Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: Oligonucleotide sequence for primers used in ChIP-qPCR analysis for TFEB binding to SMPD1 promoter.

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Sequencing, Binding Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: SMPD1 is a direct target gene for TFEB. (A) Western blots and densitometric analysis of TFEB, LAMP-1 and SMPD1 in JEG3 cells following transient transfection with TFEB siRNA or a control (ss) scrambled sequence ( N = 3 separate experiments run in duplicate; * P < 0.05 compared to ss control). (B) Luciferase reporter assay showing SMPD1 expression in JEG3 cells following overexpression of TFEB (OE TFEB) or empty vector (OE EV). ( N = 3 separate experiments; * P < 0.05 compared to empty vector control). (C) qPCR of SMPD1 promoter regions –200 to 300 bp and –900 to 1,000 bp after chromatin immunoprecipitation with TFEB in E-PE and PTC placentae ( N = 4 for each group). (D) qPCR of SMPD1 promoter region –200 to 300 bp after chromatin immunoprecipitation with TFEB in JEG3 cells exposed to 20 μM CER16:0 or EtOH vehicle.

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Western Blot, Transfection, Control, Sequencing, Luciferase, Reporter Assay, Expressing, Over Expression, Plasmid Preparation, Chromatin Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: Ceramide triggers lysosomal exocytosis in JEG3 cells. (A) IF images for LAMP-1 and SMPD1 in JEG3 cells following exposure to 20 μM CER 16:0 or EtOH vehicle. (B) IF images of FITC-dextran (green) loaded JEG3 cells following exposure to 20 μM CER 16:0 or EtOH vehicle. Nuclei: DAPI (blue). (C) Fold change in fluorescence intensity of FITC-dextran in JEG3 cells treated with 20 μM CER 16:0 or EtOH vehicle ( N = 3 separate experiments; * P < 0.05 compared to vehicle). (D) Fold change in fluorescence intensity of released FITC-dextran in media of JEG3 cells treated with 20 μM CER 16:0 or EtOH vehicle ( N = 3 separate experiments; ** P < 0.01 compared to vehicle). (E) Fold change variation of intracellular Ca 2+ content of JEG3 cells treated with 20 μM CER 16:0 versus EtOH vehicle. Data are expressed as mean ± SEM.

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Fluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: L-SMPD1 localizes to lipid rafts of apical syncytial membranes in E-PE placentae and is released into the maternal circulation via exosomes. (A) IF images depicting LAMP-1 and ceramide localization in E-PE and PTC placentae. LAMP-1 (green); Ceramide (red); nuclear DAPI (blue). ST, syncytiotrophoblast. (B) WB and associated densitometry for L-SMPD1 in lysates of apical syncytial membranes (AM) of PTC and E-PE placentae (PTC, N = 3; PE, N = 5; ** P < 0.01 vs. PTC). PLAP was used as AM marker. (C) Total ceramide levels, measured by LC-MS/MS, in syncytial membranes extracts of TC and E-PE placentae ( N = 3 placentae per group; * P < 0.05 compared to control). ( D-left panel ) Distribution of L-SMPD1 in detergent insoluble (Ins) and soluble (Sol) fractions of AM from TC and E-PE placentae. PLAP was used as lipid raft (detergent insoluble fraction) marker. ( D-right panel ) Total ceramide levels measured by LC-MS/MS in extracts of AM Ins and Sol fractions of TC and E-PE placentae ( N = 3 samples per group; * P < 0.05 compared to TC). (E) WB for L-SMPD1, CD63 and PLAP of exosomes isolated from PTC ( N = 3) and E-PE ( N = 3) maternal plasma. (F) WB for L-SMPD1 in PLAP-precipitated exosomes from PTC ( N = 3) and E-PE ( N = 3) maternal plasma. ( F-right panel ) LC-MS/MS quantification of total ceramide in PTC and E-PE PLAP-precipitated exosomes (E-PE, N = 4; PTC, N = 3; * P < 0.05 vs. to PTC). Data are expressed as mean ± SEM. Dotted line: non-contiguous lanes run on the same gel.

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Marker, Liquid Chromatography with Mass Spectroscopy, Control, Isolation, Clinical Proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: SMPD1 enriched exosomes induces endothelial cells activation. (A) WB for L-SMPD1, CD63 and corresponding densitometry of exosomes isolated from conditioned media of JEG3 cells treated with 20 mM CER 16:0 or EtOH ( N = 3 separate experiments). (B) SMPD1 enzyme activity of exosomes from conditioned media of JEG3 cells treated with CER 16:0 or EtOH ( N = 4 separate experiments). RFU, relative fluorescence units. (C) Representative WB for L-SMPD1 of exosomes from JEG3 cells treated with CER 16:0 or EtOH vehicle in conjunction with TFEB siRNA or control (ss) scrambled sequence treatment ( N = 3 separate experiments). (D) WB and densitometry of ICAM-1 in lysates of HMVEC cells treated for 3 h with ExoCer or ExoV ( N = 3 separate experiments). (E) IF images and mean fluorescence intensity quantification of ICAM-1 (Green) in HMVEC cells treated with ExoCer or ExoV ( N = 3 separate experiments). Nuclei: DAPI (blue). ( F-left panels) Representative flow cytometry density plots of CD54 (ICAM-1) and CD146 of HMVEC cells exposed for 3 h to either 2 × 10 6 ExoV or ExoCer, or 20 mM CER 16:0. ( F-right panel ) Quantification of CD54 + /CD146 + HMVEC cells after treatments ( N = 4 experiments). (G) Densitometry of WB for ICAM-1 normalized to stain free gel in HMVEC lysates treated for 3 h with ExoV or ExoCer in presence or absence of 25 μM Imipramine (I) ( N = 3 separate experiments).

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Activation Assay, Isolation, Activity Assay, Fluorescence, Control, Sequencing, Flow Cytometry, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Ceramide-Induced Lysosomal Biogenesis and Exocytosis in Early-Onset Preeclampsia Promotes Exosomal Release of SMPD1 Causing Endothelial Dysfunction

doi: 10.3389/fcell.2021.652651

Figure Lengend Snippet: Active SMPD1 in exosomes from JEG3 cells exposed to ceramide affect endothelial angiogenesis. (A) Angiogenesis assay of HMVEC treated with 2 × 10 6 ExoV or ExoCer ( N = 4 separate experiments). Tube formation was documented after 3 h of treatment by quantification of number of branches and the total length of the segments of the network. Arrows indicates the main branches. Data are expressed as mean ± SEM ( N = 4 separate experiments). (B) Tube formation assay of HMVEC cells treated for 3 h with ExoV or ExoCer in presence or absence of 25 μM Imipramine (I) or 10 μM Fluoxetine (F) and accompanying quantification (C) of tubular branches. Data are expressed as mean ± SEM ( N ≥ 3 separate experiments).

Article Snippet: Antibodies against ICAM-1 (mouse, IF 1:200 WB: 1:500; sc-18853), ACTB (mouse, WB 1:2000; sc-47778), SMPD1 (rabbit, IF 1:200, WB 1:350; sc-11352), CD63 (mouse, WB 1:100; sc-5275) and CD34 (mouse, IHC 1:100) were obtained from Santa Cruz Biotechnology ® (Dallas, TX, United States).

Techniques: Angiogenesis Assay, Tube Formation Assay

Journal: Journal of Advanced Research

Article Title: A novel peptide MIB1-223aa encoded by exosomal circMIB1 from cancer-associated fibroblasts drives triple-negative breast cancer metastasis and stemness via stabilizing MIB1 to activate Notch signaling

doi: 10.1016/j.jare.2025.06.023

Figure Lengend Snippet: CAF enhanced stemness and metastasis of TNBC cells through exosome-transmitted circMIB1. A–B. Knockdown efficiency of circMIB1 in CAFs (A) and the content of circMIB1 in corresponding exosomes (B) detected by qRT-PCR. C. FISH assay showed the expression of circMIB1 in TNBC cells treated with or without indicated exosomes and the scale indicates the length of 20 μm (left). The relative mean fluorescence intensity was analyzed by Image J (right). D. The relative expression of circMIB1 and MIB1 in TNBC cells treated with or without indicated exosomes detected by qRT-PCR. E. Statistical analysis of the relative number of migrated or invaded TNBC cells treated with or without indicated exosomes in transwell assay. F. The expression of EMT-associated markers in TNBC cells treated with or without indicated exosomes was detected by western blot. G–H. Sphere formation assay (G) and Extreme Limiting Dilution Analysis (ELDA) (H) were performed to assess the stemness of TNBC cells treated with or without indicated exosomes. Scale bar: 100 μm. I. Flow cytometry analysis was performed to evaluate the proportion of stem cells in MDA-MB-231 and MDA-MB-468 treated with or without indicated exosomes. J. The expression of stemness-associated markers in TNBC cells treated with or without indicated exosomes was detected by western blot. 1: control; 2: +NF-1 EXO; 3: +CAF-1 si-NC EXO; 4: +CAF-1 si-circMIB1 EXO. K–M. Subcutaneous tumor formation of MDA-MB-231 injected with or without indicated stably transfected CAFs in BALB/C nude mice. Excised tumors were imaged (K) and measured in volume (L) and weight (M). The scale indicates the length of 1 cm. N. HE staining showed the histological morphology of excised tumors in each group. Scale bar: 100 μm. O. The expression of circMIB1 was detected by ISH and α-SMA, MIB1-223aa, Ki67, E-cad, OCT4 detected by IHC (left). Scale bar: 100 μm. Quantitative analysis on the indicated factors in ISH and IHC was performed by H-score (right). (ns, no significance, * P < 0.05, ** P < 0.01).

Article Snippet: TNBC cell lines, MDA-MB-231 (RRID: CVCL_0062) and MDA-MB-468 (RRID: CVCL_0419), and human embryonic kidney cells (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Fluorescence, Transwell Assay, Western Blot, Tube Formation Assay, Flow Cytometry, Control, Injection, Stable Transfection, Transfection, Staining

Journal: Journal of Advanced Research

Article Title: A novel peptide MIB1-223aa encoded by exosomal circMIB1 from cancer-associated fibroblasts drives triple-negative breast cancer metastasis and stemness via stabilizing MIB1 to activate Notch signaling

doi: 10.1016/j.jare.2025.06.023

Figure Lengend Snippet: circMIB1 encoded a novel protein MIB1-223aa. A. Detection of the combination between circMIB1, MIB1 mRNA and ribosome fractions by qRT-PCR. B. Statistical analysis for the distribution proportion of circMIB1, MIB1 mRNA, circPMS1 and Actin mRNA in polysome fractions and non-polysome fractions. C. The wild-type or mutant sequence of circMIB1 IRES was inserted into the dual-luciferase reporter gene vectors (left). The translation initiation activity of circMIB1 IRES in TNBC cells was detected using dual-luciferase reporter gene assay (right). D. The wild-type or mutant sequence of circMIB1 IRES was inserted into the fluorescence vectors (left). The translation initiation activity of circMIB1 IRES in TNBC cells was detected using immunofluorescence assay (right). Scale bar: 100 μm. E. Schematic illustration of endogenous circMIB1, Flag-labelled circMIB1 overexpressing circular vector (circMIB1-Flag), Flag-labelled circMIB1 overexpressing circular vector with mutant stop codon (circMIB1-Flag-TGAmut), and Flag-labelled MIB1-223aa overexpressing linear vector (MIB1-223aa-Flag). F. Expression of Flag tagged MIB1-223aa in differentially transfected HEK293T was detected by western blot. G. Expression of Flag tagged MIB1-223aa in differentially transfected MDA-MB-231 showed by immunofluorescence assay. Scale bar: 50 μm. H. MIB1-223aa was detected using Flag antibody through co-IP assay in differentially transfected MDA-MB-231 (upper). The peptide sequence of MIB1-223aa was identified by LC-MS/MS (lower). I. Expression of MIB1 and MIB1-223aa in 4 pairs of TNBC and corresponding normal tissues. J. Kaplan-Meier curve respectively shows the OS and DFS of TNBC patients with different MIB1-223aa expression levels in TNBC cells. K. Receiver Operating Characteristic (ROC) Curve for the prognostic analysis models. (ns, no significance, * P < 0.05, ** P < 0.01).

Article Snippet: TNBC cell lines, MDA-MB-231 (RRID: CVCL_0062) and MDA-MB-468 (RRID: CVCL_0419), and human embryonic kidney cells (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Quantitative RT-PCR, Mutagenesis, Sequencing, Luciferase, Activity Assay, Reporter Gene Assay, Fluorescence, Immunofluorescence, Plasmid Preparation, Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Advanced Research

Article Title: A novel peptide MIB1-223aa encoded by exosomal circMIB1 from cancer-associated fibroblasts drives triple-negative breast cancer metastasis and stemness via stabilizing MIB1 to activate Notch signaling

doi: 10.1016/j.jare.2025.06.023

Figure Lengend Snippet: circMIB1 enhanced stemness and metastasis of TNBC cells through encoding MIB1-223aa. A. Transfection efficiency of the indicated vectors in TNBC cells assessed by qRT-PCR (upper) and western blot (lower). B. Statistical analysis of the relative number of migrated and invaded cells in transwell assay. C. The expression of EMT-associated markers in differentially transfected TNBC cells was detected by western blot. D. ELDA was performed to assess the stemness of differentially transfected TNBC cells. E. Flow cytometry analysis was performed to evaluate the proportion of stem cells in differentially transfected MDA-MB-231 and MDA-MB-468. F. The expression of stemness-associated markers in differentially transfected TNBC cells was detected by western blot. G. Images of dissected tumors in subcutaneous tumor formation assay established by injection of MDA-MB-231 cells stably expressing indicated plasmids (left). Scale bar: 1 cm. Statistical analysis of the excised tumor volume (upper) and weight (lower) in each group. H. Representative images of dissected lungs from mice in pulmonary metastatic models established by tail vein injection of MDA-MB-231 cells stably expressing indicated plasmids. The metastatic nodes were indicated by red arrows (left). The number of pulmonary metastatic nodes in each group (right). 1: pLCDH-ciR; 2: circMIB1 overexpression; 3: circMIB1-TGAmut; 4: pCDH; 5: MIB1-223aa overexpression. I. IHC was performed to evaluate the expression of Ki67, E-cad, OCT4 in tumor tissues formed by indicated MDA-MB-231 cells in subcutaneous tumor formation assay. Scale bar: 100 μm. 1: pLCDH-ciR; 2: circMIB1 overexpression; 3: circMIB1-TGAmut; 4: pCDH; 5: MIB1-223aa overexpression. J. Quantitative analysis on the indicated factors in IHC by H-score. 1: pLCDH-ciR; 2: circMIB1 overexpression; 3: circMIB1-TGAmut; 4: pCDH; 5: MIB1-223aa overexpression. (ns, no significance, * P < 0.05, ** P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: TNBC cell lines, MDA-MB-231 (RRID: CVCL_0062) and MDA-MB-468 (RRID: CVCL_0419), and human embryonic kidney cells (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Transwell Assay, Expressing, Flow Cytometry, Tube Formation Assay, Injection, Stable Transfection, Over Expression

Journal: Journal of Advanced Research

Article Title: A novel peptide MIB1-223aa encoded by exosomal circMIB1 from cancer-associated fibroblasts drives triple-negative breast cancer metastasis and stemness via stabilizing MIB1 to activate Notch signaling

doi: 10.1016/j.jare.2025.06.023

Figure Lengend Snippet: circMIB1 promoted metastasis and stemness of TNBC cells through activating MIB1/DLL4/Notch pathway. A. Western blot was used to detect the expression of Notch1 (300 kDa), cleaved Notch1 (120 kDa) and HES1 in differentially transfected TNBC cells. 1: pLCDH-ciR; 2: circMIB1 overexpression; 3: circMIB1-TGAmut; 4: pCDH; 5: MIB1-223aa overexpression. B. Dual-luciferase reporter gene assay was used to evaluate the activity of Notch signaling in differentially transfected TNBC cells. C. Statistical analysis of the relative number of differently transfected migrated and invaded cells with or without DAPT (10 μM) treatment in transwell assay. D. Detection of EMT-associated markers in indicated treated TNBC cells by western blot. E. Representative images of dissected lungs from mice in pulmonary metastatic models established by tail vein injection of MDA-MB-231 cells stably expressing indicated plasmids. Normal saline or DAPT (20 mg/kg) were injected intraperitoneally once every 5 days. The metastatic nodes were indicated by red arrows. 1: pLCDH-ciR + control; 2: circMIB1 overexpression + control; 3: pLCDH-ciR + DAPT; 4: circMIB1 overexpression + DAPT. F. HE staining showed the histological morphology of pulmonary metastatic nodules in each group. Scale bar: 100 μm. 1: pLCDH-ciR + control; 2: circMIB1 overexpression + control; 3: pLCDH-ciR + DAPT; 4: circMIB1 overexpression + DAPT. G. The number of pulmonary metastatic nodes in each group. H. Statistical analysis of the relative diameter of spheres formed by indicated treated TNBC cells. I. ELDA was performed to assess the stemness of differentially treated TNBC cells. J. Detection of stemness-associated markers in differentially treated TNBC cells by western blot. K. Flow cytometry analysis was performed to evaluate the proportion of stem cells in differentially treated MDA-MB-231 and MDA-MB-468. L. The expression of Notch1 (300 kDa) and cleaved Notch1 (120 kDa) in differentially transfected TNBC cells during rescue assay. M. Dual-luciferase reporter gene assay was used to evaluate the activity of Notch signaling in differentially transfected TNBC cells during rescue assay. N. Co-IP and western blot were used to identify the specific binding region of MIB1 to DLL4 using anti-Flag antibody (left) and anti-Myc antibody (right), respectively. O. Co-IP and western blot were used to evaluate the effect of MIB1 overexpression on the ubiquitination level of DLL4 protein. (ns, no significance, * P < 0.05, ** P < 0.01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: TNBC cell lines, MDA-MB-231 (RRID: CVCL_0062) and MDA-MB-468 (RRID: CVCL_0419), and human embryonic kidney cells (HEK293T) were obtained from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Luciferase, Reporter Gene Assay, Activity Assay, Transwell Assay, Injection, Stable Transfection, Saline, Control, Staining, Flow Cytometry, Rescue Assay, Co-Immunoprecipitation Assay, Binding Assay, Ubiquitin Proteomics

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Characterization of CSCs and their sEVs. (A) Schematic representation of the workflow for CSC enrichment, characterization, and proteomic analysis. Tumor-bearing mice were used to generate single-cell suspensions, followed by sphere culture to enrich CSCs. Enriched CSCs were used for subsequent stemness characterization and drug resistance comparison. Proteomic profiling was conducted via LC-MS. (B) Flow cytometry analysis of CD44 and CD133 expression in A549, A549CR, and CSC-enriched populations. (C) Quantification of CD44- and CD133-positive cells across different populations. Data are presented as mean ± SD ( *p < 0.05, ** *p < 0.001, n = 3). Comparisons between groups were analyzed using one-way ANOVA, followed by Tukey's post-hoc test. (D) Dose-response curves for cisplatin and paclitaxel treatment in A549, A549CR, and CSCs. Data are presented as mean ± SD (n = 3). (E) GO enrichment analysis of proteins identified from proteomic profiling, focusing on extracellular vesicle-related pathways. (F) Diagram showing the isolation of sEVs from different cell populations and their subsequent co-culture with A549 cells for functional characterization. (G) SEM images of sEVs derived from A549, A549CR, and CSCs. Scale bar: 200 nm. (H) NTA of sEVs from A549, A549CR, and CSCs showing size distribution. (I) Confocal microscopy images of A549 cells incubated with PKH67-labeled sEVs showing sEV uptake. Nuclei are stained with DAPI (blue), cytoskeletons with phalloidin (red), and sEVs with PKH67 (green). Scale bar: 20 µm. (J) Western blot analysis of stem cell markers in A549 cells after incubation with sEVs from different origins. (K) Flow cytometry analysis of CD44 and CD133 expression in A549 cells treated with sEVs. (L) Quantification of cells positive for CD44 and CD133 after treatment with sEVs from different sources. Data are presented as mean ± SD (ns: not significant, * p < 0.05, *** p < 0.001, n = 3).

Article Snippet: Human lung cancer cell line A549 (ATCC, Cat# CCL-185) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yeasen Biotechnology, Shanghai, China) and 1% penicillin-streptomycin (Gibco, Cat# 15140122, MA, USA).

Techniques: Comparison, Liquid Chromatography with Mass Spectroscopy, Flow Cytometry, Expressing, Isolation, Co-Culture Assay, Functional Assay, Derivative Assay, Confocal Microscopy, Incubation, Labeling, Staining, Western Blot

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: In vivo effects of sEVs on tumor growth and stemness. (A) Schematic diagram of the in vivo experimental design. A549 cells were injected subcutaneously into nude mice to establish tumors. sEVs derived from A549, A549CR, and A549CSC were injected peritumorally every three days. Tumor growth was monitored using IVIS imaging, and histological analyses were performed post-harvest. (B) Representative IVIS imaging showing fluorescence signals in tumors treated with Dir-labeled sEVs from different origins. The images include in vivo and ex vivo fluorescence signals after sEV injection. (C) Tumor growth curves showing tumor volume changes over time in mice treated with sEVs from different sources. Data are presented as mean ± SD (ns: not significant, ** p < 0.01, *** p < 0.001, n = 5). Comparisons between groups were analyzed using one-way ANOVA followed by Tukey's post-hoc test. (D) Representative IVIS images showing in vivo bioluminescent signal intensity in tumor-bearing mice treated with sEVs derived from A549, A549CR, or A549CSC. (E) Representative images of harvested tumors from each group after treatment with sEVs. (F) Histological and immunohistochemical analyses of tumors. H&E staining shows tumor morphology, while immunohistochemical staining shows PKM2, pY105-PKM2, SOX2, and OCT4. Scale bar: 25 µm.

Article Snippet: Human lung cancer cell line A549 (ATCC, Cat# CCL-185) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yeasen Biotechnology, Shanghai, China) and 1% penicillin-streptomycin (Gibco, Cat# 15140122, MA, USA).

Techniques: In Vivo, Injection, Derivative Assay, Imaging, Fluorescence, Labeling, Ex Vivo, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Analysis of PKM2 and its phosphorylation in NSCLC cells and tissues. (A) KEGG pathway enrichment analysis of differentially expressed proteins in A549 chemosensitive cells and CSCs. (B) Heatmap depicting proteomic analysis of A549 chemosensitive cells and CSCs. (C) Western blot analysis of PKM2 and pY105-PKM2 in A549, A549CR, and CSCs, with or without TEPP-46 treatment. Crosslinking experiments show PKM2 configurations, and immunoprecipitation (IP) indicates phosphorylation at Y105. (D) Western blot analysis of selected kinases (YES1, Src, JAK3, FAK, ITK, AXL) in A549, A549CR, and CSCs. (E) Representative immunohistochemical staining of PKM2 and pY105-PKM2 in tumor and peritumoral tissues. Scale bars: 50 µm. (F) Box plots showing IHC scores of PKM2 and pY105-PKM2 in tumor and peritumoral tissues. (G) Kaplan-Meier survival curves comparing overall survival (OS) of NSCLC patients based on PKM2 and pY105-PKM2 expression levels. (H) Representative immunohistochemistry staining of CD133 in tumor and peritumoral tissues. Scale bars: 50 µm. (I) Scatter plots showing the relationship between CD133 expression and PKM2 or pY105-PKM2 in tumor tissues.

Article Snippet: Human lung cancer cell line A549 (ATCC, Cat# CCL-185) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yeasen Biotechnology, Shanghai, China) and 1% penicillin-streptomycin (Gibco, Cat# 15140122, MA, USA).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Immunohistochemical staining, Staining, Expressing, Immunohistochemistry

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Generation and characterization of PKM2 WT and PKM2 Y105F cell lines and their impact on NSCLC stemness and tumorigenicity. (A) Schematic of CRISPR-Cas9-based PKM2 knockout in A549 cells followed by lentiviral transduction of PKM2 WT or PKM2 Y105F plasmids and western blot validation of PKM2 knockout.(B) Workflow of lentiviral packaging, transduction, and selection of PKM2 WT and PKM2 Y105F stable cell lines. (C) Western blot analysis showing expression of PKM2 and pY105-PKM2 in PKM2 WT and PKM2 Y105F cell lines. (D) Western blot analysis of stemness markers (OCT4, SOX2, and NANOG) in PKM2 WT and PKM2 Y105F cell lines. (E) Cell viability assays of PKM2 WT and PKM2 Y105F cells following cisplatin treatment. Data are presented as mean ± SD (ns: not significant, * p < 0.05, ** p < 0.01, n = 3). (F) Colony formation assay of PKM2 WT and PKM2 Y105F cells. Representative images and quantified colony numbers are shown. Data are presented as mean ± SD (** p < 0.01, n = 3). (G) Sphere formation assay of PKM2 WT and PKM2 Y105F cells. Representative images and quantified sphere numbers are shown. Data are presented as mean ± SD (** p < 0.01, n = 3). (H) Representative images of harvested subcutaneous xenograft tumors derived from PKM2 WT and PKM2 Y105F cells. (I) Tumor growth curves showing tumor volume over time for PKM2 WT and PKM2 Y105F xenografts. Data are presented as mean ± SD (** p < 0.01, n = 6). (J) Limiting dilution assay using bioluminescence imaging of subcutaneous xenografts with decreasing cell numbers (1×10⁶ to 5×10³) of PKM2 WT and PKM2 Y105F cells. (K) Immunohistochemical analysis of xenograft tumors showing H&E staining, PKM2, pY105-PKM2, and stemness markers (OCT4, SOX2). Scale bar = 25 μm.

Article Snippet: Human lung cancer cell line A549 (ATCC, Cat# CCL-185) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yeasen Biotechnology, Shanghai, China) and 1% penicillin-streptomycin (Gibco, Cat# 15140122, MA, USA).

Techniques: CRISPR, Knock-Out, Transduction, Western Blot, Biomarker Discovery, Selection, Stable Transfection, Expressing, Colony Assay, Tube Formation Assay, Derivative Assay, Limiting Dilution Assay, Imaging, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: sEV-mediated pY105-PKM2 induces slow cell cycle, metabolic remodeling, and promotes chemoresistance and stemness in NSCLC. (A) Schematic representation of the experimental setup. A549 cells were treated with sEVs derived from A549-PKM2 WT and A549-PKM2 Y105F cells, followed by transcriptomic sequencing. (B) Glucose consumption and lactate production in A549 cells treated with Vec-sEV, PKM2^WT-sEV, or PKM2^Y105F-sEV. Data are presented as mean ± SD (ns, not significant, ** p < 0.01, *** p < 0.001, n = 3). (C) OCR of A549 cells treated with Vec-sEV, PKM2 WT -sEV, or PKM2 Y105F -sEV under sequential injections of oligomycin, FCCP, and rotenone. (D) Western blot analysis of Cyclin B1, CDC25B, total CDK1, and CDK1 phosphorylated at Thr14 and Tyr15 in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV. (E) Schematic representation of Apcin treatment experiments designed to assess the role of APC/CDC20 in sEV-induced cell cycle regulation, stemness, and chemoresistance. (F) Flow cytometry analysis of cell cycle distribution in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV, with or without Apcin treatment. (G) Quantification of cell populations in different phases of the cell cycle (Sub G1, G1, S, G2/M) based on flow cytometry analysis. Data are presented as mean ± SD (ns, not significant, * p < 0.05, ** p < 0.01, n = 3). (H) Flow cytometry analysis of CD44 and CD133 expression in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV, with or without Apcin treatment. (I) Quantification of CD44- and CD133-positive cells in A549 cells treated as described in Panel H. Data are presented as mean ± SD (ns, not significant, *** p < 0.001, n = 3). (J) Flow cytometry analysis of apoptotic cells in A549 cells treated with sEVs, followed by cisplatin or paclitaxel treatment, with or without Apcin. (K) Quantification of apoptotic cells in A549 cells treated as described in Panel J. Data are presented as mean ± SD (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3).

Article Snippet: Human lung cancer cell line A549 (ATCC, Cat# CCL-185) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yeasen Biotechnology, Shanghai, China) and 1% penicillin-streptomycin (Gibco, Cat# 15140122, MA, USA).

Techniques: Derivative Assay, Sequencing, Western Blot, Flow Cytometry, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: In vivo assessment of the effects of sEVs derived from PKM2 WT and PKM2 Y105F cells on tumor growth, glycolytic activity, and stemness. (A) Schematic representation of the in vivo experimental design. Chemosensitive A549 cells were subcutaneously implanted into nude mice, and upon tumor volumes reaching ~100 mm³, sEVs derived from Vec, PKM2 WT , or PKM2 Y105F cells were administered peritumorally every three days. Tumor growth was monitored, followed by ¹⁸F-FDG PET-CT and IVIS imaging and histological analysis of harvested tumors. (B) Representative IVIS imaging showing fluorescence signals in tumors treated with Dir-labeled sEVs from different origins. The images include in vivo and ex vivo fluorescence signals after sEV injection. (C) Tumor growth curves depicting tumor volumes over time in mice treated with different sEVs. Data are presented as mean ± SD (ns, not significant, *** p < 0.001, n = 5). (D) Representative IVIS images showing bioluminescent signals from subcutaneous tumors at the experimental endpoint in mice treated with Vec-sEV, PKM2 WT -sEV, or PKM2 Y105F -sEV. (E) Images of harvested tumors from mice treated with different sEVs. (F) Representative ¹⁸F-FDG PET-CT scans showing glycolytic activity in tumors treated with different sEVs. (G) Histological analysis of tumor tissues, including hematoxylin and eosin (HE) staining and immunohistochemistry for PKM2, pY105-PKM2, stemness markers OCT4 and SOX2, and cell cycle regulators CDC25B, Cyclin B1, and CDK1. Scale bar: 25 µm.

Article Snippet: Human lung cancer cell line A549 (ATCC, Cat# CCL-185) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yeasen Biotechnology, Shanghai, China) and 1% penicillin-streptomycin (Gibco, Cat# 15140122, MA, USA).

Techniques: In Vivo, Derivative Assay, Activity Assay, Positron Emission Tomography-Computed Tomography, Imaging, Fluorescence, Labeling, Ex Vivo, Injection, Staining, Immunohistochemistry

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: IQGAP1 links phosphorylated PKM2 to TSG101. (A) Experimental workflow for immunoprecipitation and mass spectrometry analysis. Flag-tagged PKM2 WT and PKM2 Y105F proteins were immunoprecipitated using anti-Flag magnetic beads, followed by liquid chromatography-mass spectrometry. The Venn diagram illustrates the overlap and unique binding partners of PKM2 WT and PKM2 Y105F . (B) Mass spectrometry results highlighting the binding of IQGAP1 exclusively to PKM2 WT . (C) Co-immunoprecipitation of IQGAP1, pY105-PKM2, and TSG101 in A549-PKM2 WT and A549- PKM2 Y105F cells. Total protein input is shown for comparison. (D) Immunoprecipitation analysis of interactions between IQGAP1, TSG101, and PKM2 in A549- PKM2 WT and A549- PKM2 Y105F cells. (E) Co-immunoprecipitation results following siRNA-mediated knockdown of IQGAP1 in A549-PKM2 WT and A549-PKM2 Y105F cells. (F) Total protein input levels of IQGAP1, TSG101, and PKM2 in A549- PKM2 WT and A549- PKM2 Y105F cells, with or without IQGAP1 knockdown. (G) Immunoprecipitation analysis showing the effect of TEPP-46 treatment on interactions between IQGAP1, PKM2, and TSG101 in A549- PKM2 WT and A549- PKM2 Y105F cells. (H) DuoLink proximity ligation assay detecting interactions between PKM2 and TSG101. Signals (yellow) are shown in A549- PKM2 WT and A549- PKM2 Y105F cells, with and without IQGAP1 knockdown. DAPI (blue) marks nuclei, and phalloidin (red) labels actin filaments. Scale bar: 10 µm. (I) Immunofluorescence analysis showing colocalization of Flag-tagged PKM2 WT and PKM2 Y105F with TSG101 in A549 cells, with and without IQGAP1 knockdown. Scale bar: 10 µm.

Article Snippet: Human lung cancer cell line A549 (ATCC, Cat# CCL-185) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yeasen Biotechnology, Shanghai, China) and 1% penicillin-streptomycin (Gibco, Cat# 15140122, MA, USA).

Techniques: Immunoprecipitation, Mass Spectrometry, Magnetic Beads, Liquid Chromatography, Binding Assay, Comparison, Knockdown, Proximity Ligation Assay, Immunofluorescence

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: IQGAP1 mediates the sorting of pY105-PKM2 into sEVs and synergistically promotes stemness and drug resistance. (A) Western blot analysis of pY105-PKM2 in sEVs derived from A549-PKM2 WT or A549-PKM2 Y105F cells, with or without IQGAP1 silencing using siRNA (siRNA-NC, siRNA-IQGAP1#3, or siRNA-IQGAP1#4). (B) Flow cytometry analysis of apoptosis in recipient A549 cells treated with sEVs from IQGAP1-silenced or control A549-PKM2 WT cells, followed by treatment with cisplatin (5 µg/mL) or paclitaxel (200 ng/mL). (C) Western blot analysis of pY105-PKM2 in sEVs derived from A549-PKM2 WT or A549-PKM2 Y105F cells, with or without TEPP-46 treatment, which inhibits PKM2 phosphorylation at Y105. (D) Flow cytometry analysis of apoptosis in recipient A549 cells treated with sEVs from TEPP-46-treated A549-PKM2 WT cells, followed by cisplatin or paclitaxel treatment. (E) Sphere formation assay of A549 cells overexpressing IQGAP1, PKM2 WT , PKM2 Y105F , or combinations thereof. Representative images and quantification of sphere numbers are shown. (F) Cell viability assay of A549 cells overexpressing IQGAP1, PKM2 WT , PKM2 Y105F , or their combinations, following cisplatin treatment (5 µg/mL). (G) Correlation between IQGAP1 and PKM2 expression in CD133-high and CD133-low tumor samples. (H) Correlation between IQGAP1 and pY105-PKM2 expression in CD133-high and CD133-low tumor samples. Data (B-F) are presented as mean ± SD (n = 3). Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human lung cancer cell line A549 (ATCC, Cat# CCL-185) was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Yeasen Biotechnology, Shanghai, China) and 1% penicillin-streptomycin (Gibco, Cat# 15140122, MA, USA).

Techniques: Western Blot, Derivative Assay, Flow Cytometry, Control, Phospho-proteomics, Tube Formation Assay, Viability Assay, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: In vivo effects of sEVs on tumor growth and stemness. (A) Schematic diagram of the in vivo experimental design. A549 cells were injected subcutaneously into nude mice to establish tumors. sEVs derived from A549, A549CR, and A549CSC were injected peritumorally every three days. Tumor growth was monitored using IVIS imaging, and histological analyses were performed post-harvest. (B) Representative IVIS imaging showing fluorescence signals in tumors treated with Dir-labeled sEVs from different origins. The images include in vivo and ex vivo fluorescence signals after sEV injection. (C) Tumor growth curves showing tumor volume changes over time in mice treated with sEVs from different sources. Data are presented as mean ± SD (ns: not significant, ** p < 0.01, *** p < 0.001, n = 5). Comparisons between groups were analyzed using one-way ANOVA followed by Tukey's post-hoc test. (D) Representative IVIS images showing in vivo bioluminescent signal intensity in tumor-bearing mice treated with sEVs derived from A549, A549CR, or A549CSC. (E) Representative images of harvested tumors from each group after treatment with sEVs. (F) Histological and immunohistochemical analyses of tumors. H&E staining shows tumor morphology, while immunohistochemical staining shows PKM2, pY105-PKM2, SOX2, and OCT4. Scale bar: 25 µm.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: In Vivo, Injection, Derivative Assay, Imaging, Fluorescence, Labeling, Ex Vivo, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Analysis of PKM2 and its phosphorylation in NSCLC cells and tissues. (A) KEGG pathway enrichment analysis of differentially expressed proteins in A549 chemosensitive cells and CSCs. (B) Heatmap depicting proteomic analysis of A549 chemosensitive cells and CSCs. (C) Western blot analysis of PKM2 and pY105-PKM2 in A549, A549CR, and CSCs, with or without TEPP-46 treatment. Crosslinking experiments show PKM2 configurations, and immunoprecipitation (IP) indicates phosphorylation at Y105. (D) Western blot analysis of selected kinases (YES1, Src, JAK3, FAK, ITK, AXL) in A549, A549CR, and CSCs. (E) Representative immunohistochemical staining of PKM2 and pY105-PKM2 in tumor and peritumoral tissues. Scale bars: 50 µm. (F) Box plots showing IHC scores of PKM2 and pY105-PKM2 in tumor and peritumoral tissues. (G) Kaplan-Meier survival curves comparing overall survival (OS) of NSCLC patients based on PKM2 and pY105-PKM2 expression levels. (H) Representative immunohistochemistry staining of CD133 in tumor and peritumoral tissues. Scale bars: 50 µm. (I) Scatter plots showing the relationship between CD133 expression and PKM2 or pY105-PKM2 in tumor tissues.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Western Blot, Immunoprecipitation, Immunohistochemical staining, Staining, Expressing, Immunohistochemistry

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Generation and characterization of PKM2 WT and PKM2 Y105F cell lines and their impact on NSCLC stemness and tumorigenicity. (A) Schematic of CRISPR-Cas9-based PKM2 knockout in A549 cells followed by lentiviral transduction of PKM2 WT or PKM2 Y105F plasmids and western blot validation of PKM2 knockout.(B) Workflow of lentiviral packaging, transduction, and selection of PKM2 WT and PKM2 Y105F stable cell lines. (C) Western blot analysis showing expression of PKM2 and pY105-PKM2 in PKM2 WT and PKM2 Y105F cell lines. (D) Western blot analysis of stemness markers (OCT4, SOX2, and NANOG) in PKM2 WT and PKM2 Y105F cell lines. (E) Cell viability assays of PKM2 WT and PKM2 Y105F cells following cisplatin treatment. Data are presented as mean ± SD (ns: not significant, * p < 0.05, ** p < 0.01, n = 3). (F) Colony formation assay of PKM2 WT and PKM2 Y105F cells. Representative images and quantified colony numbers are shown. Data are presented as mean ± SD (** p < 0.01, n = 3). (G) Sphere formation assay of PKM2 WT and PKM2 Y105F cells. Representative images and quantified sphere numbers are shown. Data are presented as mean ± SD (** p < 0.01, n = 3). (H) Representative images of harvested subcutaneous xenograft tumors derived from PKM2 WT and PKM2 Y105F cells. (I) Tumor growth curves showing tumor volume over time for PKM2 WT and PKM2 Y105F xenografts. Data are presented as mean ± SD (** p < 0.01, n = 6). (J) Limiting dilution assay using bioluminescence imaging of subcutaneous xenografts with decreasing cell numbers (1×10⁶ to 5×10³) of PKM2 WT and PKM2 Y105F cells. (K) Immunohistochemical analysis of xenograft tumors showing H&E staining, PKM2, pY105-PKM2, and stemness markers (OCT4, SOX2). Scale bar = 25 μm.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: CRISPR, Knock-Out, Transduction, Western Blot, Selection, Stable Transfection, Expressing, Colony Assay, Tube Formation Assay, Derivative Assay, Limiting Dilution Assay, Imaging, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: sEV-mediated pY105-PKM2 induces slow cell cycle, metabolic remodeling, and promotes chemoresistance and stemness in NSCLC. (A) Schematic representation of the experimental setup. A549 cells were treated with sEVs derived from A549-PKM2 WT and A549-PKM2 Y105F cells, followed by transcriptomic sequencing. (B) Glucose consumption and lactate production in A549 cells treated with Vec-sEV, PKM2^WT-sEV, or PKM2^Y105F-sEV. Data are presented as mean ± SD (ns, not significant, ** p < 0.01, *** p < 0.001, n = 3). (C) OCR of A549 cells treated with Vec-sEV, PKM2 WT -sEV, or PKM2 Y105F -sEV under sequential injections of oligomycin, FCCP, and rotenone. (D) Western blot analysis of Cyclin B1, CDC25B, total CDK1, and CDK1 phosphorylated at Thr14 and Tyr15 in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV. (E) Schematic representation of Apcin treatment experiments designed to assess the role of APC/CDC20 in sEV-induced cell cycle regulation, stemness, and chemoresistance. (F) Flow cytometry analysis of cell cycle distribution in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV, with or without Apcin treatment. (G) Quantification of cell populations in different phases of the cell cycle (Sub G1, G1, S, G2/M) based on flow cytometry analysis. Data are presented as mean ± SD (ns, not significant, * p < 0.05, ** p < 0.01, n = 3). (H) Flow cytometry analysis of CD44 and CD133 expression in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV, with or without Apcin treatment. (I) Quantification of CD44- and CD133-positive cells in A549 cells treated as described in Panel H. Data are presented as mean ± SD (ns, not significant, *** p < 0.001, n = 3). (J) Flow cytometry analysis of apoptotic cells in A549 cells treated with sEVs, followed by cisplatin or paclitaxel treatment, with or without Apcin. (K) Quantification of apoptotic cells in A549 cells treated as described in Panel J. Data are presented as mean ± SD (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3).

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Derivative Assay, Sequencing, Western Blot, Flow Cytometry, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: In vivo assessment of the effects of sEVs derived from PKM2 WT and PKM2 Y105F cells on tumor growth, glycolytic activity, and stemness. (A) Schematic representation of the in vivo experimental design. Chemosensitive A549 cells were subcutaneously implanted into nude mice, and upon tumor volumes reaching ~100 mm³, sEVs derived from Vec, PKM2 WT , or PKM2 Y105F cells were administered peritumorally every three days. Tumor growth was monitored, followed by ¹⁸F-FDG PET-CT and IVIS imaging and histological analysis of harvested tumors. (B) Representative IVIS imaging showing fluorescence signals in tumors treated with Dir-labeled sEVs from different origins. The images include in vivo and ex vivo fluorescence signals after sEV injection. (C) Tumor growth curves depicting tumor volumes over time in mice treated with different sEVs. Data are presented as mean ± SD (ns, not significant, *** p < 0.001, n = 5). (D) Representative IVIS images showing bioluminescent signals from subcutaneous tumors at the experimental endpoint in mice treated with Vec-sEV, PKM2 WT -sEV, or PKM2 Y105F -sEV. (E) Images of harvested tumors from mice treated with different sEVs. (F) Representative ¹⁸F-FDG PET-CT scans showing glycolytic activity in tumors treated with different sEVs. (G) Histological analysis of tumor tissues, including hematoxylin and eosin (HE) staining and immunohistochemistry for PKM2, pY105-PKM2, stemness markers OCT4 and SOX2, and cell cycle regulators CDC25B, Cyclin B1, and CDK1. Scale bar: 25 µm.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: In Vivo, Derivative Assay, Activity Assay, Positron Emission Tomography-Computed Tomography, Imaging, Fluorescence, Labeling, Ex Vivo, Injection, Staining, Immunohistochemistry

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: IQGAP1 links phosphorylated PKM2 to TSG101. (A) Experimental workflow for immunoprecipitation and mass spectrometry analysis. Flag-tagged PKM2 WT and PKM2 Y105F proteins were immunoprecipitated using anti-Flag magnetic beads, followed by liquid chromatography-mass spectrometry. The Venn diagram illustrates the overlap and unique binding partners of PKM2 WT and PKM2 Y105F . (B) Mass spectrometry results highlighting the binding of IQGAP1 exclusively to PKM2 WT . (C) Co-immunoprecipitation of IQGAP1, pY105-PKM2, and TSG101 in A549-PKM2 WT and A549- PKM2 Y105F cells. Total protein input is shown for comparison. (D) Immunoprecipitation analysis of interactions between IQGAP1, TSG101, and PKM2 in A549- PKM2 WT and A549- PKM2 Y105F cells. (E) Co-immunoprecipitation results following siRNA-mediated knockdown of IQGAP1 in A549-PKM2 WT and A549-PKM2 Y105F cells. (F) Total protein input levels of IQGAP1, TSG101, and PKM2 in A549- PKM2 WT and A549- PKM2 Y105F cells, with or without IQGAP1 knockdown. (G) Immunoprecipitation analysis showing the effect of TEPP-46 treatment on interactions between IQGAP1, PKM2, and TSG101 in A549- PKM2 WT and A549- PKM2 Y105F cells. (H) DuoLink proximity ligation assay detecting interactions between PKM2 and TSG101. Signals (yellow) are shown in A549- PKM2 WT and A549- PKM2 Y105F cells, with and without IQGAP1 knockdown. DAPI (blue) marks nuclei, and phalloidin (red) labels actin filaments. Scale bar: 10 µm. (I) Immunofluorescence analysis showing colocalization of Flag-tagged PKM2 WT and PKM2 Y105F with TSG101 in A549 cells, with and without IQGAP1 knockdown. Scale bar: 10 µm.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Immunoprecipitation, Mass Spectrometry, Magnetic Beads, Liquid Chromatography, Binding Assay, Comparison, Knockdown, Proximity Ligation Assay, Immunofluorescence

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: IQGAP1 mediates the sorting of pY105-PKM2 into sEVs and synergistically promotes stemness and drug resistance. (A) Western blot analysis of pY105-PKM2 in sEVs derived from A549-PKM2 WT or A549-PKM2 Y105F cells, with or without IQGAP1 silencing using siRNA (siRNA-NC, siRNA-IQGAP1#3, or siRNA-IQGAP1#4). (B) Flow cytometry analysis of apoptosis in recipient A549 cells treated with sEVs from IQGAP1-silenced or control A549-PKM2 WT cells, followed by treatment with cisplatin (5 µg/mL) or paclitaxel (200 ng/mL). (C) Western blot analysis of pY105-PKM2 in sEVs derived from A549-PKM2 WT or A549-PKM2 Y105F cells, with or without TEPP-46 treatment, which inhibits PKM2 phosphorylation at Y105. (D) Flow cytometry analysis of apoptosis in recipient A549 cells treated with sEVs from TEPP-46-treated A549-PKM2 WT cells, followed by cisplatin or paclitaxel treatment. (E) Sphere formation assay of A549 cells overexpressing IQGAP1, PKM2 WT , PKM2 Y105F , or combinations thereof. Representative images and quantification of sphere numbers are shown. (F) Cell viability assay of A549 cells overexpressing IQGAP1, PKM2 WT , PKM2 Y105F , or their combinations, following cisplatin treatment (5 µg/mL). (G) Correlation between IQGAP1 and PKM2 expression in CD133-high and CD133-low tumor samples. (H) Correlation between IQGAP1 and pY105-PKM2 expression in CD133-high and CD133-low tumor samples. Data (B-F) are presented as mean ± SD (n = 3). Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Western Blot, Derivative Assay, Flow Cytometry, Control, Tube Formation Assay, Viability Assay, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Graphical summary of IQGAP1-mediated sorting of phos-PKM2 in CSC-derived sEVs and its role in chemoresistance in NSCLC. This schematic illustrates the role of CSC-derived sEVs in promoting stemness and chemoresistance in NSCLC. In CSCs (left panel), surface markers CD44 and CD133 are expressed. Phosphorylation of PKM2 at Y105 (phos-PKM2) is induced by receptor tyrosine kinases (YES1, Src, JAK3, FAK, ITK, AXL), facilitating its transition from the tetrameric to the dimeric form. phos-PKM2 translocates to the nucleus to enhance the expression of stemness-related transcription factors (SOX2, NANOG, and OCT4). IQGAP1 mediates the selective incorporation of phos-PKM2 into sEVs through interactions with the ESCRT component TSG101, enabling the secretion of sEVs into the tumor microenvironment. In chemosensitive cancer cells (right panel), CSC-derived sEV uptake delivers phos-PKM2, inducing metabolic reprogramming (enhanced glycolysis and suppressed oxidative phosphorylation) and slowing the cell cycle via APC/C-CDC20 inhibition and reduced Cyclin B degradation. These processes collectively promote stemness and chemoresistance, replenishing the CSC pool. The lower section illustrates a lung tumor model, depicting how CSC-derived sEVs expand the CSC population within the tumor microenvironment, thereby driving chemotherapy resistance and malignant progression.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Derivative Assay, Expressing, Inhibition

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: In vivo effects of sEVs on tumor growth and stemness. (A) Schematic diagram of the in vivo experimental design. A549 cells were injected subcutaneously into nude mice to establish tumors. sEVs derived from A549, A549CR, and A549CSC were injected peritumorally every three days. Tumor growth was monitored using IVIS imaging, and histological analyses were performed post-harvest. (B) Representative IVIS imaging showing fluorescence signals in tumors treated with Dir-labeled sEVs from different origins. The images include in vivo and ex vivo fluorescence signals after sEV injection. (C) Tumor growth curves showing tumor volume changes over time in mice treated with sEVs from different sources. Data are presented as mean ± SD (ns: not significant, ** p < 0.01, *** p < 0.001, n = 5). Comparisons between groups were analyzed using one-way ANOVA followed by Tukey's post-hoc test. (D) Representative IVIS images showing in vivo bioluminescent signal intensity in tumor-bearing mice treated with sEVs derived from A549, A549CR, or A549CSC. (E) Representative images of harvested tumors from each group after treatment with sEVs. (F) Histological and immunohistochemical analyses of tumors. H&E staining shows tumor morphology, while immunohistochemical staining shows PKM2, pY105-PKM2, SOX2, and OCT4. Scale bar: 25 µm.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: In Vivo, Injection, Derivative Assay, Imaging, Fluorescence, Labeling, Ex Vivo, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Analysis of PKM2 and its phosphorylation in NSCLC cells and tissues. (A) KEGG pathway enrichment analysis of differentially expressed proteins in A549 chemosensitive cells and CSCs. (B) Heatmap depicting proteomic analysis of A549 chemosensitive cells and CSCs. (C) Western blot analysis of PKM2 and pY105-PKM2 in A549, A549CR, and CSCs, with or without TEPP-46 treatment. Crosslinking experiments show PKM2 configurations, and immunoprecipitation (IP) indicates phosphorylation at Y105. (D) Western blot analysis of selected kinases (YES1, Src, JAK3, FAK, ITK, AXL) in A549, A549CR, and CSCs. (E) Representative immunohistochemical staining of PKM2 and pY105-PKM2 in tumor and peritumoral tissues. Scale bars: 50 µm. (F) Box plots showing IHC scores of PKM2 and pY105-PKM2 in tumor and peritumoral tissues. (G) Kaplan-Meier survival curves comparing overall survival (OS) of NSCLC patients based on PKM2 and pY105-PKM2 expression levels. (H) Representative immunohistochemistry staining of CD133 in tumor and peritumoral tissues. Scale bars: 50 µm. (I) Scatter plots showing the relationship between CD133 expression and PKM2 or pY105-PKM2 in tumor tissues.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Western Blot, Immunoprecipitation, Immunohistochemical staining, Staining, Expressing, Immunohistochemistry

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Generation and characterization of PKM2 WT and PKM2 Y105F cell lines and their impact on NSCLC stemness and tumorigenicity. (A) Schematic of CRISPR-Cas9-based PKM2 knockout in A549 cells followed by lentiviral transduction of PKM2 WT or PKM2 Y105F plasmids and western blot validation of PKM2 knockout.(B) Workflow of lentiviral packaging, transduction, and selection of PKM2 WT and PKM2 Y105F stable cell lines. (C) Western blot analysis showing expression of PKM2 and pY105-PKM2 in PKM2 WT and PKM2 Y105F cell lines. (D) Western blot analysis of stemness markers (OCT4, SOX2, and NANOG) in PKM2 WT and PKM2 Y105F cell lines. (E) Cell viability assays of PKM2 WT and PKM2 Y105F cells following cisplatin treatment. Data are presented as mean ± SD (ns: not significant, * p < 0.05, ** p < 0.01, n = 3). (F) Colony formation assay of PKM2 WT and PKM2 Y105F cells. Representative images and quantified colony numbers are shown. Data are presented as mean ± SD (** p < 0.01, n = 3). (G) Sphere formation assay of PKM2 WT and PKM2 Y105F cells. Representative images and quantified sphere numbers are shown. Data are presented as mean ± SD (** p < 0.01, n = 3). (H) Representative images of harvested subcutaneous xenograft tumors derived from PKM2 WT and PKM2 Y105F cells. (I) Tumor growth curves showing tumor volume over time for PKM2 WT and PKM2 Y105F xenografts. Data are presented as mean ± SD (** p < 0.01, n = 6). (J) Limiting dilution assay using bioluminescence imaging of subcutaneous xenografts with decreasing cell numbers (1×10⁶ to 5×10³) of PKM2 WT and PKM2 Y105F cells. (K) Immunohistochemical analysis of xenograft tumors showing H&E staining, PKM2, pY105-PKM2, and stemness markers (OCT4, SOX2). Scale bar = 25 μm.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: CRISPR, Knock-Out, Transduction, Western Blot, Selection, Stable Transfection, Expressing, Colony Assay, Tube Formation Assay, Derivative Assay, Limiting Dilution Assay, Imaging, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: sEV-mediated pY105-PKM2 induces slow cell cycle, metabolic remodeling, and promotes chemoresistance and stemness in NSCLC. (A) Schematic representation of the experimental setup. A549 cells were treated with sEVs derived from A549-PKM2 WT and A549-PKM2 Y105F cells, followed by transcriptomic sequencing. (B) Glucose consumption and lactate production in A549 cells treated with Vec-sEV, PKM2^WT-sEV, or PKM2^Y105F-sEV. Data are presented as mean ± SD (ns, not significant, ** p < 0.01, *** p < 0.001, n = 3). (C) OCR of A549 cells treated with Vec-sEV, PKM2 WT -sEV, or PKM2 Y105F -sEV under sequential injections of oligomycin, FCCP, and rotenone. (D) Western blot analysis of Cyclin B1, CDC25B, total CDK1, and CDK1 phosphorylated at Thr14 and Tyr15 in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV. (E) Schematic representation of Apcin treatment experiments designed to assess the role of APC/CDC20 in sEV-induced cell cycle regulation, stemness, and chemoresistance. (F) Flow cytometry analysis of cell cycle distribution in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV, with or without Apcin treatment. (G) Quantification of cell populations in different phases of the cell cycle (Sub G1, G1, S, G2/M) based on flow cytometry analysis. Data are presented as mean ± SD (ns, not significant, * p < 0.05, ** p < 0.01, n = 3). (H) Flow cytometry analysis of CD44 and CD133 expression in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV, with or without Apcin treatment. (I) Quantification of CD44- and CD133-positive cells in A549 cells treated as described in Panel H. Data are presented as mean ± SD (ns, not significant, *** p < 0.001, n = 3). (J) Flow cytometry analysis of apoptotic cells in A549 cells treated with sEVs, followed by cisplatin or paclitaxel treatment, with or without Apcin. (K) Quantification of apoptotic cells in A549 cells treated as described in Panel J. Data are presented as mean ± SD (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3).

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Derivative Assay, Sequencing, Western Blot, Flow Cytometry, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: In vivo assessment of the effects of sEVs derived from PKM2 WT and PKM2 Y105F cells on tumor growth, glycolytic activity, and stemness. (A) Schematic representation of the in vivo experimental design. Chemosensitive A549 cells were subcutaneously implanted into nude mice, and upon tumor volumes reaching ~100 mm³, sEVs derived from Vec, PKM2 WT , or PKM2 Y105F cells were administered peritumorally every three days. Tumor growth was monitored, followed by ¹⁸F-FDG PET-CT and IVIS imaging and histological analysis of harvested tumors. (B) Representative IVIS imaging showing fluorescence signals in tumors treated with Dir-labeled sEVs from different origins. The images include in vivo and ex vivo fluorescence signals after sEV injection. (C) Tumor growth curves depicting tumor volumes over time in mice treated with different sEVs. Data are presented as mean ± SD (ns, not significant, *** p < 0.001, n = 5). (D) Representative IVIS images showing bioluminescent signals from subcutaneous tumors at the experimental endpoint in mice treated with Vec-sEV, PKM2 WT -sEV, or PKM2 Y105F -sEV. (E) Images of harvested tumors from mice treated with different sEVs. (F) Representative ¹⁸F-FDG PET-CT scans showing glycolytic activity in tumors treated with different sEVs. (G) Histological analysis of tumor tissues, including hematoxylin and eosin (HE) staining and immunohistochemistry for PKM2, pY105-PKM2, stemness markers OCT4 and SOX2, and cell cycle regulators CDC25B, Cyclin B1, and CDK1. Scale bar: 25 µm.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: In Vivo, Derivative Assay, Activity Assay, Positron Emission Tomography-Computed Tomography, Imaging, Fluorescence, Labeling, Ex Vivo, Injection, Staining, Immunohistochemistry

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: IQGAP1 links phosphorylated PKM2 to TSG101. (A) Experimental workflow for immunoprecipitation and mass spectrometry analysis. Flag-tagged PKM2 WT and PKM2 Y105F proteins were immunoprecipitated using anti-Flag magnetic beads, followed by liquid chromatography-mass spectrometry. The Venn diagram illustrates the overlap and unique binding partners of PKM2 WT and PKM2 Y105F . (B) Mass spectrometry results highlighting the binding of IQGAP1 exclusively to PKM2 WT . (C) Co-immunoprecipitation of IQGAP1, pY105-PKM2, and TSG101 in A549-PKM2 WT and A549- PKM2 Y105F cells. Total protein input is shown for comparison. (D) Immunoprecipitation analysis of interactions between IQGAP1, TSG101, and PKM2 in A549- PKM2 WT and A549- PKM2 Y105F cells. (E) Co-immunoprecipitation results following siRNA-mediated knockdown of IQGAP1 in A549-PKM2 WT and A549-PKM2 Y105F cells. (F) Total protein input levels of IQGAP1, TSG101, and PKM2 in A549- PKM2 WT and A549- PKM2 Y105F cells, with or without IQGAP1 knockdown. (G) Immunoprecipitation analysis showing the effect of TEPP-46 treatment on interactions between IQGAP1, PKM2, and TSG101 in A549- PKM2 WT and A549- PKM2 Y105F cells. (H) DuoLink proximity ligation assay detecting interactions between PKM2 and TSG101. Signals (yellow) are shown in A549- PKM2 WT and A549- PKM2 Y105F cells, with and without IQGAP1 knockdown. DAPI (blue) marks nuclei, and phalloidin (red) labels actin filaments. Scale bar: 10 µm. (I) Immunofluorescence analysis showing colocalization of Flag-tagged PKM2 WT and PKM2 Y105F with TSG101 in A549 cells, with and without IQGAP1 knockdown. Scale bar: 10 µm.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Immunoprecipitation, Mass Spectrometry, Magnetic Beads, Liquid Chromatography, Binding Assay, Comparison, Knockdown, Proximity Ligation Assay, Immunofluorescence

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: IQGAP1 mediates the sorting of pY105-PKM2 into sEVs and synergistically promotes stemness and drug resistance. (A) Western blot analysis of pY105-PKM2 in sEVs derived from A549-PKM2 WT or A549-PKM2 Y105F cells, with or without IQGAP1 silencing using siRNA (siRNA-NC, siRNA-IQGAP1#3, or siRNA-IQGAP1#4). (B) Flow cytometry analysis of apoptosis in recipient A549 cells treated with sEVs from IQGAP1-silenced or control A549-PKM2 WT cells, followed by treatment with cisplatin (5 µg/mL) or paclitaxel (200 ng/mL). (C) Western blot analysis of pY105-PKM2 in sEVs derived from A549-PKM2 WT or A549-PKM2 Y105F cells, with or without TEPP-46 treatment, which inhibits PKM2 phosphorylation at Y105. (D) Flow cytometry analysis of apoptosis in recipient A549 cells treated with sEVs from TEPP-46-treated A549-PKM2 WT cells, followed by cisplatin or paclitaxel treatment. (E) Sphere formation assay of A549 cells overexpressing IQGAP1, PKM2 WT , PKM2 Y105F , or combinations thereof. Representative images and quantification of sphere numbers are shown. (F) Cell viability assay of A549 cells overexpressing IQGAP1, PKM2 WT , PKM2 Y105F , or their combinations, following cisplatin treatment (5 µg/mL). (G) Correlation between IQGAP1 and PKM2 expression in CD133-high and CD133-low tumor samples. (H) Correlation between IQGAP1 and pY105-PKM2 expression in CD133-high and CD133-low tumor samples. Data (B-F) are presented as mean ± SD (n = 3). Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Western Blot, Derivative Assay, Flow Cytometry, Control, Tube Formation Assay, Viability Assay, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Graphical summary of IQGAP1-mediated sorting of phos-PKM2 in CSC-derived sEVs and its role in chemoresistance in NSCLC. This schematic illustrates the role of CSC-derived sEVs in promoting stemness and chemoresistance in NSCLC. In CSCs (left panel), surface markers CD44 and CD133 are expressed. Phosphorylation of PKM2 at Y105 (phos-PKM2) is induced by receptor tyrosine kinases (YES1, Src, JAK3, FAK, ITK, AXL), facilitating its transition from the tetrameric to the dimeric form. phos-PKM2 translocates to the nucleus to enhance the expression of stemness-related transcription factors (SOX2, NANOG, and OCT4). IQGAP1 mediates the selective incorporation of phos-PKM2 into sEVs through interactions with the ESCRT component TSG101, enabling the secretion of sEVs into the tumor microenvironment. In chemosensitive cancer cells (right panel), CSC-derived sEV uptake delivers phos-PKM2, inducing metabolic reprogramming (enhanced glycolysis and suppressed oxidative phosphorylation) and slowing the cell cycle via APC/C-CDC20 inhibition and reduced Cyclin B degradation. These processes collectively promote stemness and chemoresistance, replenishing the CSC pool. The lower section illustrates a lung tumor model, depicting how CSC-derived sEVs expand the CSC population within the tumor microenvironment, thereby driving chemotherapy resistance and malignant progression.

Article Snippet: For immunohistochemistry, tissues were quenched with 3% H 2 O 2 solution, blocked with 3% bovine serum albumin, and incubated with primary antibodies against PKM2 (Bioss, Cat#bs-0102M), phospho-PKM2 (Tyr105) (Bioss, Cat# bs-3334R,), OCT4 (Proteintech, Cat#, 11263-1-AP,), SOX2 (Proteintech, Cat# 11064-1-AP,), CDC25B (Zen Bioscience, Cat# R381486,), CyclinB1 (Cat# R23324, Zen Bioscience), and CDK1 (Zen Bioscience, Cat# R23884,).

Techniques: Derivative Assay, Expressing, Inhibition

Journal: Advanced Science

Article Title: QSOX2‐Mediated Disulfide Bond Modification Enhances Tumor Stemness and Chemoresistance by Activating TSC2/mTOR/c‐Myc Feedback Loop in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202500597

Figure Lengend Snippet: High QSOX2 enhances stemness, resulting in poor prognosis in ESCC. A) Heat maps showed that QSOX2 was significantly upregulated in ESCC tumors compared to normal esophageal tissues. B) Western blot was used to assess the protein level of QSOX2 in five pairs of ESCC tumors (E) and adjacent normal tissues (N). β‐Tubulin was used as a control. C) IHC staining was employed to analyze the protein level of QSOX2 in adjacent normal tissues, ESCC tumors, and metastatic lymph nodes. D) Kaplan–Meier survival curves demonstrated that a high level of QSOX2 was associated with poor prognosis in ESCC patients. E) Western blot was used to assess the protein level of QSOX2 in immortalized esophageal epithelial cell HET‐1A and six ESCC cell lines. F) Western blot was performed to confirm the overexpression or silence of QSOX2 in ESCC cells. G, H) Western blot was used to detect the expression of stemness markers after QSOX2 overexpression or knockdown. I, J) Flow Cytometry was conducted to measure the level of CD271 in ESCC cells after QSOX2 overexpression or silence. MFI, Mean Fluorescence Intensity. K) Tumor incidence was evaluated in BALB/c‐nude mice one month after injection of ESCC cells with a gradient cell count. L, M) Sphere formation assay was performed to assess the stemness of ESCC cells following QSOX2 overexpression or knockdown. In all panels, data are presented as the mean ± SD; In panels B and C, data were analyzed using paired two‐tailed Student's t‐test with Welch's correction; In panels I‐M, data were analyzed using unpaired two‐tailed Student's t‐test with Welch's correction; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The human immortalized esophageal epithelial cell line (HET‐1A) and six ESCC cell lines (KYSE30, KYSE140, KYSE150, KYSE180, KYSE410, and KYSE510) were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Western Blot, Control, Immunohistochemistry, Over Expression, Expressing, Knockdown, Flow Cytometry, Fluorescence, Injection, Cell Counting, Tube Formation Assay, Two Tailed Test

Journal: Advanced Science

Article Title: QSOX2‐Mediated Disulfide Bond Modification Enhances Tumor Stemness and Chemoresistance by Activating TSC2/mTOR/c‐Myc Feedback Loop in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202500597

Figure Lengend Snippet: QSOX2 promotes chemoresistance, proliferation, and metastasis in ESCC cells. A) Cell apoptosis assays evaluated the chemosensitivity of ESCC cells with QSOX2 overexpression. B) Calcein AM/PI double staining was performed to test the apoptotic cell rate of QSOX2 overexpressed ESCC cells under treatments with Cisplatin or Paclitaxel. C) Cell apoptosis assays showed the increased chemosensitivity of ESCC cells with QSOX2 silence. D) Calcein AM/PI double staining was used to analyze the apoptotic cell rate of QSOX2‐silenced ESCC cells treated with Cisplatin or Paclitaxel. E) Co‐expression analysis between QSOX2 and MKI67 in ESCA using the TCGA cohort. F, G) EdU staining was performed to test the cell proliferation rate of ESCC cells with QSOX2 overexpression or silence. H) Xenograft tumor experiment was performed using KYSE510‐Vector and KYSE510‐QSOX2 cells, and tumor volume was calculated. I, J) IHC staining with antibodies against QSOX2 and Ki67 was performed on xenograft tumors derived from QSOX2‐overexpressed KYSE510 cells (I) or QSOX2‐silenced KYSE180 cells (J). K) Transwell assay showed the enhanced cell migration and invasion abilities of ESCC cells with QSOX2 overexpression. L) A lung metastasis experiment was performed to analyze the metastatic ability of KYSE510‐Vector and KYSE510‐QSOX2. In I and J panels, data are presented as the mean ± SEM; other panels, data are presented as the mean ± SD; unpaired two‐tailed Student's t‐test with Welch's correction; * p < 0.05, ** p < 0.01, and *** p < 0.001. ns, no significant difference.

Article Snippet: The human immortalized esophageal epithelial cell line (HET‐1A) and six ESCC cell lines (KYSE30, KYSE140, KYSE150, KYSE180, KYSE410, and KYSE510) were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Over Expression, Double Staining, Expressing, Staining, Plasmid Preparation, Immunohistochemistry, Derivative Assay, Transwell Assay, Migration, Two Tailed Test

Journal: Advanced Science

Article Title: QSOX2‐Mediated Disulfide Bond Modification Enhances Tumor Stemness and Chemoresistance by Activating TSC2/mTOR/c‐Myc Feedback Loop in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202500597

Figure Lengend Snippet: QSOX2 enhances tumor stemness by upregulating c‐Myc. A) Correlation between QSOX2 and MsigDB hallmark gene sets in the TCGA cohort. B) Co‐expression analysis between QSOX2 and cell stemness markers in ESCA using the TCGA cohort. C) Volcano plots illustrate the differentially expressed genes between QSOX2 + and QSOX2 − ESCC cell populations using the GEO dataset ( GSE188955 ). D) Quantification of c‐Myc and QSOX2 staining intensity in ESCC patient tissues ( n = 200 cells) using ImageJ software, followed by linear regression analysis. E) Western blot analysis of c‐Myc expression after QSOX2 overexpression in ESCC cells. F) Percentage of c‐Myc positive tumor cells in xenograft tumors derived from QSOX2‐ or vector‐transfected KYSE510 cells was analyzed by multiplex IF staining. G) Xenograft tumors derived from KYSE510‐QSOX2 cells were treated with Ebselen (5 mg kg −1 , i.p.) every three days for three times 12 days after cell injection, and tumor volume and weight were measured. H) Double IF staining with antibodies against QSOX2 and c‐Myc was performed on KYSE510‐QSOX2‐derived xenograft tumors after treatment with Ebselen. In F and H panels, data are presented as the mean ± SEM; in panel G, data are presented as the mean ± SD; unpaired two‐tailed Student's t‐test with Welch's correction; ** p < 0.01, and *** p < 0.001.

Article Snippet: The human immortalized esophageal epithelial cell line (HET‐1A) and six ESCC cell lines (KYSE30, KYSE140, KYSE150, KYSE180, KYSE410, and KYSE510) were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Expressing, Staining, Software, Western Blot, Over Expression, Derivative Assay, Plasmid Preparation, Transfection, Multiplex Assay, Injection, Two Tailed Test

Journal: Advanced Science

Article Title: QSOX2‐Mediated Disulfide Bond Modification Enhances Tumor Stemness and Chemoresistance by Activating TSC2/mTOR/c‐Myc Feedback Loop in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202500597

Figure Lengend Snippet: QSOX2 activates the mTOR/c‐Myc signaling by promoting the phosphorylation of TSC2 at Ser939 site. A) LC‐MS/MS analysis of QSOX2 binding proteins. B) Protein IP assay was performed with QSOX2 antibody on KYSE30 and KYSE180 cells. C) Multiplex IF staining showed co‐localization of QSOX2, TSC1, and TSC2 in KYSE30 and KYSE180 cells. D) Multiplex IF staining showed co‐localization of QSOX2, TSC1, and TSC2 in ESCC patient tissues. E) Western blot was used to analyze the activation or inactivation of TSC2/mTOR/4E‐BP1/c‐Myc signaling after QSOX2 overexpression or silence in ESCC cells. F) The levels of QSOX2, phosphorylated TSC2 (p‐TSC2 Ser939 ), and phosphorylated mTOR (p‐mTOR Ser2448 ) in ESCC patient tissues with high or low QSOX2 expression were analyzed by multiplex IF staining. G) Multiplex IF staining showed the co‐expression of QSOX2, p‐mTOR Ser2448 and c‐Myc in ESCC patient tissues. H) Multiplex IF staining showed the levels of QSOX2, p‐TSC2 Ser939 , p‐mTOR Ser2448 and c‐Myc in xenograft tumors. I) Western blot was performed to test the levels of p‐mTOR Ser2448 and c‐Myc in KYSE140‐Vector and KYSE140‐QSOX2 cells treated with different concentrations of Rapamycin (24 h). J) Sphere formation assay to evaluate stemness in QSOX2‐overexpressing ESCC cells treated with Rapamycin (10 µM, 24 h). K) Calcein AM/PI double staining was performed to analyze the effect of Rapamycin on the apoptotic cell rate of KYSE140‐Vector and KYSE140‐QSOX2 cells treated with Cisplatin or Paclitaxel. In J and K panels, data are presented as the mean ± SD; unpaired two‐tailed Student's t‐test with Welch's correction; *** p < 0.001. ns, no significant difference.

Article Snippet: The human immortalized esophageal epithelial cell line (HET‐1A) and six ESCC cell lines (KYSE30, KYSE140, KYSE150, KYSE180, KYSE410, and KYSE510) were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Multiplex Assay, Staining, Western Blot, Activation Assay, Over Expression, Expressing, Plasmid Preparation, Tube Formation Assay, Double Staining, Two Tailed Test

Journal: Advanced Science

Article Title: QSOX2‐Mediated Disulfide Bond Modification Enhances Tumor Stemness and Chemoresistance by Activating TSC2/mTOR/c‐Myc Feedback Loop in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202500597

Figure Lengend Snippet: QSOX2 promotes disulfide bond formation and phosphorylation of TSC2 by binding Akt. A) Western blot analysis confirming the activation of p‐TSC2 Ser939 after QSOX2 overexpression and the inhibition of p‐TSC2 Ser939 by Akt inhibitor MK‐2206 treatment (5 µM, 24 h). B, C) Western blot analysis showing the effect of QSOX2 on the protein levels of total Akt and p‐Akt Ser473 in the indicated ESCC cells. D) Protein IP assay was performed with TSC2 antibody on ESCC cells with or without QSOX2 overexpression. E) Protein IP assay was performed with TSC2 antibody on ESCC cells with or without QSOX2 silence. F) Multiplex IF demonstrated co‐localization of TSC2, Akt, and QSOX2 in KYSE140‐Vector and KYSE140‐QSOX2 cells. G) Western blot showed the protein band location of TSC2 from Vector‐ or QSOX2‐transfected ESCC cells. Protein lysates were treated with polyethylene glycol maleimide (PEG‐mal) or N‐ethylmaleimide (NEM) as indicated above each blot. PEG‐mal can alkylate free cysteine residues that have not formed disulfide bonds, thereby increasing the molecular weight of the protein. H) ESCC cells were treated with Akt inhibitor MK‐2206 (5 µM, 24 h), and protein lysates were treated with PEG‐mal as indicated above each blot. Western blot analyzed the protein band location of TSC2 from Vector‐ or QSOX2‐transfected ESCC cells. I, J) Mass spectrometry analysis coupled with quantitative bar chart visualization revealed alterations in the disulfide bond formation between C189 and C977/C1026 residues of TSC2 protein in KYSE510‐Vector versus KYSE510‐QSOX2 cells.

Article Snippet: The human immortalized esophageal epithelial cell line (HET‐1A) and six ESCC cell lines (KYSE30, KYSE140, KYSE150, KYSE180, KYSE410, and KYSE510) were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Phospho-proteomics, Binding Assay, Western Blot, Activation Assay, Over Expression, Inhibition, Multiplex Assay, Plasmid Preparation, Transfection, Molecular Weight, Mass Spectrometry

Journal: Advanced Science

Article Title: QSOX2‐Mediated Disulfide Bond Modification Enhances Tumor Stemness and Chemoresistance by Activating TSC2/mTOR/c‐Myc Feedback Loop in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202500597

Figure Lengend Snippet: CAFs‐secreted IGF‐1 upregulates the Akt/mTOR/c‐Myc/QSOX2 signaling. A) CAFs‐conditioned media upregulated the levels of QSOX2, p‐mTOR, p‐4E‐BP1, and c‐Myc in KYSE180 cells. B) Western blot was used to test the levels of QSOX2, p‐mTOR, p‐4E‐BP1, and c‐Myc in KYSE180 cells treated with fibroblasts‐conditioned media. NFs, normal fibroblasts; CAFs, cancer‐associated fibroblasts. C) Multiplex IF staining confirmed that CAFs (α‐SMA positive) were adjacent to QSOX2‐expressed ESCC cells. D) Single‐cell RNA sequencing from GEO datasets ( GSE160269 and GSE188955 ) was performed to identify the main cell populations expressing IGF‐1. E) The levels of QSOX2, p‐mTOR, p‐4E‐BP1, and c‐Myc in KYSE180 cells treated with different concentrations of IGF‐1 (24 h) were analyzed by western blot. F) Western blot confirmed the activation of IGF1R/Akt/mTOR/c‐Myc/QSOX2 signaling in KYSE180 cells by CAFs‐conditioned media, and this stimulation was inhibited by IGF1R inhibitor Linsitinib (5 µM, 24 h). G) Western blot showed the activation of IGF1R/AKT/mTOR/c‐Myc/QSOX2 signaling by IGF‐1 treatment (10 ng µL −1 , 24 h), and this stimulation was inhibited by Linsitinib (5 µM), MK‐2206 (5 µM), or Rapamycin (10 µM). H, I) Multiplex IF staining confirmed the activation of IGF1R/c‐Myc/QSOX2 signaling by IGF‐1 treatment (10 ng µL −1 , 24 h), and this stimulation was inhibited by Linsitinib (5 µM) in KYSE180 (H) and KYSE140 (I) cells. J) The transcriptional binding site of c‐Myc in the QSOX2 gene promoter. K) ChIP‐qPCR analysis showing that IGF‐1 (10 ng µL −1 , 24 h) stimulation promoted the binding of c‐Myc to the promoter of QSOX2 gene. L) Multiplex IF staining was performed to analyze the levels of p‐IGF1R, c‐Myc and QSOX2 on KYSE180‐derived xenograft tumors treated with or without Linstinib (25 mg kg −1 , i.g.). In panel K, data are presented as the mean ± SD; unpaired two‐tailed Student's t‐test with Welch's correction; * p < 0.05.

Article Snippet: The human immortalized esophageal epithelial cell line (HET‐1A) and six ESCC cell lines (KYSE30, KYSE140, KYSE150, KYSE180, KYSE410, and KYSE510) were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Western Blot, Multiplex Assay, Staining, RNA Sequencing, Expressing, Activation Assay, Binding Assay, ChIP-qPCR, Derivative Assay, Two Tailed Test

Journal: Advanced Science

Article Title: QSOX2‐Mediated Disulfide Bond Modification Enhances Tumor Stemness and Chemoresistance by Activating TSC2/mTOR/c‐Myc Feedback Loop in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202500597

Figure Lengend Snippet: Combining Ebselen, Rapamycin, and Cisplatin inhibits ESCC progression. A) Western blot confirmed that the inhibition of mTOR/4E‐BP1/c‐Myc signaling in KYSE140/KYSE510‐QSOX2 cells by Ebselen (100 µM, 24 h) or/and Rapamycin (10 µM, 24 h) treatments. B) Calcein AM/PI double staining was performed to test the apoptotic cell rate of KYSE140‐QSOX2 cells treated with Ebselen (100 µM, 24 h) or/and Rapamycin (10 µM, 24 h). C) Cell apoptosis assays showed the sensitivity of KYSE510‐QSOX2 cells to Ebselen (100 µM, 24 h), Rapamycin (10 µM, 24 h), Cisplatin (10 µM, 24 h), or their combination. D, E) Tumors were generated by s.c. injection of KYSE510‐QSOX2 cells (3 × 10 6 cells per mouse). The mice were treated with Ebselen (5 mg kg −1 , i.p.), Rapamycin (5 mg kg −1 , i.p.), Cisplatin (5 mg kg −1 , i.p.) alone or in combination every three days for three times. Concurrently with the treatment, tumor volume was measured. F) Double IF staining showed the percentage of Ki67 or cleaved Caspase‐3 (Cl‐Casp3) positive tumor cells in KYSE510‐QSOX2‐derived xenograft tumors. G) Double IF staining was performed to analyze the percentage of c‐Myc positive tumor cells in KYSE510‐QSOX2‐derived xenograft tumors treated with the indicated treatments. H) Multiplex IF staining showed the mean fluorescence intensity of p‐p38 and p‐ERK in KYSE510‐QSOX2‐derived xenograft tumors after treatments. In B, C, and E panels, data are presented as the mean ± SD; In panels F‐H, data are presented as the mean ± SEM; unpaired two‐tailed Student's t‐test with Welch's correction; * p < 0.05, ** p < 0.01, and *** p < 0.001. ns, no significant difference.

Article Snippet: The human immortalized esophageal epithelial cell line (HET‐1A) and six ESCC cell lines (KYSE30, KYSE140, KYSE150, KYSE180, KYSE410, and KYSE510) were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Western Blot, Inhibition, Double Staining, Generated, Injection, Staining, Derivative Assay, Multiplex Assay, Fluorescence, Two Tailed Test

Journal: Advanced Science

Article Title: QSOX2‐Mediated Disulfide Bond Modification Enhances Tumor Stemness and Chemoresistance by Activating TSC2/mTOR/c‐Myc Feedback Loop in Esophageal Squamous Cell Carcinoma

doi: 10.1002/advs.202500597

Figure Lengend Snippet: Blocking QSOX2‐mTOR feedback loop, in combination with chemotherapy, reduces tumor stemness and induces tumor dormancy. CAFs secrete IGF‐1 to activate the IGF1R/Akt/mTOR/c‐Myc signaling axis in ESCC cells, leading to an increase in QSOX2 expression. High QSOX2 facilitates the formation of disulfide bonds in TSC2, thereby promoting the binding of TSC2 to Akt and subsequent phosphorylation of TSC2 at the Ser939 site. Phosphorylation of TSC2 at Ser939 relieves its inhibitory effect on mTOR/c‐Myc signaling. Therefore, this mechanism constitutes a positive feedback loop, enhancing tumor stemness, chemotherapy drug resistance, and metastasis of ESCC cells. Inhibiting QSOX2 with Ebselen, in combination with Rapamycin and chemotherapy, inhibits ESCC progression by blocking QSOX2‐mTOR feedback loop, suppressing tumor stemness, enhancing chemotherapy sensitivity, and promoting tumor dormancy.

Article Snippet: The human immortalized esophageal epithelial cell line (HET‐1A) and six ESCC cell lines (KYSE30, KYSE140, KYSE150, KYSE180, KYSE410, and KYSE510) were purchased from the DSMZ (Braunschweig, Germany).

Techniques: Blocking Assay, Expressing, Binding Assay, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Heatmap illustration of mRNA expression levels in TCGA LUAD and LUSC cohorts ( n = 1,016 tumors). An EMT score calculated for each tumor, as described previously , was correlated with each PI4K family member or, as a comparison, with the EMT-activating transcription factor using Pearson’s coefficient ( r value). ( B ) qPCR analysis of PI4K2A and PI4KB mRNA levels in human lung cancer cell lines classified as epithelial (E) or mesenchymal (M). ( C and D ) WB analysis of PI4K2A, PI4KB, and ZEB1 levels in epithelial ( C ) or mesenchymal ( D ) cells subjected to ZEB1 gain or loss of function, respectively. Relative densitometric values are shown under the gel lanes. α-Tubulin was used as a loading control. Empty vector (Vec), scrambled control (siCTL), and ZEB1 (siZEB1) siRNAs were used. ( E ) WB analysis of PI4K2A in cells transfected with miR mimics. ( F ) PI4K2A 3′-UTR reporter assays. H1299 cells were cotransfected with miR mimics and reporters containing WT or miR-182/-183 binding site mutant 3′-UTRs ( n = 4 replicates per condition). ( G – I ) PI4P ELISA in siRNA-transfected H1299 ( G ), H441 ( H ), and HCC827 ( I ) cells. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( B ); 1-way ANOVA test for multiple comparisons ( F – I ). miR-NC, negative control mimic.

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Expressing, Comparison, Control, Plasmid Preparation, Transfection, Binding Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) qPCR analysis of ACBD3 mRNA levels in the cell lines (dots) described in B. ( B and C ) WB analysis of ACBD3 protein levels in epithelial ( B ) and mesenchymal ( C ) cells subjected to ZEB1 gain or loss of function, respectively. ( D and E ) PI4P ELISA in mesenchymal ( D ) and epithelial ( E ) cells subjected to ACBD3 gain or loss of function, respectively. ( F ) Schema showing constructs containing MS2 binding sites (12X) fused downstream of a WT or mutant (MT) ACBD3 3′-UTR lacking the miR-34a–binding site (BS). ( G ) MS2-based RIP. MS2-UTR–associated miR-34a was quantified as fold enrichment values relative to MS2. miR-200b was included as a negative control. ( H and I ) WB analysis of ACBD3 and ZFP36L1 levels in cells transfected with miR mimics ( H ) or siRNAs ( I ). ( J and K ) WB analysis of ZFP36L1 levels in cell lysates (input), an MS2-based RIP complex (GFP), or a negative control IP (IgG). ( L ) Schema of the working model. ZEB1 executes a PI4KB-to-PI4K2A dependency switch by silencing miR-34a and miR-182/-183. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P values were determined by 2-tailed Student’s t test for 2-group comparisons ( A , D , and E ); 1-way ANOVA test for multiple comparisons.

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Enzyme-linked Immunosorbent Assay, Construct, Binding Assay, Mutagenesis, Negative Control, Transfection

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) WB analysis confirming target gene deletion in PI4K2A-KO H1299 cells. ( B ) Orthotopic lung tumor size (left plot) and mediastinal and contralateral lung metastasis numbers (right plot) generated in nude mice (dots) by the intrathoracic injection of cells described in A . ( C ) WB analysis confirming target gene depletion in shRNA-transfected 344SQ cells. ( D ) Tumor weights (left plot) and lung metastasis numbers (right plot) generated in syngeneic, immunocompetent mice (dots) by subcutaneous injection of the cells described in C . ( E ) Daily subcutaneous tumor volume measurements (dots) in nude mice treated with PI-273 or vehicle (DMSO). ( F and G ) Tumor tissues removed at necropsy in E were imaged ( F ) and weighed ( G ). ( H ) Orthotopic lung tumor size (left plot) and metastasis numbers (right plot) in nude mice treated with PI-273 or vehicle. ( I ) Kaplan-Meier survival analysis of mice bearing orthotopic lung tumors treated with PI-273 or DMSO. ( J ) WB analysis demonstrating reconstitution of shPI4K2A-transfected H1299 cells (shUTR) with WT or enzyme-dead mutant (D308A) PI4K2A. Empty vector (Vec). ( K ) Orthotopic lung tumors (arrows) generated in nude mice by the cells in J . Scale bars: 5 mm. ( L ) Orthotopic lung tumor size (left plot) and metastasis numbers (right plot). ( M ) Annexin V/propidium iodide flow cytometric analysis of the apoptotic fraction in siRNA-transfected cells. ( N ) Colonies formed in soft agar by siRNA-transfected cells. Values are expressed relative to siCTL. ( O ) Boyden chamber migration and invasion assays on siRNA-transfected cells. Values are expressed relative to siCTL. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P values were determined by 2-tailed Student’s t test for 2-group comparisons ( E – H ); 1-way ANOVA test for multiple comparisons ( B , D , and L – O ; and log-rank test ( I ). shCTL, control shRNA.

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Generated, Injection, shRNA, Transfection, Mutagenesis, Plasmid Preparation, Migration, Control

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Relative soft agar colony numbers. siRNA-transfected H1299 cells were treated with CM samples from siRNA-transfected H1299 cells. ( B ) Apoptosis assays on siRNA-transfected H1299 cells treated with CM samples. WB analysis of cleaved PARP1 (C-PARP1) and PI4K2A (gel). ( C ) Volcano plot illustration of proteins (dots) identified by LC-MS analysis of CM samples. P value ( y axis) and fold change ( x axis) are shown. PI4K2A-upregulated secreted proteins (pink quadrant) of interest are labeled. ( D ) Gene Ontology analysis of the pink quadrant in C . ( E ) Subcutaneous tumors (dots) were weighed (left plot) and subjected to flow cytometry to quantify CD31 + cells (middle plot) and cleaved-caspase 3 + (CC3 + ) cells (right plot). ( F ) Heatmap illustration of the correlation between mRNAs and EMT scores (Byers or Creighton) in TCGA LUAD cohort. r values were determined by Pearson’s correlation. ( G ) Kaplan-Meier survival analysis of TCGA LUAD and LUSC cohorts based on 6-gene signatures of secreted proteins. Tumors were scored as being above (high) or below (low) each cohort’s median values. ( H – J ) Apoptosis assays and WB analysis of cleaved PARP1 (gel) ( H ), soft agar colony formation assays ( I ), and Boyden chamber migration and invasion assays ( J ) were carried out on siRNA-transfected H1299 cells. ( K ) HUVEC migration in Boyden chambers. CM samples from siRNA-transfected H1299 cells were loaded into the lower wells. Scale bars: 200 μm. ( L ) HUVEC spheroid invasion assay. HUVEC spheroids were seeded in 3D collagen and treated with CM samples. Scale bar: 100 μm. ( M ) HUVEC tube formation assay in 3D Matrigel following treatment with CM samples. Scale bars: 100 μm. ( N ) HUVEC migration in Boyden chambers. CM from siRNA-transfected H1299 cells was loaded into the lower chambers. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( E ); 1-way ANOVA test for multiple comparisons ( A , B , and H – N ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Transfection, Liquid Chromatography with Mass Spectroscopy, Labeling, Flow Cytometry, Migration, Invasion Assay, HUVEC Tube Formation Assay

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Single-channel and merged confocal micrographs of total and surface VSV-G in H1299 cells cotransfected with siRNAs and EGFP–VSV-G and imaged 30 minutes after transfer to the permissive temperature. Plot shows the ratio of surface VSV-G to total VSV-G in each cell (dot) 30 or 60 minutes after transfer to 32°C. Scale bar: 20 μm. ( B ) BRET measurement of PI4P in RAB6A + vesicles in siRNA-transfected H1299 cells. Results are expressed as a ratio of the values from GSK-A1–treated and vehicle-treated (DMSO) cells at each time point ( n = 5 replicates per group). ( C ) Confocal micrographs of RAB6A + vesicles (blue arrows) and unfissioned RAB6A + tubules (red arrows) emerging from the Golgi. Scale bar: 20 μm. Dotted lines indicate the cell boundaries. Results were quantified per cell (dot plots). ( D ) Venn diagram of PI42KA-interacting proteins identified by TurboID and IP approaches. Overlapping proteins are listed on the right. Reported PI4K2A-interacting proteins ( , ) are shown in bold. ( E ) Schematic illustration of full-length and truncated PI4K2A constructs. WB assays on whole-cell lysates (WCLs) (input) or IP proteins isolated from H1299 cells transfected with MYC-tagged PI4K2A constructs (gel). Full-length (1–479) and truncated constructs are indicated under the gels. IgG was used as the control IP. ( F ) WB analysis of WCLs (WCL) and Golgi-enriched fractions (Golgi) from siRNA-transfected H1299 cells. ( G ) Confocal micrographs of SPP1 + vesicles (arrows) in siRNA-transfected H1299 cells costained with anti-SPP1 and anti–Golgin 97 antibodies. Scale bars: 50 μm. Dot plot shows the vesicle numbers per cell. ( H ) WB analysis of WCLs or enriched subcellular fractions from siRNA-transfected H1299 cells. Densitometric values were normalized to siCTL (graph). Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( B ); 1-way ANOVA test for multiple comparisons ( A , C , G , and H ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Transfection, Construct, Isolation, Control

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) Intracellular levels of biotinylated Tfn in siRNA-transfected H1299 cells were quantified at the indicated time points after a 30-minute pulse. The percentage of the internalized Tfn pool was calculated relative to the initial loading ( n = 4 samples per condition). ( B ) WB analysis of CD44, ITGB1, and AXL protein levels in WCLs (input) or streptavidin bead–enriched protein samples from H1299 cells that were transfected with PI4K2A/TurboID construct and treated with biotin. Controls included the PI4KB/TurboID construct (PI4KB) and Turbo alone (CTL). ( C ) WB analysis of WCLs (input) or anti-HA immunoprecipitates from H1299 cells transfected with HA-tagged PI4K2A. IgG was used as the control IP. ( D ) SPP1 protein-protein interaction network (STRING-db.org). ( E ) WB analysis of proteins isolated by streptavidin bead–based pulldowns carried out on H1299 cells treated with biotin-labeled recombinant SPP1. ( F ) Confocal micrographs of plasma membrane–bound ITGB1 (arrows, upper panels) and CD44 (arrows, lower panels) in nonpermeabilized siRNA-transfected H1299 cells stained with antibodies against endogenous ITGB1 or CD44. Scale bars: 10 μm. ( G ) WB analysis of cell membrane–enriched fractions (Mem.) and WCL. Densitometric values are shown under the gels. ( H ) WB analysis of cleaved PARP1 (gel) and flow cytometric analysis of annexin V/PI–stained cells (graph) to quantify apoptosis in siRNA-transfected H1299 cells. ( I ) Boyden chamber migration and invasion assays on siRNA-transfected cells. ( J ) Schematic illustration of the working model. PI4K2A coordinates exocytic and endocytic vesicular trafficking to activate an SPP1-dependent autocrine loop. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. P values were determined by 1-way ANOVA test for multiple comparisons ( A , H , and I ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Transfection, Construct, Control, Isolation, Labeling, Recombinant, Clinical Proteomics, Membrane, Staining, Migration

Journal: The Journal of Clinical Investigation

Article Title: EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer

doi: 10.1172/JCI165863

Figure Lengend Snippet: ( A ) WB analysis of parental and PI4K2A-KO H1299 cells. Densitometric values are shown under the gels. Positive (EGFR) and negative (ITGB1) controls. ( B ) Correlation between AXL and PI4K2A protein levels in cell lines (dots). ( C ) WB analysis of parental and PI4K2A-KO H1299 cells treated with the AXL ligand Gas6. pAKT, phosphorylated AKT. Graphs show densitometric analysis of pAXL and pAKT levels normalized to t = 0 minutes. ( D ) Boyden chamber migration assays on parental and PI4K2A-KO H1299 cells treated with (+) or without (–) Gas6. ( E ) WB analysis of parental and PI4K2A-KO H1299 cells stably transfected with empty vector or AXL. ( F ) Boyden chamber migration assays on cells in E . ( G ) WB analysis of parental and PI4K2A-KO H1299 cells treated with cycloheximide (CHX). Graphs show the densitometric values. ( H ) Single-channel and merged confocal micrographs of parental and PI4K2A-KO cells costained with anti-AXL and anti-LAMP1 antibodies. Lysosomal AXL (inset, arrows) was quantified as the percentage of total AXL that colocalized with LAMP1 per field ( n = 10 fields per condition). Scale bar: 10 μm. Original magnification, ×2.5 (enlarged insets). ( I ) WB analysis of PI4K2A-KO cells treated with proteasomal (MG132) or lysosomal (leupeptin or monensin) inhibitors. DMSO was used as the vehicle. l.e., long exposure duration; s.e., short exposure duration. ( J ) WB analysis of siRNA-transfected H1299 cells. Densitometric values are shown under the gel. ( K ) WB analysis of WCLs (input) and anti-HA immunoprecipitates from H1299 cells transfected with HA-tagged PI4K2A. IgG was used as the negative control IP. ( L ) WB analysis of WCLs (input) and anti-HSP90 immunoprecipitates from parental and PI4K2A-KO H1299 cells. IgG was used as the negative control IP. Data indicate the mean ± SD from a single experiment incorporating biological replicate samples ( n = 3, unless otherwise indicated) and are representative of at least 2 independent experiments. ** P < 0.01 and *** P < 0.001, by 2-tailed Student’s t test for 2-group comparisons ( C , D , G , and H ); 1-way ANOVA test for multiple comparisons ( F ).

Article Snippet: 3724 and 2367), β-actin (no. 4970), GM130 (no. 12480), Golgin-97 (no. 13192), AXL (no. 8661), and EGFR (no. 2256) from Cell Signaling Technology; antibodies against ACBD3 (sc-101277), ZEB1 (sc-25388), and PI4K2A (sc-390026) from Santa Cruz Biotechnology; antibodies against SPP1 (22952-1-AP), SEMA7A (18070-1-AP), HSP90 (11405-1-AP), ZFP36L1 (12306-1-AP), and CD44 (15675-1-AP) from Proteintech; antibodies against ITGB1 (GTX636657) from GeneTex; antibodies against RNA polymerase II (AB_2732926) from Active Motif; recombinant SPP1 proteins (ab92964) from Abcam; recombinant Gas6 (885-GSB) and SPP1 (1433-OP-050) proteins from R&D Systems; PI-273 (HY-103489) from MedChemExpress; NC03 (AOB17420) from Aobious; and miRNA mimics (HMI0508, HMI0275, and HMI0280) and control mimics (HMC0002) from MilliporeSigma.

Techniques: Migration, Stable Transfection, Transfection, Plasmid Preparation, Negative Control

Journal: Experimental & Molecular Medicine

Article Title: SLC6A14-mediated glutamine promotes SYTL4–CXCL8 axis activation to drive gemcitabine resistance and immune evasion in pancreatic cancer

doi: 10.1038/s12276-025-01596-w

Figure Lengend Snippet: a Heatmap of the SLC family population between normal and tumor cells at the RNA level. b UMAP plot of a total of eight major cell clusters in patients with PDAC ( n = 23) c Dot plot of the top cell-type-specific markers for the patients with PDAC ( n = 23). d Feature plots indicating the UMAP space in which cells were color-coded according to SLC6A14 score. e UMAP plot of ductal and acinar cell clusters in patients with PDAC (n = 23). f , g Feature plots indicating the UMAP space in which cells were color-coded according to pseudotime score ( f ) and SLC6A14 score ( g ). h H&E staining and immunohistochemistry assays were performed on normal pancreatic and PDAC tumors from patients (40× magnification). i Volcano plots. The log 2 (fold change) represents the mean expression level of each gene. Each dot represents a single gene. Black dots represent no significant DEGs between the normal and tumor groups, red dots represent upregulated genes and blue dots represent downregulated genes ( GSE183795 ). j UMAP showing gene expression in normal pancreatic tissues and pancreatic cancer tissues ( GSE183795 ). k Gemcitabine-sensitive ( n = 6) and gemcitabine-resistant ( n = 6), followed by western blot (left) and RT–qPCR (right) analysis. l Kaplan–Meier survival analyses of patients with PDAC, based on SLC6A14 expression for overall survival. Scale bars, 50 μm ( h ). Error bars, mean ± s.d., # P < 0.05, ## P < 0.01, ### P < 0.001; n.s., not significant; by Student’s t -test ( k ).

Article Snippet: SLC6A14 Human shRNA lentiviral particles (4.5 × 10 6 ) (Origene, cat. no. TL309295V) were intravenously injected into the retro-orbital venous sinus.

Techniques: Staining, Immunohistochemistry, Expressing, Gene Expression, Western Blot, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: SLC6A14-mediated glutamine promotes SYTL4–CXCL8 axis activation to drive gemcitabine resistance and immune evasion in pancreatic cancer

doi: 10.1038/s12276-025-01596-w

Figure Lengend Snippet: a ROS levels in CAPAN-1, CAPAN-1/GR and CAPAN-1/GR either transfected with si SLC6A14 or treated with α-MT, measured by flow cytometry with DCF-DA staining. b OCR in CAPAN-1/GR and CAPAN-1/GR cells transfected with si SLC6A14 . c The peak area of glutamine in CAPAN-1, CAPAN-1/GR si Con and CAPAN-1/GR si SLC6A14 incubated for 24 h with a medium containing ±glutamine was measured by LC–MS analysis. d The GSH/GSSG ratio (GSSG, oxidized glutathione) in CAPAN-1, CAPAN-1/GR si Con and CAPAN-1/GR si SLC6A14 incubated with ± glutamine for 24 h was measured by LC–MS analysis. e Invasion assay on CAPAN-1/GR si Con , CAPAN-1/GR si SLC6A14 or α-MT for 24 h, then incubated with ±glutamine for 24 h (40× magnification). f Wound-healing assay on CAPAN-1/GR si SLC6A14 or α-MT for 24 h, then incubated with ±glutamine for 24 h. g CAPAN-1/GR si Con , CAPAN-1/GR siSLC6A14 or α-MT for 24 h, then incubated with ±glutamine for 24 h, followed by western blot analysis. h WST assay on CAPAN-1, CAPAN-1/GR si Con , CAPAN-1/GR si SLC6A14 or α-MT for 24 h, then incubated with ±glutamine for 24 h. i Sphere formation assay on CAPAN-1/GR si Con , CAPAN-1/GR si SLC6A14 or α-MT for 24 h, then visualized on the indicated time points (40× magnification). j Western blot analysis of stemness-related genes in CAPAN-1/GR si Con , and CAPAN-1/GR si SLC6A14 or α-MT. Scale bars, 100 μm ( e and i ). Error bars, mean ± s.d., # P < 0.05, ## P < 0.01; ### P < 0.001; n.s., not significant; by one-way ANOVA ( e , f and i ) or Student’s t -test ( c , d and h ).

Article Snippet: SLC6A14 Human shRNA lentiviral particles (4.5 × 10 6 ) (Origene, cat. no. TL309295V) were intravenously injected into the retro-orbital venous sinus.

Techniques: Transfection, Flow Cytometry, Staining, Incubation, Liquid Chromatography with Mass Spectroscopy, Invasion Assay, Wound Healing Assay, Western Blot, WST Assay, Tube Formation Assay

Journal: Experimental & Molecular Medicine

Article Title: SLC6A14-mediated glutamine promotes SYTL4–CXCL8 axis activation to drive gemcitabine resistance and immune evasion in pancreatic cancer

doi: 10.1038/s12276-025-01596-w

Figure Lengend Snippet: a The peak area of α-KG in CAPAN-1, CAPAN-1/GR si Con and CAPAN-1/GR si SLC6A14 incubated for 24 h with a medium containing ±glutamine was measured by LC–MS analysis. b Intracellular α-KG levels in CAPAN-1, CAPAN-1/GR si Con and CAPAN-1/GR si SLC6A14 incubated for 24 h with a medium containing ±glutamine, followed by glutamine assays. c Western blot analysis of SLC6A14, p-mTOR, mTOR, p-NF-κB and NF-κB in CAPAN-1, CAPAN-1/GR si Con and CAPAN-1/GR si SLC6A14 incubated for 24 h with ±glutamine. d Cytokine expression profiles in CAPAN-1/GR si Con versus si SLC6A14 via cytokine assays. Cytokine detection (top left), heatmap (bottom left) and P values (right). e CXCL8 levels in CAPAN-1, CAPAN-1/GR si Con , CAPAN-1/GR si SLC6A14 or α-MT for 24 h, then incubated with ±glutamine for 24 h. f GSEA of downregulated genes involved in the positive regulation of secretion in CAPAN-1/GR cells si Con versus si SLC6A14 (FDR q value <0.05; top). KEGG enrichment analysis of genes downregulated in CAPAN-1/GR cells transfected with si SLC6A14 (bottom). g A set of regulated secretion, exocytosis and SLC6A14 -related genes were compared, and two common genes were identified. Correlation analyses of CXCL8 between either ANXA2 or SYTL4 were performed using TISIDB. h Kaplan–Meier survival analysis of patients with PDAC based on SLC6A14 and SYTL4 expression for overall survival graph. i Endogenous SYTL4 was immunoprecipitated with endogenous SLC6A14 and NF-κB from CAPAN-1/GR cells and subjected to immunoprecipitation, followed by immunoblotting with indicated antibodies. j CAPAN-1/GR cells si SLC6A14 were subjected to immunoprecipitation, followed by immunoblot analysis with indicated antibodies. k CXCL8 secretion levels in CAPAN-1, CAPAN-1/GR si Con and CAPAN-1/GR si SLC6A14 or si SYTL4 , and CAPAN-1/GR cells cotransfected with both si SLC6A14 and si SYTL4 . Error bars, mean ± s.d., # P < 0.05; ## P < 0.01; ### P < 0.001; n.s., not significant; by Student’s t -test ( a , b , e and h ).

Article Snippet: SLC6A14 Human shRNA lentiviral particles (4.5 × 10 6 ) (Origene, cat. no. TL309295V) were intravenously injected into the retro-orbital venous sinus.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Western Blot, Expressing, Transfection, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: SLC6A14-mediated glutamine promotes SYTL4–CXCL8 axis activation to drive gemcitabine resistance and immune evasion in pancreatic cancer

doi: 10.1038/s12276-025-01596-w

Figure Lengend Snippet: a SLC6A14 and CXCR2 gene expression in the stroma ( n = 18) versus tumor ( n = 18) tissues from patients with PDAC ( GSE164665 ). b CXCR2 and α-SMA levels in NFs and CAFs via western blot analysis. c Expression of mitochondrial fission genes ( DNM1L , FIS1 and MFF ) and fusion genes ( OPA1 , MFN1 and MFN2 ) in NF and CAF via qRT–PCR analysis. d Western blot analysis of α-SMA, p-DRP1, DRP1 and OPA1 in NF, CAF and CAF incubated with CAPAN-1/GR si Con or si SLC6A14 -conditioned medium for 24 h. e Glutamine secretion in NF and CAF cocultured with control, CAPAN-1, CAPAN-1/GR si Con and CAPAN-1/GR si SLC6A14 -conditioned medium for 24 h. f Secreted extracellular glutamine in NF or CAF cocultured with CAPAN-1/GR si Con or si SLC6A14 -conditioned medium at 24 and 48 h, calculated by subtracting the initial time point from the consecutive time points. g CXCL8 levels in control, CAPAN-1, CAPAN-1/GR si Con and CAPAN-1/GR si SLC6A14 cocultured with NF and CAF-conditioned medium for 24 h. h Secreted extracellular CXCL8 from CAPAN-1/GR si Con versus si SLC6A14 cocultured with NF- or CAF-conditioned medium at 24 and 48 h, calculated by subtracting the initial time point from the consecutive time points. i Glutamine secretion levels of NF and CAF followed by 24 h coculture with control, CAPAN-1, CAPAN-1/GR si Con , CAPAN-1/GR si SLC6A14 or si SYTL4 , and CAPAN-1/GR both si SLC6A14 and si SYTL4 . Error bars, mean ± s.d., # P < 0.05, ## P < 0.01, ### P < 0.001; n.s., not significant; by one-way ANOVA ( f , h and i ) or Student’s t -test ( a – c , e and g ).

Article Snippet: SLC6A14 Human shRNA lentiviral particles (4.5 × 10 6 ) (Origene, cat. no. TL309295V) were intravenously injected into the retro-orbital venous sinus.

Techniques: Gene Expression, Western Blot, Expressing, Quantitative RT-PCR, Incubation, Control

Journal: Experimental & Molecular Medicine

Article Title: SLC6A14-mediated glutamine promotes SYTL4–CXCL8 axis activation to drive gemcitabine resistance and immune evasion in pancreatic cancer

doi: 10.1038/s12276-025-01596-w

Figure Lengend Snippet: a Gemcitabine-sensitive ( n = 5) and gemcitabine-resistant ( n = 5) cells were analyzed by western blot. b Kaplan–Meier survival analysis of patients with PDAC based on SLC6A14 and PD-L1 expression for overall survival graph. c Expression of SLC6A14, RRM1, p-NF-κB, NF-κB and PD-L1 in BxPC-3/GR si Con , BxPC-3/GR si SLC6A14 or si NF-κB , and BxPC-3/GR cells cotransfected with both si SLC6A14 and si NF-κB , analyzed by western blot. d Expression of SLC6A14, RRM1, p-NF-κB, NF-κB and PD-L1 in CAPAN-1/GR si Con , CAPAN-1/GR si SLC6A14 or si NF-κB , and CAPAN-1/GR cells cotransfected with both si SLC6A14 and si NF-κB via western blot analysis. e Volcano plot of the Spearman correlation between CD8 + T cell infiltration and SLC6A14 in 40 cancer types. Blue points indicate cancer types in which SLC6A14 was significantly negatively correlated with CD8 + T cell infiltration (adjusted P < 0.05). f , g T cell-mediated cancer cell killing. The GR-PDAC cell lines, BxPC-3/GR ( f ) and CAPAN-1/GR ( g ), with si SLC6A14 incubated for 24 h with ±glutamine were cocultured with activated human CD8 + T cells for 48 h to observe the effects of SLC6A14 on T cells. The cancer cells that survived were stained with crystal violet. The ratio of cancer cells to T cells was 1:10. Representative images are shown on the left, and quantitative data are shown on the right. h , i Activated CD8 + T cells cocultured with BxPC-3/GR ( h ) and CAPAN-1/GR ( i ) cells subjected to ELISA for IFN-γ measurement. Error bars, mean ± s.d., # P < 0.05, ## P < 0.01, ### P < 0.001; n.s., not significant; by one-way ANOVA ( f and g ) or Student’s t -test ( h and i ).

Article Snippet: SLC6A14 Human shRNA lentiviral particles (4.5 × 10 6 ) (Origene, cat. no. TL309295V) were intravenously injected into the retro-orbital venous sinus.

Techniques: Western Blot, Expressing, Incubation, Staining, Enzyme-linked Immunosorbent Assay

Journal: Experimental & Molecular Medicine

Article Title: SLC6A14-mediated glutamine promotes SYTL4–CXCL8 axis activation to drive gemcitabine resistance and immune evasion in pancreatic cancer

doi: 10.1038/s12276-025-01596-w

Figure Lengend Snippet: a BALB/c nude mice were orthotopically injected with CAPAN-1 or CAPAN-1/GR cells (3 × 10 6 ). After 3 weeks, lentivirus particles carrying sh Control or sh SLC6A14 were injected (2 × 10 6 particles) via the retro-orbital route. After 1 week, 10 mg/kg gemcitabine ( n = 5) was administered intraperitoneally twice a week. b Tumor size. c Tumor weight. d Representative images of pancreatic tumors ( n = 5). e Immunofluorescence staining of SLC6A14 (red), SYTL4 (green) and DAPI (blue) for the nuclei. f Immunofluorescence staining of p-mTOR (red) and DAPI (blue) in the nuclei. g Immunofluorescence staining of p-NF-κB (red) and DAPI (blue) in the nuclei. h Intratumoral glutamine levels in the pancreatic tissue. i Expression levels of SLC6A14 , GLS , GOT2 and GLUD1 in the pancreatic tissues, followed by qRT–PCR analysis. j , Liver metastases ( n = 5). k Expression of SLC6A14, p-NF-κB, NF-κB, PD-L1, SYTL4, E-cadherin and N-cadherin in pancreatic tissues, followed by western blotting analysis. Scale bars, 50 μm ( e – g ). Error bars, mean ± s.d., # P < 0.05, ## P < 0.01, ### P < 0.001; n.s., not significant; by one-way ANOVA ( b , c and h ) or Student’s t -test ( i ). GS-sh Con , gemcitabine-sensitive sh Control ; GR-sh Con , gemcitabine-resistant sh Control ; GR-sh SLC6A14 , gemcitabine-resistant sh SLC6A14 .

Article Snippet: SLC6A14 Human shRNA lentiviral particles (4.5 × 10 6 ) (Origene, cat. no. TL309295V) were intravenously injected into the retro-orbital venous sinus.

Techniques: Injection, Control, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: SLC6A14-mediated glutamine promotes SYTL4–CXCL8 axis activation to drive gemcitabine resistance and immune evasion in pancreatic cancer

doi: 10.1038/s12276-025-01596-w

Figure Lengend Snippet: a BALB/c nude mice were subcutaneously injected with CAPAN-1 or CAPAN-1/GR cells (2 × 10 5 ) with or without NFs or CAFs (3 × 10 5 ). After 3 weeks, lentivirus particles carrying sh Control or sh SLC6A14 were injected (2 × 10 6 particles) via the retro-orbital route. After 1 week, 10 mg/kg gemcitabine ( n = 5) was administered intraperitoneally twice a week. b Tumor volume. c Tumor weight. d Immunofluorescence staining of PD-L1 (red), ki-67 (green) and DAPI (blue) in the nuclei. e Intratumoral glutamine levels, followed by glutamine assays. f Protein expression of SLC6A14, SYTL4, CXCR2, p-DRP1, DRP1 and OPA1 in the pancreatic tissues. g Immunofluorescence staining of CK19 (green), α-SMA (red) and DAPI (blue) in the nuclei. h Schematic representation of the mechanism by which SLC6A14 confers gemcitabine resistance in pancreatic cancer by upregulating NF-κB/PD-L1 to promote immune evasion and activating SYTL4/CXCL8–CXCR2 signaling to reprogram the gemcitabine-resistant TME. Scale bars, 50 μm ( d and g ). Error bars, mean ± s.d., # P < 0.05, ## P < 0.01, ### P < 0.001; n.s., not significant; by one-way ANOVA ( b and e ) or Student’s t -test ( c ).

Article Snippet: SLC6A14 Human shRNA lentiviral particles (4.5 × 10 6 ) (Origene, cat. no. TL309295V) were intravenously injected into the retro-orbital venous sinus.

Techniques: Injection, Control, Immunofluorescence, Staining, Expressing

Journal: Journal of Biomedical Science

Article Title: Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

doi: 10.1186/s12929-023-00957-9

Figure Lengend Snippet: M2-Exos inhibit PMN recruitment and NET formation during sepsis in vitro. a and b PMNs from healthy volunteers were preactivated by septic plasma and then cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages. After 5 h, PMNs were collected for migration capacity analysis with IL-8 as a chemokine. After a 2-h incubation, cells in the lower chamber were collected and counted under a microscope. c and d PMNs isolated from septic patients were directly cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h after isolation. Then, PMNs were transferred for the transwell assay. e – h Ex vivo NET formation assay with neutrophils isolated from healthy volunteers or septic patients activated by septic plasma (SP). Plasma from healthy volunteers (HP) was used as a negative control. Typical images of NET formation are presented in e and g using SYTOX Green (green), where white arrows indicate NETs. Scale bar, 50 μm. NET formation was quantified as the percentage of neutrophils forming NETs and the NET area per microscopic field. f and h Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. One-way analysis of variance with Tukey’s multiple comparisons test was used for the analysis. Graphs represent means ± standard deviations; * P < 0.05, ** P < 0.01 compared within two groups

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll 1.077 density gradient (Solarbio, Beijing, China) as described previously [ ].

Techniques: In Vitro, Clinical Proteomics, Derivative Assay, Migration, Incubation, Microscopy, Isolation, Transwell Assay, Ex Vivo, Tube Formation Assay, Negative Control, Cell Culture

Journal: Journal of Biomedical Science

Article Title: Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

doi: 10.1186/s12929-023-00957-9

Figure Lengend Snippet: M2-Exos lead to lipid mediator class switching of PMNs during sepsis. a , c and d PMNs isolated from healthy volunteers were preactivated upon adding 20% septic plasma (SP) to the culture medium, and 20% plasma from healthy volunteers (HP) was used as a negative control. After 1 h, the culture medium was replaced with fresh medium, and PMNs were then cocultured with PBS/M0-Exos/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h. b , e and f PMNs from septic patients were directly cocultured with M0/M2-Exos (100 μg/mL) for 5 h after isolation. a and b Targeted metabolite analysis of eicosanoids in M0/M2-Exo-treated PMNs using liquid chromatography high-resolution mass spectrometry. LXA4 ( c and e ) and LTB4 ( d and f ) concentrations in the supernatant of cocultured PMNs were detected by ELISA kits. g LTB4 levels were compared in M0-Exos and M2-Exos from PBMC-differentiated macrophages by ELISA. One-way analysis of variance with Tukey’s multiple comparisons test ( c – f ) or Student’s t test ( a , b , g ) was used for the analysis. Graphs represent means ± standard deviations, n = 3–5; * P < 0.05, ** P < 0.01 compared within two groups. TXB2, thromboxane B2; PGE2, prostaglandin E2; PGD2, prostaglandin D2; PGA2, prostaglandin A2; PGJ2, prostaglandin J2; 6-trans-LTB4,6-trans leukotriene B4; 15-HETE, 15-hydroxyeicosatetraenoic acid; 14,15-EET, 14,15-epoxy-5,8,11-eicosatrienoic acid; 5-oxo-ETE, 5-Oxo-6,8,11,14-eicosatetraenoic acid; 11,12-EET, 11,12-epoxy-5,8,14-eicosatrienoic acid; 8,9-EET, 8,9-epoxyeicosatrienoic acid

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll 1.077 density gradient (Solarbio, Beijing, China) as described previously [ ].

Techniques: Isolation, Clinical Proteomics, Negative Control, Derivative Assay, Liquid Chromatography, Mass Spectrometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biomedical Science

Article Title: Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

doi: 10.1186/s12929-023-00957-9

Figure Lengend Snippet: M2-Exos inhibit PMN recruitment and NET formation through LXA4 upregulation. a Ex vivo NET formation assay with PMNs isolated from healthy volunteers or septic patients activated by septic plasma (SP) and then cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h with or without BOC-2 (10 µM). Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. b Typical images of NET formation using SYTOX Green (green). Scale bar, 50 μm. NET formation was quantified as the percentage of neutrophils forming NETs and the NET area per microscopic field. c Transwell analysis of PMN migration capacity isolated from healthy volunteers or septic patients. One-way analysis of variance with Tukey’s multiple comparisons test was used for the analysis. n = 4. d – l WT C57BL/6 mice were administered M2-Exos (300 μg/mouse) derived from mouse Raw264.7 macrophages via intraperitoneal injection 1 h after CLP. To block the LXA4 receptor, mice were treated with 50 µg/kg BOC-2 i.p. 30 min before CLP. d and e Quantification of dsDNA and circulating NET structures in the plasma of mice using PicoGreen fluorescent dye and MPO-DNA-ELISA, respectively. f Representative images showing the presence of NETs (MPO, red; citrullinated H3, green) in the lung tissues, as indicated by white arrows. Nuclei were counterstained with DAPI (blue). Scale bar, 40 μm. g Flow cytometry detection of the percentage of systemic circulating PMNs by staining with CD11b and Gr-1. h Absolute neutrophil number in peripheral blood. i and j Ly6G + cells in the lung tissues were detected by immunofluorescence and immunohistochemistry. Scale bar, 40 μm. k Evaluation of lung histology by H&E staining (magnification × 400). Red arrows indicate neutrophils in the alveolar and interstitial space, blue arrows indicate alveolar macrophages, and green arrows indicate proteinaceous debris filling. Scale bar, 50 μm. Lung injury scores were assessed. l Detection of inflammatory cytokine mRNA (IL-1β, IL-6, TNF-α) expression in lung tissues by RT‒qPCR. Student’s t test was used for the analysis. Graphs represent means ± standard deviations, n = 6; * P < 0.05, ** P < 0.01 compared within two groups

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll 1.077 density gradient (Solarbio, Beijing, China) as described previously [ ].

Techniques: Ex Vivo, Tube Formation Assay, Isolation, Clinical Proteomics, Derivative Assay, Cell Culture, Migration, Injection, Blocking Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Immunofluorescence, Immunohistochemistry, Expressing

Journal: Journal of Biomedical Science

Article Title: Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

doi: 10.1186/s12929-023-00957-9

Figure Lengend Snippet: LXA4 increased by M2-Exos downregulates CXCR2 and ROS expressions in PMNs. PMNs isolated from healthy volunteers ( a and b ) or septic patients ( d ) were activated by septic plasma (SP) and then cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h with or without BOC-2 (10 µM) incubation. c PMNs from septic patients were directly cocultured with M0/M2-Exos (100 μg/mL) for 5 h after isolation. CXCR2 ( a and c ) and ROS ( b and d ) expressions in cocultured PMNs were detected by flow cytometry. n = 4–5. e and f WT C57BL/6 mice were administered M0/M2-Exos (300 μg/mouse) derived from mouse Raw264.7 macrophages via intraperitoneal injection 1 h after CLP. To block the LXA4 receptor, mice were treated with 50 µg/kg BOC-2 i.p. 30 min before CLP. CXCR2 ( e ) and ROS ( f ) expressions in peripheral blood neutrophils were detected by flow cytometry. One-way analysis of variance with Tukey’s multiple comparisons test was used for the analysis. Graphs represent means ± standard deviations, n = 6; * P < 0.05, ** P < 0.01 compared within two groups

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll 1.077 density gradient (Solarbio, Beijing, China) as described previously [ ].

Techniques: Isolation, Clinical Proteomics, Derivative Assay, Incubation, Flow Cytometry, Injection, Blocking Assay

Journal: Journal of Biomedical Science

Article Title: Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

doi: 10.1186/s12929-023-00957-9

Figure Lengend Snippet: M2-Exos promote LXA4 production in PMNs by increasing 15-LO expression. a – i PMNs isolated from healthy volunteers were activated by septic plasma (SP) and then cocultured with M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h with or without PD146176 (1 µM) and LXA4 (100 nM), as indicated in the figures. DMSO was used as a negative control. a Representative images of 15-LO in PMNs detected by immunofluorescence. ( b left panel) 15-LO and 5-LO expressions in PMNs were detected by Western blot. ( b right panel) PMNs isolated from septic patients were cocultured with M0/M2-Exos (100 μg/mL) derived from PBMC-differentiated macrophages for 5 h. 15-LO and 5-LO expressions in PMNs were detected by Western blot. c and d LXA4 and LTB4 concentrations in the supernatant of PMNs were detected by ELISA. e Transwell analysis of neutrophil chemotaxis towards IL-8. f Typical images of NET formation using SYTOX Green (green). Scale bar, 50 μm. NET formation was quantified as the percentage of neutrophils forming NETs and the NET area per microscopic field. g Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. Flow cytometry detection of CXCR2 ( h ) and ROS ( i ) expressions in cocultured PMNs. Student’s t test ( c and d ) or one-way analysis of variance with Tukey’s multiple comparisons test ( e – i ) was used for the analysis. Graphs represent means ± standard deviations, n = 4–6; * P < 0.05, ** P < 0.01 compared within two groups

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll 1.077 density gradient (Solarbio, Beijing, China) as described previously [ ].

Techniques: Expressing, Isolation, Clinical Proteomics, Derivative Assay, Negative Control, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, Chemotaxis Assay, Cell Culture, Flow Cytometry

Journal: Journal of Biomedical Science

Article Title: Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

doi: 10.1186/s12929-023-00957-9

Figure Lengend Snippet: Exosomal PGE2 from M2 macrophages is necessary for 15-LO upregulation and PMN inhibition. a PGE2 levels in M0-Exos/M2-Exos/exosomes from celecoxib (20 µM)-treated PBMC-differentiated M2 macrophages (Cel-M2-Exos) were examined by ELISA. PMNs isolated from healthy volunteers were activated by septic plasma (SP) and then cocultured with M2-Exos/Cel-M2-Exos/Cel-M2-Exos + PGE2 (100 nM) for 5 h. b Immunoblot analysis of 15-LO in PMNs. c and d LXA4 and LTB4 concentrations in the supernatant of PMNs were detected by ELISA. e Typical images of NET formation using SYTOX Green (green). Scale bar, 50 μm. NET formation was quantified as the percentage of neutrophils forming NETs and the NET area per microscopic field. f Quantification of dsDNA in the supernatant of cultured PMNs using PicoGreen fluorescent dye. g Transwell analysis of neutrophil chemotaxis towards IL-8. Flow cytometry detection of CXCR2 ( h ) and ROS ( i ) expressions in cocultured PMNs. One-way analysis of variance with Tukey’s multiple comparisons test was used for the analysis. n = 4–6. j – u WT C57BL/6 mice were administered M2-Exos/Cel-M2-Exos (300 μg/mouse) derived from mouse Raw264.7 macrophages via intraperitoneal injection 1 h after CLP. j Representative images showing the presence of NETs (MPO, red; citrullinated H3, green) in the lung tissues, as indicated by white arrows. Nuclei were counterstained with DAPI (blue). Scale bar, 40 μm. k and l Quantification of dsDNA and circulating NET structures in the plasma of mice using PicoGreen fluorescent dye and MPO-DNA-ELISA, respectively. m Flow cytometry detection of the percentage of systemic circulating neutrophils by staining with CD11b and Gr-1. n Absolute neutrophil number in peripheral blood. o and p Ly6G + cells in the lung tissues were detected by immunohistochemistry and immunofluorescence. Scale bar, 40 μm. CXCR2 ( q ) and ROS ( r ) expressions in peripheral blood neutrophils were detected by flow cytometry. s Evaluation of lung histology by H&E staining (magnification × 400). Red arrows indicate neutrophils in the alveolar and interstitial space, and black arrows indicate thickening of the alveolar walls. Scale bar, 50 μm. Lung injury scores were assessed. t Detection of inflammatory cytokine mRNA (IL-1β, IL-6, TNF-α) expression in lung tissues by RT‒qPCR. Student’s t test was used for the analysis. Graphs represent means ± standard deviations, n = 6. u Survival rate of CLP mice treated with M2-Exos or Cel-M2-Exos (n = 8); the log-rank test was used for the analysis. * P < 0.05, ** P < 0.01 compared within two groups

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll 1.077 density gradient (Solarbio, Beijing, China) as described previously [ ].

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Isolation, Clinical Proteomics, Western Blot, Cell Culture, Chemotaxis Assay, Flow Cytometry, Derivative Assay, Injection, Staining, Immunohistochemistry, Immunofluorescence, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: In vivo effects of sEVs on tumor growth and stemness. (A) Schematic diagram of the in vivo experimental design. A549 cells were injected subcutaneously into nude mice to establish tumors. sEVs derived from A549, A549CR, and A549CSC were injected peritumorally every three days. Tumor growth was monitored using IVIS imaging, and histological analyses were performed post-harvest. (B) Representative IVIS imaging showing fluorescence signals in tumors treated with Dir-labeled sEVs from different origins. The images include in vivo and ex vivo fluorescence signals after sEV injection. (C) Tumor growth curves showing tumor volume changes over time in mice treated with sEVs from different sources. Data are presented as mean ± SD (ns: not significant, ** p < 0.01, *** p < 0.001, n = 5). Comparisons between groups were analyzed using one-way ANOVA followed by Tukey's post-hoc test. (D) Representative IVIS images showing in vivo bioluminescent signal intensity in tumor-bearing mice treated with sEVs derived from A549, A549CR, or A549CSC. (E) Representative images of harvested tumors from each group after treatment with sEVs. (F) Histological and immunohistochemical analyses of tumors. H&E staining shows tumor morphology, while immunohistochemical staining shows PKM2, pY105-PKM2, SOX2, and OCT4. Scale bar: 25 µm.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies: PKM2 (Cat# 3198S), phospho-PKM2 (Tyr105) (Cat# 3827), and IQGAP1 (Cat# 20648) from Cell Signaling Technology; NANOG (Cat# 14295-1-AP), OCT4 (Cat# 11263-1-AP), SOX2 (Cat# 11064-1-AP), Calnexin (Cat# 10427-2-AP ), CD44 ( Cat#60224-1-Ig), CD133 (Cat# 18470-1-AP), and TSG101 (Cat# 67381-1) from Proteintech; CDK1 (phospho T14 + Y15) (Cat# ab277772) from Abcam (MA, USA); JAK3 (Cat# CY7016) and ITK (Cat# CY6933) from Abways (Shanghai, China); Yes1 (Cat# R26126), Src (Cat# R25792), AXL (Cat# R23576), FAK (Cat# R24277), Cyclin B1 (Cat# R23324), CDK1 (Cat# R23884), and CDC25B (Cat# R381486) from Zen Bioscience (Chengdu, China); β-actin (Cat# AC206) from ABclonal (MA, USA); and FLAG (Cat# LF304) from Epizyme.

Techniques: In Vivo, Injection, Derivative Assay, Imaging, Fluorescence, Labeling, Ex Vivo, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Analysis of PKM2 and its phosphorylation in NSCLC cells and tissues. (A) KEGG pathway enrichment analysis of differentially expressed proteins in A549 chemosensitive cells and CSCs. (B) Heatmap depicting proteomic analysis of A549 chemosensitive cells and CSCs. (C) Western blot analysis of PKM2 and pY105-PKM2 in A549, A549CR, and CSCs, with or without TEPP-46 treatment. Crosslinking experiments show PKM2 configurations, and immunoprecipitation (IP) indicates phosphorylation at Y105. (D) Western blot analysis of selected kinases (YES1, Src, JAK3, FAK, ITK, AXL) in A549, A549CR, and CSCs. (E) Representative immunohistochemical staining of PKM2 and pY105-PKM2 in tumor and peritumoral tissues. Scale bars: 50 µm. (F) Box plots showing IHC scores of PKM2 and pY105-PKM2 in tumor and peritumoral tissues. (G) Kaplan-Meier survival curves comparing overall survival (OS) of NSCLC patients based on PKM2 and pY105-PKM2 expression levels. (H) Representative immunohistochemistry staining of CD133 in tumor and peritumoral tissues. Scale bars: 50 µm. (I) Scatter plots showing the relationship between CD133 expression and PKM2 or pY105-PKM2 in tumor tissues.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies: PKM2 (Cat# 3198S), phospho-PKM2 (Tyr105) (Cat# 3827), and IQGAP1 (Cat# 20648) from Cell Signaling Technology; NANOG (Cat# 14295-1-AP), OCT4 (Cat# 11263-1-AP), SOX2 (Cat# 11064-1-AP), Calnexin (Cat# 10427-2-AP ), CD44 ( Cat#60224-1-Ig), CD133 (Cat# 18470-1-AP), and TSG101 (Cat# 67381-1) from Proteintech; CDK1 (phospho T14 + Y15) (Cat# ab277772) from Abcam (MA, USA); JAK3 (Cat# CY7016) and ITK (Cat# CY6933) from Abways (Shanghai, China); Yes1 (Cat# R26126), Src (Cat# R25792), AXL (Cat# R23576), FAK (Cat# R24277), Cyclin B1 (Cat# R23324), CDK1 (Cat# R23884), and CDC25B (Cat# R381486) from Zen Bioscience (Chengdu, China); β-actin (Cat# AC206) from ABclonal (MA, USA); and FLAG (Cat# LF304) from Epizyme.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Immunohistochemical staining, Staining, Expressing, Immunohistochemistry

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Generation and characterization of PKM2 WT and PKM2 Y105F cell lines and their impact on NSCLC stemness and tumorigenicity. (A) Schematic of CRISPR-Cas9-based PKM2 knockout in A549 cells followed by lentiviral transduction of PKM2 WT or PKM2 Y105F plasmids and western blot validation of PKM2 knockout.(B) Workflow of lentiviral packaging, transduction, and selection of PKM2 WT and PKM2 Y105F stable cell lines. (C) Western blot analysis showing expression of PKM2 and pY105-PKM2 in PKM2 WT and PKM2 Y105F cell lines. (D) Western blot analysis of stemness markers (OCT4, SOX2, and NANOG) in PKM2 WT and PKM2 Y105F cell lines. (E) Cell viability assays of PKM2 WT and PKM2 Y105F cells following cisplatin treatment. Data are presented as mean ± SD (ns: not significant, * p < 0.05, ** p < 0.01, n = 3). (F) Colony formation assay of PKM2 WT and PKM2 Y105F cells. Representative images and quantified colony numbers are shown. Data are presented as mean ± SD (** p < 0.01, n = 3). (G) Sphere formation assay of PKM2 WT and PKM2 Y105F cells. Representative images and quantified sphere numbers are shown. Data are presented as mean ± SD (** p < 0.01, n = 3). (H) Representative images of harvested subcutaneous xenograft tumors derived from PKM2 WT and PKM2 Y105F cells. (I) Tumor growth curves showing tumor volume over time for PKM2 WT and PKM2 Y105F xenografts. Data are presented as mean ± SD (** p < 0.01, n = 6). (J) Limiting dilution assay using bioluminescence imaging of subcutaneous xenografts with decreasing cell numbers (1×10⁶ to 5×10³) of PKM2 WT and PKM2 Y105F cells. (K) Immunohistochemical analysis of xenograft tumors showing H&E staining, PKM2, pY105-PKM2, and stemness markers (OCT4, SOX2). Scale bar = 25 μm.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies: PKM2 (Cat# 3198S), phospho-PKM2 (Tyr105) (Cat# 3827), and IQGAP1 (Cat# 20648) from Cell Signaling Technology; NANOG (Cat# 14295-1-AP), OCT4 (Cat# 11263-1-AP), SOX2 (Cat# 11064-1-AP), Calnexin (Cat# 10427-2-AP ), CD44 ( Cat#60224-1-Ig), CD133 (Cat# 18470-1-AP), and TSG101 (Cat# 67381-1) from Proteintech; CDK1 (phospho T14 + Y15) (Cat# ab277772) from Abcam (MA, USA); JAK3 (Cat# CY7016) and ITK (Cat# CY6933) from Abways (Shanghai, China); Yes1 (Cat# R26126), Src (Cat# R25792), AXL (Cat# R23576), FAK (Cat# R24277), Cyclin B1 (Cat# R23324), CDK1 (Cat# R23884), and CDC25B (Cat# R381486) from Zen Bioscience (Chengdu, China); β-actin (Cat# AC206) from ABclonal (MA, USA); and FLAG (Cat# LF304) from Epizyme.

Techniques: CRISPR, Knock-Out, Transduction, Western Blot, Biomarker Discovery, Selection, Stable Transfection, Expressing, Colony Assay, Tube Formation Assay, Derivative Assay, Limiting Dilution Assay, Imaging, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: sEV-mediated pY105-PKM2 induces slow cell cycle, metabolic remodeling, and promotes chemoresistance and stemness in NSCLC. (A) Schematic representation of the experimental setup. A549 cells were treated with sEVs derived from A549-PKM2 WT and A549-PKM2 Y105F cells, followed by transcriptomic sequencing. (B) Glucose consumption and lactate production in A549 cells treated with Vec-sEV, PKM2^WT-sEV, or PKM2^Y105F-sEV. Data are presented as mean ± SD (ns, not significant, ** p < 0.01, *** p < 0.001, n = 3). (C) OCR of A549 cells treated with Vec-sEV, PKM2 WT -sEV, or PKM2 Y105F -sEV under sequential injections of oligomycin, FCCP, and rotenone. (D) Western blot analysis of Cyclin B1, CDC25B, total CDK1, and CDK1 phosphorylated at Thr14 and Tyr15 in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV. (E) Schematic representation of Apcin treatment experiments designed to assess the role of APC/CDC20 in sEV-induced cell cycle regulation, stemness, and chemoresistance. (F) Flow cytometry analysis of cell cycle distribution in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV, with or without Apcin treatment. (G) Quantification of cell populations in different phases of the cell cycle (Sub G1, G1, S, G2/M) based on flow cytometry analysis. Data are presented as mean ± SD (ns, not significant, * p < 0.05, ** p < 0.01, n = 3). (H) Flow cytometry analysis of CD44 and CD133 expression in A549 cells treated with PKM2 WT -sEV or PKM2 Y105F -sEV, with or without Apcin treatment. (I) Quantification of CD44- and CD133-positive cells in A549 cells treated as described in Panel H. Data are presented as mean ± SD (ns, not significant, *** p < 0.001, n = 3). (J) Flow cytometry analysis of apoptotic cells in A549 cells treated with sEVs, followed by cisplatin or paclitaxel treatment, with or without Apcin. (K) Quantification of apoptotic cells in A549 cells treated as described in Panel J. Data are presented as mean ± SD (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3).

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies: PKM2 (Cat# 3198S), phospho-PKM2 (Tyr105) (Cat# 3827), and IQGAP1 (Cat# 20648) from Cell Signaling Technology; NANOG (Cat# 14295-1-AP), OCT4 (Cat# 11263-1-AP), SOX2 (Cat# 11064-1-AP), Calnexin (Cat# 10427-2-AP ), CD44 ( Cat#60224-1-Ig), CD133 (Cat# 18470-1-AP), and TSG101 (Cat# 67381-1) from Proteintech; CDK1 (phospho T14 + Y15) (Cat# ab277772) from Abcam (MA, USA); JAK3 (Cat# CY7016) and ITK (Cat# CY6933) from Abways (Shanghai, China); Yes1 (Cat# R26126), Src (Cat# R25792), AXL (Cat# R23576), FAK (Cat# R24277), Cyclin B1 (Cat# R23324), CDK1 (Cat# R23884), and CDC25B (Cat# R381486) from Zen Bioscience (Chengdu, China); β-actin (Cat# AC206) from ABclonal (MA, USA); and FLAG (Cat# LF304) from Epizyme.

Techniques: Derivative Assay, Sequencing, Western Blot, Flow Cytometry, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: In vivo assessment of the effects of sEVs derived from PKM2 WT and PKM2 Y105F cells on tumor growth, glycolytic activity, and stemness. (A) Schematic representation of the in vivo experimental design. Chemosensitive A549 cells were subcutaneously implanted into nude mice, and upon tumor volumes reaching ~100 mm³, sEVs derived from Vec, PKM2 WT , or PKM2 Y105F cells were administered peritumorally every three days. Tumor growth was monitored, followed by ¹⁸F-FDG PET-CT and IVIS imaging and histological analysis of harvested tumors. (B) Representative IVIS imaging showing fluorescence signals in tumors treated with Dir-labeled sEVs from different origins. The images include in vivo and ex vivo fluorescence signals after sEV injection. (C) Tumor growth curves depicting tumor volumes over time in mice treated with different sEVs. Data are presented as mean ± SD (ns, not significant, *** p < 0.001, n = 5). (D) Representative IVIS images showing bioluminescent signals from subcutaneous tumors at the experimental endpoint in mice treated with Vec-sEV, PKM2 WT -sEV, or PKM2 Y105F -sEV. (E) Images of harvested tumors from mice treated with different sEVs. (F) Representative ¹⁸F-FDG PET-CT scans showing glycolytic activity in tumors treated with different sEVs. (G) Histological analysis of tumor tissues, including hematoxylin and eosin (HE) staining and immunohistochemistry for PKM2, pY105-PKM2, stemness markers OCT4 and SOX2, and cell cycle regulators CDC25B, Cyclin B1, and CDK1. Scale bar: 25 µm.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies: PKM2 (Cat# 3198S), phospho-PKM2 (Tyr105) (Cat# 3827), and IQGAP1 (Cat# 20648) from Cell Signaling Technology; NANOG (Cat# 14295-1-AP), OCT4 (Cat# 11263-1-AP), SOX2 (Cat# 11064-1-AP), Calnexin (Cat# 10427-2-AP ), CD44 ( Cat#60224-1-Ig), CD133 (Cat# 18470-1-AP), and TSG101 (Cat# 67381-1) from Proteintech; CDK1 (phospho T14 + Y15) (Cat# ab277772) from Abcam (MA, USA); JAK3 (Cat# CY7016) and ITK (Cat# CY6933) from Abways (Shanghai, China); Yes1 (Cat# R26126), Src (Cat# R25792), AXL (Cat# R23576), FAK (Cat# R24277), Cyclin B1 (Cat# R23324), CDK1 (Cat# R23884), and CDC25B (Cat# R381486) from Zen Bioscience (Chengdu, China); β-actin (Cat# AC206) from ABclonal (MA, USA); and FLAG (Cat# LF304) from Epizyme.

Techniques: In Vivo, Derivative Assay, Activity Assay, Positron Emission Tomography-Computed Tomography, Imaging, Fluorescence, Labeling, Ex Vivo, Injection, Staining, Immunohistochemistry

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: IQGAP1 links phosphorylated PKM2 to TSG101. (A) Experimental workflow for immunoprecipitation and mass spectrometry analysis. Flag-tagged PKM2 WT and PKM2 Y105F proteins were immunoprecipitated using anti-Flag magnetic beads, followed by liquid chromatography-mass spectrometry. The Venn diagram illustrates the overlap and unique binding partners of PKM2 WT and PKM2 Y105F . (B) Mass spectrometry results highlighting the binding of IQGAP1 exclusively to PKM2 WT . (C) Co-immunoprecipitation of IQGAP1, pY105-PKM2, and TSG101 in A549-PKM2 WT and A549- PKM2 Y105F cells. Total protein input is shown for comparison. (D) Immunoprecipitation analysis of interactions between IQGAP1, TSG101, and PKM2 in A549- PKM2 WT and A549- PKM2 Y105F cells. (E) Co-immunoprecipitation results following siRNA-mediated knockdown of IQGAP1 in A549-PKM2 WT and A549-PKM2 Y105F cells. (F) Total protein input levels of IQGAP1, TSG101, and PKM2 in A549- PKM2 WT and A549- PKM2 Y105F cells, with or without IQGAP1 knockdown. (G) Immunoprecipitation analysis showing the effect of TEPP-46 treatment on interactions between IQGAP1, PKM2, and TSG101 in A549- PKM2 WT and A549- PKM2 Y105F cells. (H) DuoLink proximity ligation assay detecting interactions between PKM2 and TSG101. Signals (yellow) are shown in A549- PKM2 WT and A549- PKM2 Y105F cells, with and without IQGAP1 knockdown. DAPI (blue) marks nuclei, and phalloidin (red) labels actin filaments. Scale bar: 10 µm. (I) Immunofluorescence analysis showing colocalization of Flag-tagged PKM2 WT and PKM2 Y105F with TSG101 in A549 cells, with and without IQGAP1 knockdown. Scale bar: 10 µm.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies: PKM2 (Cat# 3198S), phospho-PKM2 (Tyr105) (Cat# 3827), and IQGAP1 (Cat# 20648) from Cell Signaling Technology; NANOG (Cat# 14295-1-AP), OCT4 (Cat# 11263-1-AP), SOX2 (Cat# 11064-1-AP), Calnexin (Cat# 10427-2-AP ), CD44 ( Cat#60224-1-Ig), CD133 (Cat# 18470-1-AP), and TSG101 (Cat# 67381-1) from Proteintech; CDK1 (phospho T14 + Y15) (Cat# ab277772) from Abcam (MA, USA); JAK3 (Cat# CY7016) and ITK (Cat# CY6933) from Abways (Shanghai, China); Yes1 (Cat# R26126), Src (Cat# R25792), AXL (Cat# R23576), FAK (Cat# R24277), Cyclin B1 (Cat# R23324), CDK1 (Cat# R23884), and CDC25B (Cat# R381486) from Zen Bioscience (Chengdu, China); β-actin (Cat# AC206) from ABclonal (MA, USA); and FLAG (Cat# LF304) from Epizyme.

Techniques: Immunoprecipitation, Mass Spectrometry, Magnetic Beads, Liquid Chromatography, Binding Assay, Comparison, Knockdown, Proximity Ligation Assay, Immunofluorescence

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: IQGAP1 mediates the sorting of pY105-PKM2 into sEVs and synergistically promotes stemness and drug resistance. (A) Western blot analysis of pY105-PKM2 in sEVs derived from A549-PKM2 WT or A549-PKM2 Y105F cells, with or without IQGAP1 silencing using siRNA (siRNA-NC, siRNA-IQGAP1#3, or siRNA-IQGAP1#4). (B) Flow cytometry analysis of apoptosis in recipient A549 cells treated with sEVs from IQGAP1-silenced or control A549-PKM2 WT cells, followed by treatment with cisplatin (5 µg/mL) or paclitaxel (200 ng/mL). (C) Western blot analysis of pY105-PKM2 in sEVs derived from A549-PKM2 WT or A549-PKM2 Y105F cells, with or without TEPP-46 treatment, which inhibits PKM2 phosphorylation at Y105. (D) Flow cytometry analysis of apoptosis in recipient A549 cells treated with sEVs from TEPP-46-treated A549-PKM2 WT cells, followed by cisplatin or paclitaxel treatment. (E) Sphere formation assay of A549 cells overexpressing IQGAP1, PKM2 WT , PKM2 Y105F , or combinations thereof. Representative images and quantification of sphere numbers are shown. (F) Cell viability assay of A549 cells overexpressing IQGAP1, PKM2 WT , PKM2 Y105F , or their combinations, following cisplatin treatment (5 µg/mL). (G) Correlation between IQGAP1 and PKM2 expression in CD133-high and CD133-low tumor samples. (H) Correlation between IQGAP1 and pY105-PKM2 expression in CD133-high and CD133-low tumor samples. Data (B-F) are presented as mean ± SD (n = 3). Statistical significance: ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies: PKM2 (Cat# 3198S), phospho-PKM2 (Tyr105) (Cat# 3827), and IQGAP1 (Cat# 20648) from Cell Signaling Technology; NANOG (Cat# 14295-1-AP), OCT4 (Cat# 11263-1-AP), SOX2 (Cat# 11064-1-AP), Calnexin (Cat# 10427-2-AP ), CD44 ( Cat#60224-1-Ig), CD133 (Cat# 18470-1-AP), and TSG101 (Cat# 67381-1) from Proteintech; CDK1 (phospho T14 + Y15) (Cat# ab277772) from Abcam (MA, USA); JAK3 (Cat# CY7016) and ITK (Cat# CY6933) from Abways (Shanghai, China); Yes1 (Cat# R26126), Src (Cat# R25792), AXL (Cat# R23576), FAK (Cat# R24277), Cyclin B1 (Cat# R23324), CDK1 (Cat# R23884), and CDC25B (Cat# R381486) from Zen Bioscience (Chengdu, China); β-actin (Cat# AC206) from ABclonal (MA, USA); and FLAG (Cat# LF304) from Epizyme.

Techniques: Western Blot, Derivative Assay, Flow Cytometry, Control, Phospho-proteomics, Tube Formation Assay, Viability Assay, Expressing

Journal: Theranostics

Article Title: A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles

doi: 10.7150/thno.103722

Figure Lengend Snippet: Graphical summary of IQGAP1-mediated sorting of phos-PKM2 in CSC-derived sEVs and its role in chemoresistance in NSCLC. This schematic illustrates the role of CSC-derived sEVs in promoting stemness and chemoresistance in NSCLC. In CSCs (left panel), surface markers CD44 and CD133 are expressed. Phosphorylation of PKM2 at Y105 (phos-PKM2) is induced by receptor tyrosine kinases (YES1, Src, JAK3, FAK, ITK, AXL), facilitating its transition from the tetrameric to the dimeric form. phos-PKM2 translocates to the nucleus to enhance the expression of stemness-related transcription factors (SOX2, NANOG, and OCT4). IQGAP1 mediates the selective incorporation of phos-PKM2 into sEVs through interactions with the ESCRT component TSG101, enabling the secretion of sEVs into the tumor microenvironment. In chemosensitive cancer cells (right panel), CSC-derived sEV uptake delivers phos-PKM2, inducing metabolic reprogramming (enhanced glycolysis and suppressed oxidative phosphorylation) and slowing the cell cycle via APC/C-CDC20 inhibition and reduced Cyclin B degradation. These processes collectively promote stemness and chemoresistance, replenishing the CSC pool. The lower section illustrates a lung tumor model, depicting how CSC-derived sEVs expand the CSC population within the tumor microenvironment, thereby driving chemotherapy resistance and malignant progression.

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies: PKM2 (Cat# 3198S), phospho-PKM2 (Tyr105) (Cat# 3827), and IQGAP1 (Cat# 20648) from Cell Signaling Technology; NANOG (Cat# 14295-1-AP), OCT4 (Cat# 11263-1-AP), SOX2 (Cat# 11064-1-AP), Calnexin (Cat# 10427-2-AP ), CD44 ( Cat#60224-1-Ig), CD133 (Cat# 18470-1-AP), and TSG101 (Cat# 67381-1) from Proteintech; CDK1 (phospho T14 + Y15) (Cat# ab277772) from Abcam (MA, USA); JAK3 (Cat# CY7016) and ITK (Cat# CY6933) from Abways (Shanghai, China); Yes1 (Cat# R26126), Src (Cat# R25792), AXL (Cat# R23576), FAK (Cat# R24277), Cyclin B1 (Cat# R23324), CDK1 (Cat# R23884), and CDC25B (Cat# R381486) from Zen Bioscience (Chengdu, China); β-actin (Cat# AC206) from ABclonal (MA, USA); and FLAG (Cat# LF304) from Epizyme.

Techniques: Derivative Assay, Phospho-proteomics, Expressing, Inhibition