Journal: Journal of Nanobiotechnology

Article Title: Momordica charantia L.-derived exosome-like nanovesicles stabilize p62 expression to ameliorate doxorubicin cardiotoxicity

doi: 10.1186/s12951-024-02705-z

Figure Lengend Snippet: MC-ELNs prevent p62 from DOX-induced ubiquitination degradation in cardiomyocytes. H9c2 cells were pre-treated with 10 µg/mL MC-ELNs for 6 h, and then cultured with or without 1 µM doxorubicin (DOX) for another 24 h. Western blot images ( A ) and quantitative analysis ( B ) on the p62 protein levels. ( C ) The p62 mRNA levels in H9c2 cells treated with 1 µM DOX in the absence or presence of 10 µg/mL MC-ELNs. ( D ) H9c2 cells were treated with PBS or 1 µM DOX in the presence or absence of 2 µM Lactacystin (LACTA) for 24 h. ( E ) H9c2 cells were incubated in the absence or presence of MC-ELNs (10 µg/mL) in combination with the protein synthesis inhibitor cycloheximide (CHX, 10 µg/mL) for 2–4 h. Western blot analysis of the expressions of p62. ( F ) Immunoprecipitation assay (IP) and western blot analysis of cell lysate derived from PBS- or DOX-treated H9c2 cells in the absence or presence of 10 µg/mL of MC-ELNs (DOX, DOX + MC-ELNs). IP was performed with an antibody against p62. The blot was probed with antibodies against p62, Ub, K48-Ub, and K63-Ub. β-actin was employed as a loading control. Western blot analysis on the identified E3 ligase enzyme bound with p62 including Parkin ( G ), TRIM21 ( H ), and TRAF6 ( I ) in H9c2 cells treated with 1 µM DOX in the absence or presence of 10 µg/mL MC-ELNs. All data are presented as mean ± SD ( n = 3 experiments per group for A - E , G - I ; n = 1 experiment per group for F ). Comparisons among more than two groups were performed by ordinary one-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparisons test

Article Snippet: The primary antibodies included Cleaved caspase3 (9664S, CST), Caspase3 (9665S, CST), Cleaved caspase7 (8438S, CST), Caspase7 (12827T, CST), Cleaved PARP (9545, CST), PARP (9532S, CST), γ-H2A.X (ab2893, Abcam), Cyclin D1 (2978S, CST), Cyclin E1 (20808S, CST), Cyclin B1 (4138S, CST), Nrf2 (SAB4501984, Sigma), HO-1 (10701-1AP, proteintech), SQSTM1/p62 (ab109012, Abcam), Keap1 (8047S, CST), Ubiquitin (ab140601, Abcam), K48-Ub (ab140601, Abcam), K63-Ub (ab179434, Abcam), TRIM21 (ab207728, Abcam), TRAF6 (HY-P80919, MCE), Parkin (ab77924, Abcam), PCNA (ab29, Abcam), LC3B (NB100-2220, NOVUS), NQO1 (67240-1-lg, proteintech), Catalase (21260-1-AP, proteintech), SOD2 (24127-1-AP, proteintech), GPX4 (ab125066, Abcam), β-actin (HRP-600008, proteintech).

Techniques: Ubiquitin Proteomics, Cell Culture, Western Blot, Incubation, Immunoprecipitation, Derivative Assay, Control

Journal: Inflammation

Article Title: Caveolin-2 Knockdown Alleviated Sepsis-induced Acute Lung Injury Via Promoting Macrophage M2 Polarization and Inhibiting Apoptosis by Hippo Signaling Pathway

doi: 10.1007/s10753-025-02378-2

Figure Lengend Snippet: Cav2 knockdown inhibited pulmonary apoptosis and promoted M2 polarization of sepsis-induced ALI in vivo. ( A ) Western blotting analysis of the expression of Bax, Bak, Caspase-3, Bcl-2, CD86, iNOS, CD80, CD206, CD163 and Arg1 protein in lung tissues, GAPDH serves as an internal control ( N = 3 per group). ( B , C , D , E ) The mRNA expression of Bax, Bak, Caspase-3 and Bcl-2 protein in lung tissues, detected by RT-qPCR ( N = 8 per group). ( F, G , H , I , J , K ) RT-qPCR analysis of the expression of CD86, iNOS, CD80, CD206, Arg1 and CD163 in lung tissues ( N = 8 per group). Mice were euthanized 24 h post-CLP, above data of animal models were expressed as median and interquartile range. * P < 0.05, ** P < 0.01, *** P < 0.001vs. Control group. # P < 0.05, ## P < 0.01 vs. CLP group

Article Snippet: Antibodies against Bax (14796S, CST, USA, dilution ratio of WB,1:1000, IHC,1:200), Bak (12150S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), Caspase-3 (9662S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:1000), Bcl-2 (15071S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:500), Lats1 (ab243656, Abcam, USA, dilution ratio of WB,1:1000), Mst1 (ab317410, Abcam, UK,dilution ratio of WB,1:1000), Sav1 (ab307698, Abcam, UK,dilution ratio of WB,1:1000), p -YAP (ab313464, Abcam, USA, dilution ratio of WB,1:500), YAP (HY- P86386 , MCE, USA, dilution ratio of WB,1:1000, IF,1:200), p -TAZ (Sc-17610-R, Santa Cruz, USA, dilution ratio of WB,1:200), TAZ (83669S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), iNOS (22226-1-AP, Proteintech, China, dilution ratio of WB,1:1000), CD86 (13395-1-AP, Proteintech, China,dilution ratio of WB,1:2000), CD80 (66406-1-lg, Proteintech, China, dilution ratio of WB,1:1000), CD163 (HY- P81177 , MCE, USA, dilution ratio of WB,1:2000), CD206 (32647-1-AP, Proteintech, China, dilution ratio of WB,1:2000), Arg1 (16001-1-AP, Proteintech, China, dilution ratio of WB,1:5000), GAPDH (2118S, CST, USA, dilution ratio of WB,1:1000), Histone H3 (HY- P86672 , MCE, USA, dilution ratio of WB,1:10000), VT02956 (MCE, USA), MY-875 (MCE, USA), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8001, MCE, USA, dilution ratio of WB,1:8000), HRP-conjugated AffiniPure Goat Anti-Mouse IgG H&L (HY-P8004, MCE, USA,dilution ratio of WB,1:10000), APC-anti CD80 antibody (17-0801-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), APC-anti CD206 (17-2061-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), PE-MHC II (12-5322-81, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:200), FITC-anti-CD163 (11-1631-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:50).

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Control, Quantitative RT-PCR

Journal: Inflammation

Article Title: Caveolin-2 Knockdown Alleviated Sepsis-induced Acute Lung Injury Via Promoting Macrophage M2 Polarization and Inhibiting Apoptosis by Hippo Signaling Pathway

doi: 10.1007/s10753-025-02378-2

Figure Lengend Snippet: Cav2 knockdown mitigated macrophage apoptosis and promoted M2 polarization in MHS cells induced by LPS. ( A ) Western blotting analysis the expression of Bak, Bax, Caspase-3, Bcl-2, iNOS, CD86, CD80, CD206, Arg1 and CD163 proteins in MHS cells, GAPDH serves as an internal control ( N = 3 per group). ( B , C , D , E ) RT-qPCR analysis the mRNA expression of Bak, Bax, Caspase-3 and Bcl-2 protein in MHS cells ( N = 8 per group). ( F , G , H , I , J , K ) RT-qPCR analysis of the mRNA expression of CD86, iNOS, CD80, CD206, Arg1 and CD163 in MHS cells ( N = 8 per group). ( L , M ) Flow cytometry was used to detect M1 and M2 polarization related phenotype in each MHS cells groups ( N = 3 per group). Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. Control group. # P < 0.05, ## P < 0.01, ### P < 0.001vs. LPS group

Article Snippet: Antibodies against Bax (14796S, CST, USA, dilution ratio of WB,1:1000, IHC,1:200), Bak (12150S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), Caspase-3 (9662S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:1000), Bcl-2 (15071S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:500), Lats1 (ab243656, Abcam, USA, dilution ratio of WB,1:1000), Mst1 (ab317410, Abcam, UK,dilution ratio of WB,1:1000), Sav1 (ab307698, Abcam, UK,dilution ratio of WB,1:1000), p -YAP (ab313464, Abcam, USA, dilution ratio of WB,1:500), YAP (HY- P86386 , MCE, USA, dilution ratio of WB,1:1000, IF,1:200), p -TAZ (Sc-17610-R, Santa Cruz, USA, dilution ratio of WB,1:200), TAZ (83669S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), iNOS (22226-1-AP, Proteintech, China, dilution ratio of WB,1:1000), CD86 (13395-1-AP, Proteintech, China,dilution ratio of WB,1:2000), CD80 (66406-1-lg, Proteintech, China, dilution ratio of WB,1:1000), CD163 (HY- P81177 , MCE, USA, dilution ratio of WB,1:2000), CD206 (32647-1-AP, Proteintech, China, dilution ratio of WB,1:2000), Arg1 (16001-1-AP, Proteintech, China, dilution ratio of WB,1:5000), GAPDH (2118S, CST, USA, dilution ratio of WB,1:1000), Histone H3 (HY- P86672 , MCE, USA, dilution ratio of WB,1:10000), VT02956 (MCE, USA), MY-875 (MCE, USA), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8001, MCE, USA, dilution ratio of WB,1:8000), HRP-conjugated AffiniPure Goat Anti-Mouse IgG H&L (HY-P8004, MCE, USA,dilution ratio of WB,1:10000), APC-anti CD80 antibody (17-0801-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), APC-anti CD206 (17-2061-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), PE-MHC II (12-5322-81, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:200), FITC-anti-CD163 (11-1631-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:50).

Techniques: Knockdown, Western Blot, Expressing, Control, Quantitative RT-PCR, Flow Cytometry

Journal: Inflammation

Article Title: Caveolin-2 Knockdown Alleviated Sepsis-induced Acute Lung Injury Via Promoting Macrophage M2 Polarization and Inhibiting Apoptosis by Hippo Signaling Pathway

doi: 10.1007/s10753-025-02378-2

Figure Lengend Snippet: Cav2 knockdown promoted M2 polarization and attenuated macrophage apoptosis by regulating Hippo signaling pathway in vitro. ( A ) Immunofluorescence analysis assessed the nuclear expression and localization of Nucleus YAP and Nucleus TAZ. ( B ) Western blotting analysis the expression of Nucleus YAP, Nucleus TAZ, Lats1, Mst1, Sav1, Cytoplasm p-YAP, Cytoplasm p-TAZ, Bak, Bax, Caspase-3, Bcl-2, iNOS, CD86, CD80, CD206, Arg1 and CD163 proteins in MHS cells, Histone H3 and GAPDH serves as an internal control of nuclear and cytoplasmic proteins ( N = 3 per group). ( B , C ) The expression of M1 and M2 polarization related phenotype in MHS cells, detected by flow cytometry. ( D , E , F , G ) The mRNA expression of Bak, Bax, Caspase-3 and Bcl-2. ( H , I , J , K ) The mRNA expression of iNOS, CD86, CD206 and Arg1. ( L , M , N ) The mRNA expression of IL-1β, IL-6 and TNF-α in MHS cells, detected by RT-qPCR. Data were expressed as mean ± SD, * P < 0.05, ** P < 0.01 vs. Control group. # P < 0.05, ## P < 0.01, ### P < 0.001 vs. LPS group. $ P < 0.05 vs. LPS + LV-si-Cav2 group. Ɛ P < 0.05, ƐƐ P < 0.01 vs. LPS + LV-OE-Cav2 group

Article Snippet: Antibodies against Bax (14796S, CST, USA, dilution ratio of WB,1:1000, IHC,1:200), Bak (12150S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), Caspase-3 (9662S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:1000), Bcl-2 (15071S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:500), Lats1 (ab243656, Abcam, USA, dilution ratio of WB,1:1000), Mst1 (ab317410, Abcam, UK,dilution ratio of WB,1:1000), Sav1 (ab307698, Abcam, UK,dilution ratio of WB,1:1000), p -YAP (ab313464, Abcam, USA, dilution ratio of WB,1:500), YAP (HY- P86386 , MCE, USA, dilution ratio of WB,1:1000, IF,1:200), p -TAZ (Sc-17610-R, Santa Cruz, USA, dilution ratio of WB,1:200), TAZ (83669S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), iNOS (22226-1-AP, Proteintech, China, dilution ratio of WB,1:1000), CD86 (13395-1-AP, Proteintech, China,dilution ratio of WB,1:2000), CD80 (66406-1-lg, Proteintech, China, dilution ratio of WB,1:1000), CD163 (HY- P81177 , MCE, USA, dilution ratio of WB,1:2000), CD206 (32647-1-AP, Proteintech, China, dilution ratio of WB,1:2000), Arg1 (16001-1-AP, Proteintech, China, dilution ratio of WB,1:5000), GAPDH (2118S, CST, USA, dilution ratio of WB,1:1000), Histone H3 (HY- P86672 , MCE, USA, dilution ratio of WB,1:10000), VT02956 (MCE, USA), MY-875 (MCE, USA), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8001, MCE, USA, dilution ratio of WB,1:8000), HRP-conjugated AffiniPure Goat Anti-Mouse IgG H&L (HY-P8004, MCE, USA,dilution ratio of WB,1:10000), APC-anti CD80 antibody (17-0801-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), APC-anti CD206 (17-2061-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), PE-MHC II (12-5322-81, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:200), FITC-anti-CD163 (11-1631-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:50).

Techniques: Knockdown, In Vitro, Immunofluorescence, Expressing, Western Blot, Control, Flow Cytometry, Quantitative RT-PCR

Journal: Inflammation

Article Title: Caveolin-2 Knockdown Alleviated Sepsis-induced Acute Lung Injury Via Promoting Macrophage M2 Polarization and Inhibiting Apoptosis by Hippo Signaling Pathway

doi: 10.1007/s10753-025-02378-2

Figure Lengend Snippet: Cav2 knockdown promoted M2 polarization and attenuated macrophage apoptosis by regulating Hippo signaling pathway in vivo. ( A ) Western blotting analysis the expression of Nucleus TAZ, Nucleus YAP, Lats1, Mst1, Sav1, Cytoplasm p-YAP Cytoplasm p-TAZ, Bak, Bax, Caspase-3, Bcl-2, iNOS, CD86, CD80, CD206,Arg1 and CD163 proteins in lung tissues, Histone H3 and GAPDH serves as an internal control of nuclear and cytoplasmic proteins ( N = 3 per group). ( B ) IHC results showing the expression levels of Bak, Bax, Caspase-3 and Bcl-2 (200X, Scale bar = 50 μm). ( C , D ) Percentage of IHC positive cells and positive cells counts score of IHC ( N = 8 per group). ( E ) HE staining of lung tissues (100X, Scale bar = 100 μm). ( F ) Lung tissues of HE staining injury scoring ( N = 8 per group). ( G , H , I ) The mRNA expression of IL-1β, IL-6 and TNF-α in lung tissues, detected by RT-qPCR ( N = 8 per group). Above data of animal models were expressed as median and interquartile range, * P < 0.05, ** P < 0.01, *** P < 0.001vs.Control group. # P < 0.05, ## P < 0.0 vs.CLP group. $ P < 0.05, $$ P < 0.01 vs. CLP + AAV-si-Cav2 group

Article Snippet: Antibodies against Bax (14796S, CST, USA, dilution ratio of WB,1:1000, IHC,1:200), Bak (12150S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), Caspase-3 (9662S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:1000), Bcl-2 (15071S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:500), Lats1 (ab243656, Abcam, USA, dilution ratio of WB,1:1000), Mst1 (ab317410, Abcam, UK,dilution ratio of WB,1:1000), Sav1 (ab307698, Abcam, UK,dilution ratio of WB,1:1000), p -YAP (ab313464, Abcam, USA, dilution ratio of WB,1:500), YAP (HY- P86386 , MCE, USA, dilution ratio of WB,1:1000, IF,1:200), p -TAZ (Sc-17610-R, Santa Cruz, USA, dilution ratio of WB,1:200), TAZ (83669S, CST, USA, dilution ratio of WB,1:1000, IHC, 1:200), iNOS (22226-1-AP, Proteintech, China, dilution ratio of WB,1:1000), CD86 (13395-1-AP, Proteintech, China,dilution ratio of WB,1:2000), CD80 (66406-1-lg, Proteintech, China, dilution ratio of WB,1:1000), CD163 (HY- P81177 , MCE, USA, dilution ratio of WB,1:2000), CD206 (32647-1-AP, Proteintech, China, dilution ratio of WB,1:2000), Arg1 (16001-1-AP, Proteintech, China, dilution ratio of WB,1:5000), GAPDH (2118S, CST, USA, dilution ratio of WB,1:1000), Histone H3 (HY- P86672 , MCE, USA, dilution ratio of WB,1:10000), VT02956 (MCE, USA), MY-875 (MCE, USA), HRP-conjugated AffiniPure Goat Anti-Rabbit IgG H&L (HY-P8001, MCE, USA, dilution ratio of WB,1:8000), HRP-conjugated AffiniPure Goat Anti-Mouse IgG H&L (HY-P8004, MCE, USA,dilution ratio of WB,1:10000), APC-anti CD80 antibody (17-0801-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), APC-anti CD206 (17-2061-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:100), PE-MHC II (12-5322-81, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:200), FITC-anti-CD163 (11-1631-82, Thermo Fisher Scientific, USA, dilution ratio of Flow,1:50).

Techniques: Knockdown, In Vivo, Western Blot, Expressing, Control, Staining, Quantitative RT-PCR

Journal: Journal of Nanobiotechnology

Article Title: Inverse-designed plasmonic biosensors with LSPR-SPP-wood anomaly coupling enhanced for biomolecular analysis

doi: 10.1186/s12951-025-03930-w

Figure Lengend Snippet: High-throughput affinity sensing platform for ADC drug development. ( a ) The association process of incubating PD-1 on the chip to detect Sintilimab. ( b ) The long-time dissociation process of PD-1 with Sintilimab. ( c ) Dynamic real-time and fitting curves of the association and dissociation phases of the interaction between PD-1 and Sintilimab. ( d ) Schematic diagram of the high-throughput antibody affinity detection protocol, with each group, repeated three times in the three samples tested, and the concentration of A-H in each column decreasing incrementally. ( e ) Schematic diagram of the main events in the antibody drug fishing and affinity detection process. ( f-h ) Dynamic real-time and fitting curves of the association and dissociation phases for Polatuzumab with CD79B, Adalimumab with TNF-α, and Sacituzumab govitecan with TROP2, detected using the unoptimized sensor. ( i-k ) Dynamic real-time and fitting curves of the association and dissociation phases for Polatuzumab with CD79B, Adalimumab with TNF-α, and Sacituzumab govitecan with TROP2, detected using the optimized IDMM-SPR sensor

Article Snippet: Bovine serum albumin (BSA), Phosphate-buffered saline (PBST), 11-mercaptoundecanoic acid (MUA), 1-ethyl3-(dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), Dimethyl Sulfoxide (DMSO), Fish gelatin,, ethanolamine, and phosphate-buffered saline (PBS) buffer, Polyethylene glycol 2000 (PEG) were purchased from Sigma-Aldrich; Protein A, IgG, CRP, and CRP ab, hTGFBR1 were purchased from monoclonal anti-CRP capture and detection antibodies were purchased from Beijing Sino Biological, Inc. (Beijing, China); Sintilimab, Adalimumab, Polatuzumab, Sacituzumab, PD-1, TNF-α, CD79B, TROP2, Quercetin were purchased from MedChemExpress.

Techniques: High Throughput Screening Assay, Concentration Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting CD93 on monocytes revitalizes antitumor immunity by enhancing the function and infiltration of CD8+ T cells

doi: 10.1136/jitc-2024-010148

Figure Lengend Snippet: CD93 + monocytes accumulate in the peritumor of human HCC and indicate worse patient survival. (A) Distribution of CD68 + CD93 + cells in HCC tissues was determined by IF staining. Red, CD93; Green, CD68; Light Gray, CD31; Blue, DAPI. Scale bar=50 µm, n=10. B-D, CD14 + monocytes were purified from non-tumor, peritumor, or tumor tissues of HCC. Levels of CD93 expression in/on these cells were analyzed by western blotting (B, n=3), flow cytometry (C, n=4), or Q-PCR (D, n=5). (E) CD93 expression by different cells within HCC tissues was measured using scRNA-seq data from an online database. (F) 99 HCC patients who underwent curative resection with follow-up data were divided into two groups according to the median value of the CD68 + or CD68 + CD93 + cell density in peritumoral tissues (CD68 + cells: low, ≤405.7 cells/mm 2 (n=49); high, >405.7 cells/mm 2 (n=50); CD68 + CD93 + cells: low, ≤22.1 cells/mm 2 (n=49); high, >22.1 cells/mm 2 (n=50)). The overall survival and tumor recurrence of these patients were analyzed via the Kaplan-Meier method and log-rank test. Results shown in A, C and D are expressed as mean±SEM. *p<0.05, **p<0.01. The following statistical analyses were performed: one-way ANOVA ( A, C and D ), or log-rank test ( F and G ). ANOVA, analysis of variance; HCC, hepatocellular carcinoma; IF, immunofluorescence.

Article Snippet: Medium, siNC-containing liposome, or si CD93 -containing liposome (C12-200, HY-145405, MCE; cholesterol, C8503, Sigma-Aldrich; DSPC, 850 365P, Avanti; DMG-PEG2000, 880 151P, Avanti) were prepared as previously described.

Techniques: Staining, Purification, Expressing, Western Blot, Flow Cytometry, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting CD93 on monocytes revitalizes antitumor immunity by enhancing the function and infiltration of CD8+ T cells

doi: 10.1136/jitc-2024-010148

Figure Lengend Snippet: Glycolytic switch induces CD93 upregulation on monocytes via the Erk signaling pathway. CD14 + cells were purified from the peripheral blood of healthy donors. (A–C) Cells were left untreated (Med) or treated with HepG2 TSN. The kinetics of CD93 expression by these cells were determined via Q-PCR (A, n=3), flow cytometry (B, n=4) and western blotting (C, n=4). (D, E) CD14 + cells were left untreated (Med) or treated with HepG2 TSN for 12 hours (D, n=7) or 24 hours (E, n=3) in the presence or absence of 2DG (20 mM) or 3PO (20 µM). Their expression of CD93 was measured by Q-PCR and flow cytometry. (F, G) Cells were left untreated or treated with HepG2 TSN for 24 hours in the presence or absence of NF-κB inhibitor JSH-23 (10 µM), or STAT3 inhibitor AG490 (10 µM), or JNK inhibitor SP600125 (10 µM), or Erk inhibitor U0126 (25 µM), or p38 inhibitor SB203580 (25 µM). Their levels of CD93 expression were determined by western blotting (F, n=3) or flow cytometry (G, n=4). (H) Cells were left untreated or treated with HepG2 TSN for 30 min in the presence or absence of 2DG (20 mM). Their levels of p-Erk and Erk expression were determined by western blotting. n=3. Results shown in A, B, D, E, G are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The following statistical analyses were performed: paired two-tailed Student’s t-test ( A, B ), one-way ANOVA ( D, E ), or two-way ANOVA ( G ). ANOVA, analysis of variance.

Article Snippet: Medium, siNC-containing liposome, or si CD93 -containing liposome (C12-200, HY-145405, MCE; cholesterol, C8503, Sigma-Aldrich; DSPC, 850 365P, Avanti; DMG-PEG2000, 880 151P, Avanti) were prepared as previously described.

Techniques: Purification, Expressing, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting CD93 on monocytes revitalizes antitumor immunity by enhancing the function and infiltration of CD8+ T cells

doi: 10.1136/jitc-2024-010148

Figure Lengend Snippet: CD93 mediates tumor-induced PD-L1 expression on monocytes. (A–E) CD14 + cells were purified from the peripheral blood of healthy donors. Cells were transfected with control siRNA (siNC) or si CD93 before being treated with medium (Med) or HepG2 TSN for 24 hours. Their levels of CD93 (A, n=4), IL-6, IL-1β, TNF-α (B, n=6), HLA-DR, CD86 (C, n=4) were determined by flow cytometry or ELISA. The expression levels of PD-L1 were determined by flow cytometry (D, n=6) and western blotting (E, n=4). (F) Frozen sections of HCC samples were stained with anti-human CD68 antibody (green), anti-human CD93 antibody (red), anti-human PD-L1 antibody (white), and DAPI (blue). The co-localization of cell signals in the peritumoral regions was analyzed by confocal microscopy. Scale bar=50 µm, n=3. G, CD14 + cells were purified from tumor tissues of nine patients with HCC. Correlations between the expression levels of CD93 and PD-L1 in these cells were analyzed via flow cytometry. Results shown in A–E are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The following statistical analyses were performed: two-way ANOVA ( A–E ), or Pearson’s correlation and linear regression analysis ( G ). ANOVA, analysis of variance; HCC, hepatocellular carcinoma; IF, immunofluorescence.

Article Snippet: Medium, siNC-containing liposome, or si CD93 -containing liposome (C12-200, HY-145405, MCE; cholesterol, C8503, Sigma-Aldrich; DSPC, 850 365P, Avanti; DMG-PEG2000, 880 151P, Avanti) were prepared as previously described.

Techniques: Expressing, Purification, Transfection, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Confocal Microscopy, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting CD93 on monocytes revitalizes antitumor immunity by enhancing the function and infiltration of CD8+ T cells

doi: 10.1136/jitc-2024-010148

Figure Lengend Snippet: CD93 increases levels of PD-L1 through the AKT-GSK3β signaling pathway. (A–E) CD14 + cells were purified from the peripheral blood of healthy donors. (A) Cells were transfected with siNC or si CD93 , and left untreated or treated with HepG2 TSN for 24 hours. Their levels of CD93, p-GSK3β, GSK3β, p-AKT, AKT, p-p38, p38, p-FAK, and FAK expression were determined by western blotting. n=4. (B–E) Cells were left untreated or treated with HepG2 TSN in the presence or absence of AKTi (5 µM) for 24 hours, or LiCl (20 mM) for 24 hours. Their levels of p-GSK3β, GSK3β, and p-AKT expression were determined by western blotting ( B, D ), and their PD-L1 expression was analyzed by western blotting (B, D, n=3) and flow cytometry (C, E, n=5). (F, G) CD14 + cells were purified from tumor tissues of nine patients with HCC. Correlations between levels of PD-L1 and p-AKT expression, or PD-L1 and p-GSK3β expression in these cells were analyzed by western blotting. Results shown in (C, E) are expressed as mean±SEM. *p<0.05, **p<0.01, ****p<0.0001. The following statistical analyses were performed: two-way ANOVA ( C, E ) or Pearson’s correlation and linear regression analysis ( F, G ). ANOVA, analysis of variance; HCC, hepatocellular carcinoma.

Article Snippet: Medium, siNC-containing liposome, or si CD93 -containing liposome (C12-200, HY-145405, MCE; cholesterol, C8503, Sigma-Aldrich; DSPC, 850 365P, Avanti; DMG-PEG2000, 880 151P, Avanti) were prepared as previously described.

Techniques: Purification, Transfection, Expressing, Western Blot, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting CD93 on monocytes revitalizes antitumor immunity by enhancing the function and infiltration of CD8+ T cells

doi: 10.1136/jitc-2024-010148

Figure Lengend Snippet: CD93 enhances the production of versican by monocytes. (A) CD14 + cells were purified from the peripheral blood of healthy donors, transfected with siNC or si CD93 , and then treated with HepG2 TSN for 24 hours. RNA-seq was performed with these differently-treated cells. The first 16 most downregulated genes in the si CD93 group versus the siNC control were shown. n=2. (B) Online HCC scRNA-seq data analysis revealed 145 genes upregulated in CD93 + versus CD93 − macrophages. These genes were compared with the downregulated genes in si CD93 -treated versus siNC-treated monocytes from our RNA-seq data, and the two overlapped genes were shown. (C–E) CD14 + monocytes were purified from non-tumor, peritumor, or tumor tissues of HCC. Levels of VCAN expression in these cells were analyzed by Q-PCR (C, n=5) or western blotting (D, n=3). Correlations between the mRNA levels of VCAN and CD93 in these cells were analyzed by Q-PCR (E, n=10). (F) CD14 + cells were purified from the peripheral blood of healthy donors, then left untreated (Med) or treated with HepG2 TSN. The kinetics of VCAN expression by these cells were determined via Q-PCR. n=4. (G, H) CD14 + cells purified from the peripheral blood of healthy donors were transfected with control siRNA (siNC) or si CD93 before being treated with medium (Med) or HepG2 TSN for 24 hours. Their levels of VCAN expression were determined by Q-PCR (G, n=4) and western blotting (H, n=4). Results shown in (C, F, H) are expressed as mean±SEM. *p<0.05, **p<0.01, ****p<0.0001. The following statistical analyses were performed: one-way ANOVA ( C ), Pearson’s correlation and linear regression analysis ( E ), paired two-tailed Student’s t-test ( F ) or two-way ANOVA ( G, H ). ANOVA, analysis of variance; HCC, hepatocellular carcinoma.

Article Snippet: Medium, siNC-containing liposome, or si CD93 -containing liposome (C12-200, HY-145405, MCE; cholesterol, C8503, Sigma-Aldrich; DSPC, 850 365P, Avanti; DMG-PEG2000, 880 151P, Avanti) were prepared as previously described.

Techniques: Purification, Transfection, RNA Sequencing, Control, Expressing, Western Blot, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting CD93 on monocytes revitalizes antitumor immunity by enhancing the function and infiltration of CD8+ T cells

doi: 10.1136/jitc-2024-010148

Figure Lengend Snippet: Monocyte-derived versican suppresses the migration of CD8 + T cells. (A) VCAN expression by different cells within HCC tissues was measured using scRNA-seq data from an online database. (B) Distribution of Versican + , CD68 + , α-SMA + , and CD8 + cells in HCC tumor tissues was analyzed by IF staining. Scale bar=50 µm, n=4. (C) Correlations between levels of VCAN expression and CD8 infiltration were analyzed using TCGA data. (D) Paraffin-embedded sections of human HCC tissues were stained with anti-human CD93 and anti-CD8 antibodies. The patients were then divided into two groups according to the median value of peritumoral numbers of CD93 + cells. The levels of CD8 + T cells infiltration in peritumoral tissues (right panel) and intratumoral tissues (left panel) were compared between the two groups (CD93 Low , n=6; CD93 High , n=7). (E, F) CD14 + monocytes and CD8 + T cells were purified from the peripheral blood of healthy donors. Membrane of the 24-well Boyden chamber was coated with collagen I (1 mg/mL) plus hyaluronan (50 µg/mL), in the presence or absence of versican (10 µg/mL) ( E ), or the CCM or TCM from siNC or si VCAN -treated monocytes ( F ). CD8 + T cells were added to the upper compartment of the chamber, and their transmembrane migration was measured after 24 hours of incubation. n=4. Results shown in (D–F) were expressed as mean±SEM. *p<0.05, ***p<0.001, ****p<0.0001. The following statistical analyses were performed: Pearson’s correlation and linear regression analysis ( C ), two-tailed Student’s t-test ( D, E ) or two-way ANOVA ( F ). ANOVA, analysis of variance; HCC, hepatocellular carcinoma; IF, immunofluorescence; TCGA, the Cancer Genome Atlas.

Article Snippet: Medium, siNC-containing liposome, or si CD93 -containing liposome (C12-200, HY-145405, MCE; cholesterol, C8503, Sigma-Aldrich; DSPC, 850 365P, Avanti; DMG-PEG2000, 880 151P, Avanti) were prepared as previously described.

Techniques: Derivative Assay, Migration, Expressing, Staining, Purification, Membrane, Incubation, Two Tailed Test, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting CD93 on monocytes revitalizes antitumor immunity by enhancing the function and infiltration of CD8+ T cells

doi: 10.1136/jitc-2024-010148

Figure Lengend Snippet: Targeting CD93 + monocytes inhibits tumor progression and enhances tumor sensitivity to anti-PD-1 treatment in mice in vivo. (A) C57BL/6 mice with established orthotopic, Luminescence-positive, Hepa1-6 tumors were injected with siNC-containing liposome, or si CD93 -containing liposome at indicated times via tail vein. (B, C) Real-time tumor growth and tumor size were monitored. (n=4). (D) Levels of Versican + F4/80 + cells infiltration in peritumoral tissues were measured by IF staining (n=4). (E–G) The infiltration and levels of IFN-γ and TNF-α expression of/by CD8 + T cells were measured on day 16 via flow cytometry (n=4). (H) C57BL/6 mice with established orthotopic, Luminescence-positive, Hepa1-6 tumors were injected with siNC-containing liposome or si CD93 -containing liposome via tail vein in the presence or absence of IgG or anti-PD-1 antibodies at indicated times. (I, J) Real-time tumor growth and tumor size were monitored (n=4). (K–M) Tumor infiltration and levels of IFN-γ and TNF-α expression of/by CD8 + T cells were measured on day 16 via flow cytometry (n=4). Results shown in (B, D–G, I, K–M) were expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. The following statistical analyses were performed: one-way ANOVA with Turkey’s correction ( D–G, K–M ), two-way ANOVA with Turkey’s correction ( B, I ). ANOVA, analysis of variance; IF, immunofluorescence.

Article Snippet: Medium, siNC-containing liposome, or si CD93 -containing liposome (C12-200, HY-145405, MCE; cholesterol, C8503, Sigma-Aldrich; DSPC, 850 365P, Avanti; DMG-PEG2000, 880 151P, Avanti) were prepared as previously described.

Techniques: In Vivo, Injection, Staining, Expressing, Flow Cytometry, Immunofluorescence