Journal: Gut microbes

Article Title: CD44 is a macrophage receptor for TcdB from Clostridioides difficile that via its lysine-158 succinylation contributes to inflammation.

doi: 10.1080/19490976.2025.2506192

Figure Lengend Snippet: Figure 3. CD44 functions as an independent receptor for TcdB/FBD in macrophages. (a – c) expression of known receptors (FZD1/2/7, CSPG4, PVRL3) in WT versus CD44-KO macrophages analyzed by (a) immunoblotting and (b, c) q-PCR. (d – g) competitive binding assays: (d, e) FZD1/2/7, CSPG4, and PVRL3 did not inhibit CD44 binding of (d) TcdB or (e) FBD, while (f, g) CD44 similarly did not interfere with TcdB/FBD binding to other receptors. (ELISA protocol: wells coated with 1 μM target protein [CD44-ECD or other receptors] were incubated with 1 μM TcdB/FBD, followed by competitor proteins. Binding was quantified by absorbance measure ment). (h – k) IL-1β (h, i) and IL-6 (j, k) levels in THP-1-Mφ treated with TcdB/FBD (100 pM) and receptor proteins (1 μM) for 24 h. All data in parts (b – k) are shown as the mean ± SEM (n = 3). ((ns: p > 0.05, **p < 0.01, &p < 0.001, #p < 0.0001).

Article Snippet: The other Primary antibodies used in this study were as follows: (Cat# 22915–1-AP), CSPG4 (Cat# 55027–1-AP), PVRL3 (Cat# 11213–1-AP), SUCLG2 (Cat# 14240–1-AP), FZD2 (Cat# 24272–1-AP), and FZD7 (Cat# 16974–1-AP) from Proteintech Group (Wuhan, China); FZD1 (Cat# PA5–86484) from Thermo Fisher Scientific; Flotillin-1 (Cat# ab41927) from Abcam (Cambridge, UK).

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

Journal: The Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/JCI186863

Figure Lengend Snippet: ( A ) The heatmap shows that ST6GalNAc-I is significantly overexpressed in genetically engineered LUAD KPA ( Kras G12D/+ Trp53 R172/+ Ad-Cre ) compared with KA ( Kras G12D/+ Ad-Cre ) lung tumors and normal mouse lung. ( B ) Quantification and representative images of immunohistochemistry of SNA lectin expression in normal, KA, and KPA mouse lung tumor tissues. Significance was determined by 1-way ANOVA ( n = 3). Original magnification, ×40. ( C ) TCGA dataset shows that ST6GalNAc-I is highly overexpressed in early-stage ( n = 421) and late-stage ( n = 110) LUAD compared with normal tissue adjacent to the tumor (NAT) ( n = 59). ( D ) ST6GalNAc-I is the top differentially overexpressed sialyltransferase in LUAD compared with other sialyltransferases or glycosyltransferases, which suggests that targeting ST6GalNAc-I may prevent tumor sialylation–mediated LUAD development. ( E ) The volcano plot represents the mass spectrometry–based proteomic analysis of A549 control versus ST6GalNAc-I–KO cells. NECTIN2 was significantly downregulated in ST6GalNAc-I–KO cells. Red represents significantly upregulated proteins [FDR < 0.05 and log 2 (fold-change) ≥ 1], and blue represents significantly downregulated proteins [FDR < 0.05 and log 2 (fold-change) ≤ 1]. ( F ) Gene Ontology–based pathway analysis using A549 ST6GalNAc-I–KO cells showed NK cell–mediated immune response, apoptosis signaling, protein stability, and metabolic pathways, suggesting that these pathways are associated with ST6GalNAc-I in LUAD.

Article Snippet: ST6GalNAc-I (catalog AB229816 , Abcam), MUC5AC (CLH2, catalog MAB2011, Millipore), NECTIN2 (catalog 27171-1-AP, ProteinTech), VCAN (catalog MA5-42721, Invitrogen), VCAN-V1 (catalog PA1-1748A, Invitrogen), CD31 (catalog AB222783 , Abcam), TIGIT (catalog NBP2-79794, Novus Biologicals), and Ki-67 (catalog AB15580, Abcam) antibodies were used as primary antibodies.

Techniques: Immunohistochemistry, Expressing, Mass Spectrometry, Control

Journal: The Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/JCI186863

Figure Lengend Snippet: ( A and B ) In silico analysis indicated that expression of NECTIN2 is significantly overexpressed and associated with poor survival outcomes in both early- and late-stage LUAD patients. ( C ) Protein-protein interaction networking analysis reveals that tumor cells expressing NECTIN2 induce T cell dysfunction through TIGIT binding, which is associated with immune suppression pathways. ( D ) IHC analysis shows the overexpression of NECTIN2 in LUAD. Data were analyzed using 2-tailed t test ( n = 38). Original magnification, ×10. ( E ) The expression of MUC5AC and NECTIN2 drastically decreased in ST6GalNAc-I–KO and MUC5AC-KD cells (A549 and H1437). ( F and G ) Immunoprecipitation assay shows NECTIN2 sialylation in LUAD cells. ( H ) SNA pull-down was performed on A549 cell lysates, followed by immunoblotting with NECTIN2 antibody, suggesting that NECTIN2 carries STn in LUAD cells. ( I ) Immunofluorescence assay reveals the decreased association of NECTIN2 and STn in A549 ST6GalNAc-I–KO cells, and the bar diagram represents the quantification of NECTIN2 and STn using arithmetic mean intensity. Data were analyzed using 2-tailed t test ( n = 3). Scale bars: 5 μm.

Article Snippet: ST6GalNAc-I (catalog AB229816 , Abcam), MUC5AC (CLH2, catalog MAB2011, Millipore), NECTIN2 (catalog 27171-1-AP, ProteinTech), VCAN (catalog MA5-42721, Invitrogen), VCAN-V1 (catalog PA1-1748A, Invitrogen), CD31 (catalog AB222783 , Abcam), TIGIT (catalog NBP2-79794, Novus Biologicals), and Ki-67 (catalog AB15580, Abcam) antibodies were used as primary antibodies.

Techniques: In Silico, Expressing, Binding Assay, Over Expression, Immunoprecipitation, Western Blot, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/JCI186863

Figure Lengend Snippet: ( A and B ) Schemes for the isolation of T cells and PBLs from healthy blood donors. A549 ST6GalNAc-I–KO and control cells were cocultured with PBLs and T cell subsets for tumor cell killing assays. ( C and D ) We observed that the killing of A549 ST6GalNAc-I–KO cancer cells cocultured with T cells was significantly higher than that of control cells cocultured with T cells as demonstrated by IncuCyte live imaging with CytoTox Red assay. Representative images show differences in red staining indicative of dead cells. Data were analyzed using 2-tailed Student’s t test ( n = 3). Scale bars: 300 μm. ( E and F ) Further, coculture of A549 ST6GalNAc-I–KO and control with specific T cells or PBLs also showed increased killing ability of ST6GalNAc-I–KO (48 hours) as indicated by CytoTox-Glo assays. Data were analyzed using 2-tailed Student’s t test ( n = 3). ( G ) The schematic illustrates how tumor cell–expressed NECTIN2 induces T cell dysfunction through the TIGIT receptor. ( H ) Western blot analysis showing that NECTIN2 is specifically expressed in A549 while TIGIT is expressed in T cells. ( I ) Immunoblot of TIGIT and NECTIN2 shows their interaction. The coculture lysates derived from A549 plus T cells were immunoprecipitated with NECTIN2 and probed with both antibodies. ( J ) Immunofluorescence images show the colocalization of NECTIN2 and TIGIT with T cell–specific marker CD3. NECTIN2-TIGIT interacting region (shown by white arrowheads) is represented using black-and-white image. Cancer cells are represented by red dashed lines, and T cells are represented by green dashed lines. Scale bars: 5 μm.

Article Snippet: ST6GalNAc-I (catalog AB229816 , Abcam), MUC5AC (CLH2, catalog MAB2011, Millipore), NECTIN2 (catalog 27171-1-AP, ProteinTech), VCAN (catalog MA5-42721, Invitrogen), VCAN-V1 (catalog PA1-1748A, Invitrogen), CD31 (catalog AB222783 , Abcam), TIGIT (catalog NBP2-79794, Novus Biologicals), and Ki-67 (catalog AB15580, Abcam) antibodies were used as primary antibodies.

Techniques: Isolation, Control, Imaging, Staining, Western Blot, Derivative Assay, Immunoprecipitation, Immunofluorescence, Marker

Journal: The Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/JCI186863

Figure Lengend Snippet: ( A ) We developed stable mouse St6galnac-I knockdown in mouse syngeneic KP2075 cells. Quantitative real-time PCR analysis shows that the transcript level of St6galnac-I was significantly decreased in St6galnac-I–KD cells. Significance was determined by 2-tailed t test ( n = 4). ( B ) Immunoblot shows that Nectin2 and Muc5ac were decreased upon St6galnac-I knockdown. ( C ) Schemes for the intratracheal orthotopic models. ( D – F ) Mice injected with St6galnac-I–KD cells showed reduced lung tumor incidence in H&E ( n = 6) along with decreased Ki-67, St6galnac-I, STn, Nectin2, and Tigit. Significance was determined by 2-tailed t test ( n = 3). Original magnification, ×4 for H&E and ×40 for IHC. ( G ) Left: Immunofluorescence assays indicate decreased association of Nectin2 and Tigit in St6galnac-I–KD tumors. Right: Quantification of the arithmetic mean intensity value of CD3 (green), NECTIN2 (red), and TIGIT (purple) per field of view ( n = 3). Scale bars: 5 μm.

Article Snippet: ST6GalNAc-I (catalog AB229816 , Abcam), MUC5AC (CLH2, catalog MAB2011, Millipore), NECTIN2 (catalog 27171-1-AP, ProteinTech), VCAN (catalog MA5-42721, Invitrogen), VCAN-V1 (catalog PA1-1748A, Invitrogen), CD31 (catalog AB222783 , Abcam), TIGIT (catalog NBP2-79794, Novus Biologicals), and Ki-67 (catalog AB15580, Abcam) antibodies were used as primary antibodies.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Injection, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: ST6GalNAc-I regulates tumor cell sialylation via NECTIN2/MUC5AC-mediated immunosuppression and angiogenesis in non–small cell lung cancer

doi: 10.1172/JCI186863

Figure Lengend Snippet: Our findings indicated that ST6GalNAc-I induces tumor cell sialylation through NECTIN2 and MUC5AC for immune evasion and tumor angiogenesis. Hence, targeting ST6GalNAc-I or tumor cell sialylation may prevent LUAD development and metastasis. Further targeting ST6GalNAc-I–associated NECTIN2 sialylation may enhance the immune checkpoint inhibitors to improve survival of patients with LUAD.

Article Snippet: ST6GalNAc-I (catalog AB229816 , Abcam), MUC5AC (CLH2, catalog MAB2011, Millipore), NECTIN2 (catalog 27171-1-AP, ProteinTech), VCAN (catalog MA5-42721, Invitrogen), VCAN-V1 (catalog PA1-1748A, Invitrogen), CD31 (catalog AB222783 , Abcam), TIGIT (catalog NBP2-79794, Novus Biologicals), and Ki-67 (catalog AB15580, Abcam) antibodies were used as primary antibodies.

Techniques: