Journal: Molecular Oncology

Article Title: Extracellular LGALS3BP : a potential disease marker and actionable target for antibody–drug conjugate therapy in glioblastoma

doi: 10.1002/1878-0261.13453

Figure Lengend Snippet: LGALS3BP expression levels in GBM patients. (A) Sandwich ELISA performed on GBM patient ( n = 17) and healthy donor ( n = 18) serum samples. (B) Graph showing the percentage of EVs‐associated LGALS3BP over total circulating LGALS3BP in serum. Mann–Whitney test * P = 0.03. (C) Box‐and‐whisker diagram of the distribution of LGALS3BP in GBM cases and peritumoral matched samples ( n = 53). The upper and lower ends of boxes represent 75th and 25th percentiles. The median value is shown with a solid line. Mann–Whitney test *** P < 0.001. Data are shown as mean ± standard deviation. (D, E) Representative images of IHC staining for LGALS3BP expression in GBM and peritumoral matched samples. All IHC images have been acquired using an optical microscope at a scale bar of 50 μm.

Article Snippet: Also, for LGALS3BP expression analysis of mice serum‐derived EVs in correlation studies, they were analysed using Proteintech™ Human LGALS3BP ELISA Kit (Cod.

Techniques: Expressing, Sandwich ELISA, MANN-WHITNEY, Whisker Assay, Standard Deviation, Immunohistochemistry, Microscopy

Journal: Molecular Oncology

Article Title: Extracellular LGALS3BP : a potential disease marker and actionable target for antibody–drug conjugate therapy in glioblastoma

doi: 10.1002/1878-0261.13453

Figure Lengend Snippet: LGALS3BP expression levels in EVs isolated from GBM patient‐derived cell lines. (A) LGALS3BP and EVs markers CD9, actin and CD63 expression levels in whole lysate and EVs isolated from Gch6 and Gch14 cellular supernatants ( n = 2). (B) Effect of N‐Glycosylation (kifunensine, KIF and tunicamycin, TUN) and O‐Glycosylation (OSMI‐1) inhibitors on the protein profile for LGALS3BP in Gch6 whole lysate and corresponding supernatant ( n = 2). (C) Sandwich ELISA performed on intact EVs isolated from Gch6 and Gch14 cell line supernatants ( n = 3). Data are shown as mean ± standard deviation. (D) Sandwich ELISA performed on intact EVs isolated from Gch6 treated with KIF ( n = 3). Data are shown as mean ± standard deviation. (E) Effect of KIF treatment on the protein profile in whole lysate and EVs isolated from Gch6 ( n = 2). (F) Confocal images of live GBM patient‐derived cells labelled with humanized 1959 anti‐LGALS3BP antibody followed by AlexaFluor 488 conjugated secondary anti‐human IgG antibody (green). Cell nuclei were stained with DAPI (blue) ( n = 3). Images were taken at 40× magnification. Scale bar: 20 μm. Negative controls were only incubated with secondary antibody.

Article Snippet: Also, for LGALS3BP expression analysis of mice serum‐derived EVs in correlation studies, they were analysed using Proteintech™ Human LGALS3BP ELISA Kit (Cod.

Techniques: Expressing, Isolation, Derivative Assay, Sandwich ELISA, Standard Deviation, Staining, Incubation

Journal: Molecular Oncology

Article Title: Extracellular LGALS3BP : a potential disease marker and actionable target for antibody–drug conjugate therapy in glioblastoma

doi: 10.1002/1878-0261.13453

Figure Lengend Snippet: Extracellular vesicles‐associated LGALS3BP correlation with tumour volume and therapeutic activity of 1959‐sss/DM3 in GBM patient‐derived xenograft model. (A) Graphic scheme representing ELISA of human LGALS3BP on EVs in serum samples from mice with human GBM xenograft. (B) Levels of LGALS3BP on EVs isolated from the serum samples of control nude mice or mice bearing human GCh6 GBM xenograft, measured by ELISA ( n = 5). Bar graph represents average ± standard deviation. (C) Spearman correlation between EVs‐associated LGALS3BP and tumour burden in xenograft‐bearing nude mice ( n = 9). (D, E) Cytotoxic activity of SH‐DM3, SH‐DM4 and temozolomide (TMZ) was obtained by using the MTT assay on Gch6 and Gch14 cell lines at indicated doses after 5 days. IC50 values were determined with graphpad prism software (San Diego, CA, USA) ( n = 3). Data are shown as mean ± standard deviation. (F) CD1 nude mice harbouring Gch6 xenografts were treated with vehicle (PBS, n = 4) or 1959‐sss/DM3 (10 mg·kg −1 , n = 4) twice weekly as indicated by the arrows. Data are shown as mean ± standard error. (G) (PBS, n = 4) (10 mg·kg −1 , n = 4). Kaplan–Meier survival curves. Log‐rank (Mantel–Cox) Test. ** P = 0.006.

Article Snippet: Also, for LGALS3BP expression analysis of mice serum‐derived EVs in correlation studies, they were analysed using Proteintech™ Human LGALS3BP ELISA Kit (Cod.

Techniques: Activity Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Isolation, Standard Deviation, MTT Assay, Software

Journal: Molecular Oncology

Article Title: Extracellular LGALS3BP : a potential disease marker and actionable target for antibody–drug conjugate therapy in glioblastoma

doi: 10.1002/1878-0261.13453

Figure Lengend Snippet: 1959‐sss/DM4 dose–response efficacy and biodistribution. (A) CD1 nude mice Gch6 xenografts were treated with vehicle (PBS) or 1959‐sss/DM4 at the indicated doses twice weekly (PBS, n = 5; 1 mg·kg −1 , n = 7; 3 mg·kg −1 , n = 6; 10 mg·kg −1 , n = 6). Data are shown as mean ± standard error. (B) (PBS, n = 5; 1 mg·kg −1 , n = 7; 3 mg·kg −1 , n = 6; 10 mg·kg −1 , n = 6). Kaplan–Meier survival curves. Log‐rank (Mantel–Cox) Test. *** P < 0.001. (C) Representative images of IHC staining for LGALS3BP expression in Gch6 patient primary tumour and in Gch6 xenograft ( n = 4). Scale bar: 50 μm. (D) Biodistribution of 1959‐sss/DM4 in tumour‐bearing nude mice. ADC was injected into the tail vein of nude mice bearing a xenograft of GCh6 patient‐derived cell line. After 24, 48 and 72 h, the mice were sacrificed, and the ADC uptake was determined in tissues (D) by ELISA using an anti‐DM4 antibody as coating. Each column and bar show the mean and standard deviation for three mice. Tissue uptake of ADC is expressed as percentage of injected dose per gram of tissue (%ID·g −1 ). Data are shown as mean ± standard deviation.

Article Snippet: Also, for LGALS3BP expression analysis of mice serum‐derived EVs in correlation studies, they were analysed using Proteintech™ Human LGALS3BP ELISA Kit (Cod.

Techniques: Immunohistochemistry, Expressing, Injection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nature Communications

Article Title: Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2

doi: 10.1038/s41467-023-36553-6

Figure Lengend Snippet: a Electron micrographs of APEX2-TECPR2 WT or L440Rfs expressing cells. Before embedding and ultrathin sectioning, fixed samples were incubated with DAB and H 2 O 2 . Insets show magnification of representative nuclear and vesicular areas. Scale bars 200 nm. b TECPR2 WT and MUT cells were subjected to homogenization and differential centrifugation. Cytosolic (CYTO) and total membrane (TM) fractions were analyzed by immunoblotting. GM130 and tubulin served as loading controls. TECPR2 WT and MUT cells differentially treated with an ATG7 inhibitor ( c ) or ATG8-HEXA KO HeLa cells ( d ) were subjected to homogenization and differential centrifugation. Total lysate (TL), cytosolic (CYTO) and total membrane (TM) fractions were analyzed by immunoblotting. GM130, LC3B, GABARAP and tubulin served as controls. e Schematics of two-step differential centrifugation, resulting in total lysates (TL), nuclear debris (N), cytosol (C), total membranes (TM), light sucrose membranes (L), a sucrose membrane pellet (P) and six different OptiPrep membrane fractions (1–6). f TECPR2 WT cells were subjected to homogenization followed by two-step differential centrifugation and immunoblotting. g HA-TECPR2 WT or L440Rfs were immune-isolated from homogenates and selected association partners detected by immunoblotting. DTNBP1 served as loading control. HA-IP, immune-isolated membranes. h Working model of TECPR2’s field of operations at the ER-Golgi interface and protein sorting defects caused by truncated TECPR2. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used:ADAMTS1 (Abcam, #ab39194), APEX2 (IgG2A) (custom made by Regina Feederle, HZM München), ATP1A1 (Abcam, #ab7671), B4GALT3 (Proteintech, #11041-1-AP), BAG2 (Biomol, #A304-751A), beta-ACTIN (Sigma,#A1978), BIOTIN (Pierce, #31852), BiP (Cell Signaling, #3177), CALNEXIN (Abcam, #ab22595), Calreticulin (santa cruz, #sc-6467), CD63 (abcam, #ab59479), CLPTM1L (Sigma, #HPA014791), c-myc (Bethyl, #A190-104A), CRLF1 (Novus, #NBP1-85606), DTNBP1 (Bethyl, #A303-360A), EDIL3 (Abcam, #ab190692), ERGIC1 (Proteintech, #16108-1-AP), ERGIC53 (Santa Cruz, #sc398777), EXTL2 (Abcam, #ab168391), Flag M2 (Cell Signaling, #2368), GABARAP(Abcam,#109364), Giantin (Biolegend, #924302), GM130 (Abcam, #ab52649), GOLIM4 (Abcam, #ab28049), HA.11 Clone 16B2 (Covance/Biolegend, #MMS-101P/#901501), HS6ST2 (Abcam, # ab122220), LAMP1 (IF 1:100, Abcam/DSHB, #ab24170/H4A3), LAMP2 (IF 1:100, Abcam, #ab25631), LAMTOR1 (Cell Signaling, #8975S), LAMTOR2 (Cell Signaling, #8145); LAMTOR3 (Cell Signaling, #8168), LC3B (Cell Signaling, #2775S), LGALS3BP (Proteintech, #10281-1-AP), LRP1 (Abcam, #ab92544), M6PR (Abcam, #ab2733), MTOR (Cell Signaling, #2983), myc 9E10 (custom made by Regina Feederle, HZM), NCAM1 (WB: 1:500, IF 1:100, Merck, #AB5032), NEK9 (Abcam, #ab138488), NID1 (Invitrogen, #PA5-99666), NPC1 (WB: 1:500, Abcam, #ab134113), PCNA (Santa Cruz, #sc-7907), PLXDC2 (Novus, #NBP1-76858), PLXNA1 (R&D, #AF4309), PLXNA2 (Abcam, #ab39357), RAB5C (Sigma,#HPA003426), SEC12 (Novus, #NBP1-87056), SEC13 (Novus, #AF9055-100), SEC24C (Abcam, #ab122633), SEC24D (Cell Signaling, #14687), SEC31A (BD, #612351), SLC38A9 (Abcam, #ab81687), SPG20 (Proteintech, #13791-1-AP), TECPR2 (Christian Behrends, custom made), TNC (Abcam, #ab108930), TOLLIP (Abcam, #ab187198), TOMM40 (Abcam, #ab185543), TRAPPC11 (Sigma, #HPA045427), TRAPPC8 (Sigma, #HPA041107), TRAPPC9 (Proteintech, #16014-1-AP), TSG101 (Abcam, #ab30871), TUBULIN (Abcam, #ab7291), VAPA (Sigma, # HPA009174), VAPB (Sigma, # HPA013144), VCP (Bethyl, #A300-588A), VPS11 (Abcam, #ab125083).

Techniques: Expressing, Incubation, Homogenization, Centrifugation, Membrane, Western Blot, Isolation, Control