Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus

doi: 10.1073/pnas.1606253113

Figure Lengend Snippet: Monocyte patrolling in uninflamed glomerular capillaries is dependent on CX3CR1 and β2 and α4 integrins. The role of surface receptors in monocyte trafficking within uninflamed glomerular capillaries was investigated in Cx3cr1GFP mice using intravital multiphoton microscopy. (A) Representative multiphoton image sequences of an untreated Cx3cr1GFP/+ mouse (Top) and Cx3cr1GFP/GFP mouse (Bottom) illustrating migration of a GFP+ monocyte (green) within glomerular capillaries (blue; labeled with Qtracker-655). Asterisks indicate starting position, and arrows indicate the path of migration. Time stamp is shown above the images. (Scale bar: 10 μm.) See also Movie S1. (B and C) The number of monocytes adhering in glomeruli per hour shown for total, crawling, and static cells (B) and monocyte dwell time (of total cells) (C) was assessed in untreated Cx3cr1GFP/+ (n = 8) and Cx3cr1GFP/GFP mice (n = 5). (D) Monocyte dwell time was compared in Cx3cr1GFP/+ mice pretreated with anti-CD18 and anti-CD49d blocking antibodies together (n = 4), or the respective isotype controls (n = 6). Data are presented as mean ± SEM; *P < 0.05 vs. corresponding control group.

Article Snippet: Anti-mouse CD45.2-PerCPC5.5 (104) and the blocking antibodies anti-CD18 (GAME-46), anti-LFA-1 (M17/4), and anti-Mac-1 (5C6) were purchased from BD Bioscience.

Techniques: Microscopy, Migration, Labeling, Blocking Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus

doi: 10.1073/pnas.1606253113

Figure Lengend Snippet: Inhibition of either β2 or α4 integrins alone is insufficient to inhibit monocyte patrolling in uninflamed glomerular capillaries. (A and B) The number of monocytes adhering in glomeruli (Left) and monocyte dwell time (Right) were assessed in uninflamed Cx3cr1GFP/+ mice pretreated with anti-CD18 (n = 5) (A) or anti-CD49d (n = 4) (B) blocking antibodies or the respective isotype controls (n = 4–5). Data are shown as mean ± SEM.

Article Snippet: Anti-mouse CD45.2-PerCPC5.5 (104) and the blocking antibodies anti-CD18 (GAME-46), anti-LFA-1 (M17/4), and anti-Mac-1 (5C6) were purchased from BD Bioscience.

Techniques: Inhibition, Blocking Assay

Journal: Nature cell biology

Article Title: SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.

doi: 10.1038/s41556-023-01096-x

Figure Lengend Snippet: Fig. 4 | SARS-CoV-2 ORF6 or NSP13 expression is sufficient to cause CHK1 loss. a, Immunofluorescence (IF) images of Huh7 cells expressing Strep-tagged SARS-CoV-2 ORF6 or NSP13 fixed 48 h post-transfection and stained for DDR markers; GFP was used as control; staining with anti-Strep-tag was used to label transfected cells; nuclei were stained with DAPI. Scale bar, 10 μm. b, The histograms show the percentage of CHK1- or RRM2-expressing cells among the transfected ones (Strep-Tag+) as determined in a; n = 3 independent experiments (n = 4 for GFP-expressing cells in RRM2 analysis). The dot plots show the number

Article Snippet: 3 nature portfolio | reporting sum m ary M arch 2021 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used gH2AX (Ser139) Abcam ab11174 gH2AX (Ser139) Millipore 05-636 53BP1 Bethyl A303-906A 53BP1 Novus NB100-304 ACE2 Abcam ab15348 ATM Abcam ab32420 ATR Santa Cruz sc-1887 Beclin 1 Bethyl A302-566A-T Beta-actin Sigma-Aldrich A2228 BrdU BDbioscience 347580 CD68 Abcam ab125212 CDT1 Cell Signaling #8064 cGAS Cell Signaling #15102 CHK1 Novus NB100-46 CHK1 (2G1D5) Cell Signaling #2360 CHK1 (ST57-09) ThermoFisher MA532180 CHK2 Millipore 05-649 Cleaved Caspase-3 (Asp 175) Cell Signaling 9661 DNA-PK Abcam ab32566 HA-tag Abcam ab236632 Histone H3 Abcam ab10799 Human Cytokeratin 8/18 (EP17/EP30) Dako M3652 ISceI (FL-86) Santa Cruz sc-98269 KAP1 Abcam ab10484 LC3B Sigma-Aldrich L7543 p16 Abcam ab51243 p21 Abcam ab188224 P21 Cell Signaling #2946 P38 MAPK Cell Signaling #9212 P53 Abcam ab1101 p62 Abcam ab240635 pATM (Ser1981) Rockland 200-301-400 pATR (T1989) Abcam ab223258 pCHK1 (S317) Cell Signaling #2344 pCHK2 (Thr68) Cell Signaling #2661 PCNA Bio Rad MCA1558 pDNA-PK (Ser2056) (EPR5670) Abcam ab124918 pKAP1 (S824) Bethyl A300-767A pP38 MAPK (Thr180/Tyr182) Cell Signaling #9211 pP53 (Ser15) Cell Signaling #9284 proSP-C Abcam ab3786 pRPA (S4/S8) Bethyl A300-245A pSTAT1 (Ser 727) Cell Signaling #9177 pSTAT1 (Tyr 701) (58D6) Cell Signaling #9167 RPA Calbiochem NA18-100UG RRM1 Santa Cruz sc-11733 RRM2 Novus NBP1-31661 RRM2 Santa Cruz sc-10844 SARS-CoV2 nucleocapsid Sino Biological 40588-T62 SARS-CoV2 nucleocapsid Sino Biological 40143-R019 SARS-CoV2 nucleocapsid (1A6) ThermoFisher MA5-35941 STAT1 (9H2) Cell Signaling #9176 Strep-tag II epitope Qiagen 34850 Tubulin Sigma-Aldrich T5168 Ubiquitin (P4D1) Santa Cruz sc-8017 Vinculin Sigma-Aldrich V9131 Cy3 D/M Jackson 715-165-150 Cy3 D/R Jackson 711-165-152 Cy3 D/G Jackson 705-165-147 4 nature portfolio | reporting sum m ary M arch 2021 A488 D/M ThermoFisher A21202 A488 D/R ThermoFisher A21206 A488 D/G ThermoFisher A11055 A647 D/M ThermoFisher A31571 A647 D/R ThermoFisher A31573 A647 D/G ThermoFisher A21447 Validation All antibodies were validated by the manufacturer and were previously used in peer reviewed works.

Techniques: Expressing, Immunofluorescence, Transfection, Staining, Control, Strep-tag

Journal: Cell reports

Article Title: p63 and SOX2 Dictate Glucose Reliance and Metabolic Vulnerabilities in Squamous Cell Carcinomas

doi: 10.1016/j.celrep.2019.07.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following commercial primary antibodies supplemented with 0.02% sodium azide were used for immunoblot analysis: p63 (1:1,000; Biocare Medical CM163A), GLUT1 (1:1,000; Alpha Diagnostics GT11-A), SOX2 (1:1,000; Cell Signaling Technology #3579), V5-tag (1:1,000; Cell Signaling Technology #13202), SLGT2 (1:1,000; abcam ab85626), Tyr1361-p-INSR (1:1,000; ThermoFisher Scientific PA5-38283), INSR (1:1,000; ThermoFisher Scientifc AHR0271), Ser473-p-AKT (1:1000; Cell Signaling Technology #4058), p-AKT (1:1,000; Cell Signaling Technology #9272), Ser235/236-p-S6 (1:1,000; Cell Signaling Technology #4858), S6 Ribosomal Protein (1:1,000; Cell Signaling Technology #2217), Thr37/46-p-4EBP1 (1:1,000; Cell Signaling Technology #2855), 4E-BP1 (1:1,000; Cell Signaling Technology #9644), PCNA (1:1,000; Cell Signaling Technology #13110), Cleaved Caspase-3 (1:1,000; Cell Signaling Technology #9664), and β-actin (1:5,000; Sigma A5441).

Techniques: Diagnostic Assay, Plasmid Preparation, Recombinant, Luciferase, Enzyme-linked Immunosorbent Assay, Expressing, Clone Assay, shRNA, Sequencing, Software, Real-time Polymerase Chain Reaction, Microscopy, Imaging

Journal: Journal of clinical medicine

Article Title: Liposomal Lapatinib in Combination with Low-Dose Photodynamic Therapy for the Treatment of Glioma.

doi: 10.3390/jcm8122214

Figure Lengend Snippet: Figure 1. (A) The encapsulation efficiency of lapatinib within the liposomal formulation prepared with up to 2000 nmol lapatinib addition and the corresponding liposome size of the resultant liposomes. (B) Long-term stability of the liposomal lapatinib formulation prepared with 500 nmol lapatinib addition monitored by both z-average diameter and the respective polydispersity indices. (mean ± S.D., n = 3).

Article Snippet: A chloroform suspension of lapatinib ditosylate (925.46 g/mol, 10 mg/mL; Selleckchem, Radnor, PA, USA) was prepared, and 500 nmol of lapatinib ditosylate equivalent (46.3 μL) was added to the lipid mixture.

Techniques: Encapsulation, Formulation, Liposomes

Journal: Journal of clinical medicine

Article Title: Liposomal Lapatinib in Combination with Low-Dose Photodynamic Therapy for the Treatment of Glioma.

doi: 10.3390/jcm8122214

Figure Lengend Snippet: Figure 2. Live-cell imaging-based of protoporphyrin IX (PpIX) fluorescence within mitochondria and cytoplasm and the fraction between the two cellular locations as well as LD50 data when ALA and liposome-encapsulated lapatinib treatments were combined. (A) Percentage changes in PpIX production in the mitochondria following control (+HBEGF 5 ng/mL an epidermal growth factor receptor (EGFR) ligand commonly found in the brain for activation) and liposome-encapsulated lapatinib (500 nM) (+HBEGF) (* p < 0.05) (n = 3 biological replicates). (B) The fraction of PpIX in the mitochondria compared to the cytoplasm with and without lapatinib for all cell lines (* p < 0.05) (n = 3 biological replicates. (C) LD50 values of four glioma cell lines and one purported glioma cancer stem cell line (GS2) of PDT alone and PDT + Lapatinib, note LD50 of ALA, was chosen as a surrogate for PDT dose in these studies. A significant decrease in LD50 for the two EGFR positive cell lines U87 and U87vIII was defined by p < 0.05 (n = 3).

Article Snippet: A chloroform suspension of lapatinib ditosylate (925.46 g/mol, 10 mg/mL; Selleckchem, Radnor, PA, USA) was prepared, and 500 nmol of lapatinib ditosylate equivalent (46.3 μL) was added to the lipid mixture.

Techniques: Live Cell Imaging, Control, Activation Assay

Journal: Journal of clinical medicine

Article Title: Liposomal Lapatinib in Combination with Low-Dose Photodynamic Therapy for the Treatment of Glioma.

doi: 10.3390/jcm8122214

Figure Lengend Snippet: Figure 3. (A) MR images of central slices of five different U87 tumours prior to assignment to treatment groups including (a–e) contrast-enhanced T1w; and (f–j) T2w images (B) H&E staining of tumours at endpoint for each treatment groups. (C) Survival post-treatment of animals in each cohort (solid black line—control; solid grey line—lapatinib; short, broken line—photodynamic therapy (PDT) alone; broken line—PDT + lapatinib.

Article Snippet: A chloroform suspension of lapatinib ditosylate (925.46 g/mol, 10 mg/mL; Selleckchem, Radnor, PA, USA) was prepared, and 500 nmol of lapatinib ditosylate equivalent (46.3 μL) was added to the lipid mixture.

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Neurological Disorder after Severe Pneumonia is Associated with Translocation of Bacteria from Lung to Brain

doi: 10.1101/2022.12.30.522351

Figure Lengend Snippet: ( A ) Ex vivo imaging showed biodistribution of 4-kDa FITC–conjugated dextran. ( B ) Confocal fluorescence imaging of brain tissue slices of mice after different treatments as indicated, scale bars, 50 μm. ( C ) Representative flow cytometric analysis of 4-kDa FITC–conjugated dextran signal and ( D ) corresponding quantification results of mean fluorescence intensity (MFI) of FITC. ( E ) Representative confocal images showing PV1 (red) detection in CD31 + (purple) blood vessels and ZO-1 (green) in lung of mice after different treatments as indicated, scale bars, 10 μm, and ( F, G ) corresponding quantitative analysis. ( H ) Representative confocal images showing PV1 (red) detection in CD31 + (purple) blood vessels and ZO-1 (green) in brain of mice after different treatments as indicated, scale bars, 10 μm, and ( I , J ) corresponding quantitative analysis. Data are shown as mean ± SD (n=3-5). Statistical significance was calculated by Student’s t-test (two-tailed) and one-way ANOVA using the Tukey posttest. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s.; nonsignificant. a. u., arbitrary units.

Article Snippet: 500 ug of 4 kDa FITC dextran (MCE) was injected into the tail vein of mice.

Techniques: Ex Vivo, Imaging, Fluorescence, Two Tailed Test