Journal: Frontiers in Cardiovascular Medicine

Article Title: Deficiency of Myeloid Pfkfb3 Protects Mice From Lung Edema and Cardiac Dysfunction in LPS-Induced Endotoxemia

doi: 10.3389/fcvm.2021.745810

Figure Lengend Snippet: Myeloid-specific Pfkfb3 deficiency attenuates LPS-induced inflammatory responses. (A) Representative images (left) and quantification (right) for immunohistochemical staining of the neutrophil marker Ly6G in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (B) Representative images (left) and quantification (right) for immunohistochemical staining of the macrophage marker Mac2 in lung sections of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 5). (C–E) qPCR analysis of the mRNA levels of Il1b (C) , Il6 (D) and Nos2 (E) in the lung of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 6). (F,G) ELISA analysis of Il1b (F) and Il6 (G) in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 4). (H) NO levels in serum of Pfkfb3 WT mice and Pfkfb3 ΔMϕ mice 6 h after LPS injection ( n = 3–4). All data are represented as mean ± SEM, ** P < 0.01 and *** P < 0.001 for Pfkfb3 WT vs. Pfkfb3 ΔMϕ (unpaired two-tailed Student's t test).

Article Snippet: After antigen retrieval with Antigen Unmasking Solution (H-3301, Vector Laboratories, Burlingame, CA, USA) at 98°C for 10 min, sections were blocked with avidin solution with 10% normal rabbit serum for 1 h at room temperature, and incubated in biotin blocking solution with primary antibodies against Mac2 (3 μg/mL, CL8942F, Cedarlane, Burlington, NC, USA), or Ly6G (3 μg/mL, 551459, BD biosciences, San Jose, CA, USA) at 4°C overnight.

Techniques: Immunohistochemical staining, Staining, Marker, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: ADAMTS7 promotes smooth muscle foam cell expansion in atherosclerosis

doi: 10.1172/JCI187451

Figure Lengend Snippet: ( A ) Schematic outlining experimental design and the generation of the SMC transgenic ADAMTS7 mouse. Created with BioRender.com. ( B ) ORO staining of the en face aorta and quantification of lesion area. n = 9. ( C ) Representative images of H&E-stained aortic root sections, quantification of plaque areas ( n = 6). Scale bar: 500 μm. ( D ) Representative images of H&E-stained aortic root sections with necrotic core outlined in dotted line ( n = 6 mice). Scale bar: 300 μm. ( E ) Representative images of aortic root sections stained against α-SMA (Cy3; red), MAC2 (Alexa Fluor 488 green), and cell nuclei (DAPI blue) with their subsequent quantification relative to lesion area ( n = 6). Scale bar: 300 μm. ( F ) Representative Picrosirius red staining of aortic root lesions with bars indicating fibrous cap thickness and quantification of fibrous cap thickness ( n = 6). Scale bar: 300 μm. ( G ) Schematic outlining experimental design and the generation of the EC transgenic ADAMTS7 mouse. Created with BioRender.com. ( H ) ORO staining of the en face aorta and quantification of lesion area. n = 3–4. ( I ) Representative images of H&E-stained aortic root sections and quantification of plaque areas ( n = 5–6). Scale bar: 500 μm. **** P < 0.0001, * P < 0.05. Statistics were analyzed using a 2-tailed Student’s t test.

Article Snippet: The sections then were stained using the following Abs and dilutions: 1:1,000 α-SMA-Cy3 (MilliporeSigma, C6198) and 1:500 MAC2 (Cedarlane, CL8942AP).

Techniques: Transgenic Assay, Staining

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

Journal: Cell stem cell

Article Title: Smooth muscle cell reprogramming in aortic aneurysms

doi: 10.1016/j.stem.2020.02.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Monoclonal anti-Mac2/Galectin-3-153Eu (clone M3/38) , Fluidigm , Cat# 3153026B, RRID:AB_2814900.

Techniques: Recombinant, Multiplex Assay, Staining, In Situ, Marker, Expressing, Plasmid Preparation, shRNA, Software