segmentation, quantification with CellProfiler, and data visualization in R Studio. Note: Original blots corresponding to panels (D, E) can be found in Figure .

Article Snippet: Cleaved Caspase‐3 , 9661S , Cell Signaling Technology (CST) , AB_2341188.

Techniques: Inhibition, Activation Assay, Mutagenesis, Staining, Microscopy, Colony Assay, 96 Well Plates, Control, Spheroids, Comparison, Western Blot, Expressing, Fluorescence

Journal: Advanced Science

Article Title: Fc‐Dependent Targeting of the GAS6–MERTK Axis With Monoclonal and Bispecific Antibodies Overcomes Drug Resistance Across EGFR Mutations in Exons 19, 20, and 21

doi: 10.1002/advs.78290

Figure Lengend Snippet: The EGFRxMERTK bispecific antibody combined with a TKI delays relapse in PDX models in an Fc‐dependent manner. (A, B) The patient‐derived xenograft TM00193 (E746_A750 Del19‐EGFR) was engrafted subcutaneously in NSG mice. When tumors became palpable (around 350 mm 3 ), mice were randomized in groups of 5–7 animals and treated for four weeks (shaded areas) with osimertinib (10 mg/kg/day; orally) and the indicated mAbs (100 micrograms/mouse/injection), or with Bis‐EM (200 micrograms/mouse/injection). Tumor volume and animal body weight were monitored for the next 90 days. The average tumor volume of each group is presented both longitudinally and on Day 55. Data shown are means ± SEM. Kaplan–Meier survival curves corresponding to each experimental arm are presented in B. Survival analysis was performed using Holm–Šídák's multiple comparisons test to assess statistical significance between treatment groups. The adjusted p ‐value for the comparison between osimertinib alone and osimertinib combined with Bis‐EM is smaller than 0.001. (C, D) NSG mice were implanted with a lung cancer PDX model, PDXJ6 (from The Jackson Laboratory, J000100672), harboring the exon 20 mutations S768_V769insSVD. Once tumors reached approximately 300 mm 3 , mice were randomized into groups of 5–7 animals per group. They were later treated for 3 weeks (shaded areas), twice a week, with the indicated mAbs (100 µg of each mAb/injection) or with Bis‐EM (200 µg/injection). Alternatively, mice were treated with poziotinib (5 mg/kg), an exon 20–specific TKI that was administered orally once daily. Tumor growth and body weights were monitored twice per week. Shown are the average tumor volumes, including day 47 statistics, and animal survival times. Data shown are means ± SEM. Survival analysis was performed as in (B). The adjusted p ‐value for the comparison between POZ+CTX and POZ+CTX+mAb745 is smaller than 0.01. Similarly, the adjusted p ‐value for the comparison between POZ+CTX and POZ + Bis‐EM is less than 0.05. POZ, poziotinib; CTX, cetuximab. (E, F) NSG mice were implanted subcutaneously with fragments derived from the PDX model harboring an EGFR exon 20 mutation S768_V769insSVD. Once tumors reached approximately 300 mm 3 , mice were randomized into treatment groups (3 mice per group; M1 through M3). Next, mice were treated for 7 days with Bis‐EM (250 µg per injection), which was administered twice weekly. Where indicated, mice received the exon 20–selective tyrosine kinase inhibitor poziotinib (POZ; 5 mg/kg). This TKI was administered orally once daily, either alone or in combination with Bis‐EM. Seven days after treatment initiation, tumors were measured, excised, and processed for immunoblotting. Extracts were resolved using electrophoresis and analyzed by immunoblotting. Vinculin and beta‐actin were used as loading controls. (G and H) PC9 cells (2.5 × 10 6 cells per animal) were injected subcutaneously into the flanks of CD1‐nu/nu mice. Once tumors became palpable, mice ( n = 7–9 per group) were randomized and treated for 3 weeks with the indicated antibodies, either Bis‐EM or the respective Fc‐mutated Bis‐EM, which were administered at 250 µg per injection, three times per week. Tumor volumes were measured twice weekly, and body weight was monitored weekly throughout the treatment period. Shaded areas indicate the treatment period. Both longitudinal tumor volume changes and the volumes on day 7 are shown. (I, J) NSG mice were implanted with fragments of the J6 patient‐derived xenograft (PDX) model harboring the EGFR exon 20 insertion mutation S768_V769insSVD. When tumors reached approximately 300 mm 3 , mice were randomized into the indicated treatment groups (7 mice per group). Mice were treated for 3 weeks (shaded areas) with Bis‐EM or with the Fc‐mutated Bis‐EM antibody, at 250 µg per injection, administered three times weekly. Tumor volumes and body weight were monitored twice weekly throughout the treatment period. Both longitudinal tumor volume changes and the volumes on day 17 are shown. Data are presented as mean ± SEM. Note: Original blots corresponding to panels (E, F) can be found in Figure .

Article Snippet: Cleaved Caspase‐3 , 9661S , Cell Signaling Technology (CST) , AB_2341188.

Techniques: Derivative Assay, Xenograft Assay, Injection, Analysis, Comparison, Mutagenesis, Western Blot, Electrophoresis

Journal: Advanced Science

Article Title: Fc‐Dependent Targeting of the GAS6–MERTK Axis With Monoclonal and Bispecific Antibodies Overcomes Drug Resistance Across EGFR Mutations in Exons 19, 20, and 21

doi: 10.1002/advs.78290

Figure Lengend Snippet:

Article Snippet: Cleaved Caspase‐3 , 9661S , Cell Signaling Technology (CST) , AB_2341188.

Techniques: Cell-Signaling

Journal: Advanced Science

Article Title: Fc‐Dependent Targeting of the GAS6–MERTK Axis With Monoclonal and Bispecific Antibodies Overcomes Drug Resistance Across EGFR Mutations in Exons 19, 20, and 21

doi: 10.1002/advs.78290

Figure Lengend Snippet:

Article Snippet: Caspase‐3 , 9662S , Cell Signaling Technology (CST) , AB_331439.

Techniques: Cell-Signaling

Journal: CNS Neuroscience & Therapeutics

Article Title: Tumor Treating Fields Enhance Temozolomide‐Associated Growth Suppression in Glioblastoma Cells and Patient‐Derived Organoids Through Cell‐Cycle Blockade, Apoptosis, and SMAD ‐Associated Signaling

doi: 10.1002/cns.71195

Figure Lengend Snippet: TMZ+TTF treatment enhances apoptotic responses in glioblastoma cells. (A) Immunofluorescence staining of cleaved caspase‐3 (red) and nuclei (blue, DAPI) in U251 and SHG140 cells under different treatment conditions. (B) Quantification of cleaved caspase‐3 fluorescence intensity. (C, E) Representative Annexin V‐FITC/PI flow cytometry plots showing the distribution of viable and apoptotic cell populations at 24 and 48 h in U251 and SHG140 cells, respectively. Lower left: Viable cells; lower right: Early apoptotic cells; upper right: Late apoptotic cells; upper left: PI‐positive non‐apoptotic/dead cells. (D, F) Quantitative analysis of viable‐cell fractions derived from Annexin V‐FITC/PI flow cytometry in U251 and SHG140 cells at 24 and 48 h. For cleaved caspase‐3 quantification, three random 20× fields were averaged within each independent experiment. At least 10,000 events were acquired per flow‐cytometry sample. Data are presented as mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test within each time point. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For cleaved caspase‐3 immunofluorescence, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, blocked with 5% BSA, and incubated overnight at 4°C with anti‐cleaved caspase‐3 antibody (Proteintech, USA; Cat. No. 25546–1‐AP; 1:200).

Techniques: Immunofluorescence, Staining, Fluorescence, Flow Cytometry, Analysis, Derivative Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Tumor Treating Fields Enhance Temozolomide‐Associated Growth Suppression in Glioblastoma Cells and Patient‐Derived Organoids Through Cell‐Cycle Blockade, Apoptosis, and SMAD ‐Associated Signaling

doi: 10.1002/cns.71195

Figure Lengend Snippet: ALK5/TGFβRI inhibition partially attenuates TTF‐associated SMAD phosphorylation, growth suppression, and cleaved caspase‐3 induction. (A) Western blot analysis of phosphorylated SMAD2 (p‐SMAD2), phosphorylated SMAD3 (p‐SMAD3), and total SMAD2 in U251 and SHG140 cells treated with vehicle control, TTF, SB525334, or TTF+SB525334. (B) Quantification of p‐SMAD2, p‐SMAD3, and total SMAD2 protein expression levels. Densitometric values for p‐SMAD2, p‐SMAD3, and SMAD2 were normalized to GAPDH. (C, D) CCK‐8 assays measuring cell viability in U251 and SHG140 cells treated with vehicle control, TTF, SB525334, or TTF+SB525334 under the same 72 h assay conditions used in Figure . SB525334 was used at 10 μM as a selective ALK5/TGFβRI inhibitor. (E) Immunofluorescence staining of cleaved caspase‐3 (red) and nuclei (blue, DAPI) in U251 and SHG140 cells under the indicated treatment conditions; merged images are shown. (F) Quantification of cleaved caspase‐3 fluorescence intensity. For cleaved caspase‐3 quantification, three random 20× fields were averaged within each independent experiment. Data are presented as mean ± SD ( n = 3 independent experiments). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For cleaved caspase‐3 immunofluorescence, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, blocked with 5% BSA, and incubated overnight at 4°C with anti‐cleaved caspase‐3 antibody (Proteintech, USA; Cat. No. 25546–1‐AP; 1:200).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Analysis, Control, Expressing, CCK-8 Assay, Immunofluorescence, Staining, Fluorescence