Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: N-glycosylation Modification of CTSD Affects Liver Metastases in Colorectal Cancer.

doi: 10.1002/advs.202411740

Figure Lengend Snippet: Figure 5. Identification of N-glycosyltransferase for CTSD. A) CTSD with a FLAG label was enriched using FLAG magnetic beads and silver-stained gel showed that proteins, such as DDOST, were enriched by CTSD. B) 191 proteins were enriched by CTSD N263Q and 235 proteins were enriched by CTSD WT, while 45 proteins exclusively interacted with CTSD WT. C) Expression levels of STT3A, STT3B, and DDOST were upregulated in colon cancer relative to normal tissues. Boxplots show median (central line), upper and lower quartiles (box limits), and 1.5 × interquartile range (whiskers). D) N-glycosylated modification at residue 263 of CTSD was regulated by STT3B and DDOST. When STT3B and DDOST were knocked out, molecular weights of CTSD decreased significantly. E) Co-immunoprecipitation and western blot analysis demonstrated that CTSD formed complexes with STT3B and DDOST. F) Immunofluorescence analysis illustrated the co-localization of DDOST and STT3B with CTSD, whereas no such co-localization was observed with STT3A.

Article Snippet: [26] Anti-rabbit CTSD (AB-75811, Abcam), Anti-mouse CTSD (AB-302649, Abcam), Anti-rabbit CTSD (AB-75852, Abcam), Anti-mouse GAPDH (60004-1-Ig, Proteintech), Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology), Anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology), Anti-mouse ACADM (67742-1-Ig, Proteintech), Anti-rabbit DDOST (14916-1-AP, Proteintech), Anti-mouse STT3A (66581- 1-Ig, Proteintech), Anti-mouse STT3B (15323-1-AP, Proteintech), Antirabbit ACSL4 (AB-155282, Abcam), Anti-rabbit SLC7A11 (AB-307601, Abcam) and Anti-rabbit GPX4 (AB-125066, Abcam) were used.

Techniques: Magnetic Beads, Staining, Expressing, Residue, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Elevation of spermine remodels immunosuppressive microenvironment through driving the modification of PD-L1 in hepatocellular carcinoma

doi: 10.1186/s12964-022-00981-6

Figure Lengend Snippet: Spermine promotes PD-L1 protein N-glycosylation and stability through inducing STT3A expression in HCC. A Effects of PNGase F on the molecular weight of PD-L1 in the lysates of SNU-368 cells treated with or without spermine (200 µM) for 24 h. B , C Effects of spermine (200 µM) on the expression of STT3A mRNA ( B ) and protein ( C ) in SNU-368 cells. * p < 0.05, ** p < 0.01, one-way ANOVA, n = 5 independent experiments per group. D , E Effects of spermine (200 µM) on the expression of STT3B mRNA ( D ) and protein ( E ) in SNU-368 cells. one-way ANOVA, n = 5 independent experiments per group. F , G Effects of STT3A ( F ) and STT3B ( G ) knockdown on spermine-induced PD-L1 protein expression in SNU-368 cells. 24 h after two specific siRNA transfection, cells were incubated in the presence or absence of spermine (200 µM) for 24 h. Scr: Scramble; 1 # siA: 1 # STT3A siRNA; 2 # siA: 2 # STT3A siRNA; 1 # siB: 1 # STT3B siRNA; 2 # siB: 2 # STT3B siRNA; * p < 0.05, *** p < 0.001, one-way ANOVA, n = 5 independent experiments per group. H , I The expression of CD274 mRNA ( H ) and PD-L1 protein ( I ) in SNU-368 cells expressing STT3A or STT3B for 24 h. *** p < 0.001 compared with Vector group, one-way ANOVA, n = 4 independent experiments per group. J IHC staining of STT3A and PD-L1 in 24 human HCC specimens. Scale bar, 50 μm. K Spearman correlation test between blood spermine concentration and tumor STT3A IHC scores. L Spearman correlation analysis of STT3A and PD-L1 IHC scores. Note that the scores of some samples overlap

Article Snippet: Mouse monoclonal anti-STT3A (sc-100796) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA); Rabbit polyclonal anti-STT3B (PA5-106380) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Expressing, Molecular Weight, Transfection, Incubation, Plasmid Preparation, Immunohistochemistry, Concentration Assay

Journal: Cancer discovery

Article Title: Pharmacological suppression of B7-H4 glycosylation restores antitumor immunity in immune-cold breast cancers

doi: 10.1158/2159-8290.CD-20-0402

Figure Lengend Snippet: (A) Stable expression of Flag-hB7-H4 was engineered to MDA-MB-468 cells (MDA-MB-468-Flag-hB7-H4). B7-H4 complex was then purified followed by mass spectrometry analysis. Coomassie blue staining of the purified B7-H4 immunocomplex is shown. The ubiquitin E3 ligase AMFR and several glycosyltransferases including STT3A, RPN1, RPN2, and UGGG1 were identified, and the representative spectra of AMFR, STT3A and UGGG1 are shown. (B) Validation of biochemical interactions of B7-H4 with AMFR, STT3A, UGGG1 as well as HSP90. MDA-MB-468-Flag-hB7-H4 cells were utilized for immunoprecipitation using anti-Flag M2-beads in the presence or absence of MG132 and/or tunicamycin. The interactions of AMFR, STT3A, UGGG1 as well as HSP90 with B7-H4 were measured by immunoblot. (C) Double immunofluorescence staining hB7-H4-Flag with AMFR or STT3A in MDA-MB-468-Flag-hB7-H4 cells followed by the confocal microscope (Scale bar =10 μm). (D) MDA-MB-468-Flag-hB7-H4 cells were subjected to duolink in situ PLA assay with specific Flag mouse antibody and AMFR or STT3A rabbit antibody (Scale bar = 100 μm). Red dots indicate the binding of the indicated two proteins. (E) AMFR knockdown results in upregulation of B7-H4. Stable knockdown of AMFR in MDA-MB-468 and SKBR3 were established. The expression of AMFR and B7-H4 were examined by immunoblotting. (F) STT3A knockdown leads to downregulation of B7-H4. STT3A stable knockdowns in MDA-MB-468 cells were established. The expression of STT3A and B7-H4 were examined by immunoblotting. (G) Decreased membrane B7-H4 in STT3A knockdown cells. MDA-MB-468-shVector, MDA-MB-468-shSTT3A, SKBR3-shVector, and SKBR3-shSTT3A cells were stained with PE anti-human B7-H4 antibody followed by flow cytometry. Representative images are shown. (H) The quantification of membrane staining of B7-H4 in MDA-MB-468-shVector, MDA-MB-468-shSTT3A, SKBR3-shVector, and SKBR3-shSTT3A cells are shown. (I) MDA-MB-468 and SKBR3 cells were treated with 10 μM OST inhibitor NGI-1 for 24 h. The expression of B7-H4 was examined by immunoblotting. (J) Blockade of B7-H4 glycosylation by NGI-1 enhances B7-H4 ubiquitination. 293T cells were transfected with Flag-hB7-H4 in the presence or absence of 10 μM NGI-1 for 24 h. Then Flag-hB7-H4 was immunoprecipitated followed by immunoblotting using antibody against ubiquitin.

Article Snippet: The following antibodies were used: B7-H4 (Cell signaling,14572) Flag (Cell signaling, 8146 or 14793), Myc (Cell signaling, 2272), eIF2a (Cell signaling, 5324), p-eIF2a (Cell signaling, 3398), HSP70 (Cell signaling, 4873), Hsp90 (Cell signaling, 4877), CALR (Cell signaling, 12238), Normal IgG (Cell signaling, 2729), ubiquitin (Cell signaling, 43124), AMFR (Cell signaling, 9590), PERK (Cell signaling, 5683), eIF2a (Sigma, HPA084885), AMFR (Sigma,HPA077835), B7-H4 (Sigma, HPA054200), STT3A (proteintech, 12034), mouse B7-H4 (R&D, AF2154), RPN1 (Bethyl, A302-026A), RPN2 (Bethyl, A304-265A), STT3A (Bethyl, A305-531A) and UGGG1 (Bethyl, A305-530A), CD45 Rat anti Mouse, Brilliant Violet 711 (BD, ThermoFisher Scientific, 563709), IFNγ Rat anti Mouse, Brilliant Violet 711 (BD, ThermoFisher Scientific, 564336), CD8a Rat anti Mouse, APC, (BD, ThermoFisher Scientific, 553035), I-A/I-E Rat anti-Mouse, Brilliant Violet 711 (BD, Thermo Fisher Scientific, 563414), CD86 Rat anti-Mouse, PE-Cy7 (BD, Thermo Fisher Scientific, 560582), CD11c Hamster anti-Mouse, APC, (Thermo Fisher Scientific, 550261), PE Mouse anti-Human B7-H4 (BD, 562507), FITC Rat anti-Mouse CD4 (BD, 553046), APC-Cy7 labeled anti-Mouse CD11b (BD, 557657), PE-Cy7 labeled anti-Mouse Ly-6G and Ly-6C (Gr-1) (BD, 552985), Anti-rat IgG (H+L), (Alexa Fluor(R) 647 Conjugate) (Cell Signaling Technology, 4418), Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor(R) 647 Conjugate) (Cell Signaling Technology, 4414), Rabbit IgG Isotype Control (Cell signaling Technology, 3452), (Alexa Fluor(R) 488 Conjugate) (Cell Signaling Technology, 4340S), Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor(R) 488 Conjugate (Cell Signaling Technology, 4412S).

Techniques: Expressing, Purification, Mass Spectrometry, Staining, Immunoprecipitation, Western Blot, Double Immunofluorescence Staining, Microscopy, In Situ, Binding Assay, Flow Cytometry, Transfection