Journal: Neuro-Oncology Advances

Article Title: The brain-penetrant cell-cycle inhibitor p28 sensitizes brain metastases to DNA-damaging agents

doi: 10.1093/noajnl/vdad042

Figure Lengend Snippet: p28 crosses the BBB and preferentially localizes to Brain metastases (BMs). (A) Confocal images of the penetration of normal and cancer cells by p28. Human cancer cell lines (MDA-231BR, BCA-1, Mel-7, and A549) and normal cells (MCF-10A and fibroblasts) were cultured with Alexa Fluor 568-labeled p28 at 37°C for 2 hours, and images were obtained by confocal microscopy. Red, p28; blue, DAPI (nucleus). (B) MDA-231BR brain-specific metastatic triple-negative breast cancer, BCA-1 breast cancer, Mel-7 melanoma, or A549 lung cancer cells were injected into the left cardiac ventricle of athymic mice. ICG-labeled p28 was intravenously injected into the mice. Near-infrared fluorescence imaging of the ICG-p28 signal (gray) in coronal brain sections (yellow dotted line on the anterior-dorsal view) of mice injected with MDA-231BR, BCA-1 or Mel-7 cells or in the anterior-dorsal view of the brain of mice injected with A549 cells. H&E staining of brain sections confirmed the presence of BMs (Tu).

Article Snippet: A549 human lung cancer cells, MCF10A, and fibroblasts were purchased from American Type Culture Collection.

Techniques: Cell Culture, Labeling, Confocal Microscopy, Injection, Fluorescence, Imaging, Staining

Journal: Redox Biology

Article Title: Identification of tyrosine brominated extracellular matrix proteins in normal and fibrotic lung tissues

doi: 10.1016/j.redox.2024.103102

Figure Lengend Snippet: PXDN and laminin expression are modulated in human lung fibroblasts (HLF) and bone-marrow-derived macrophages (BMDM) upon TGF-β1 stimulation. (A – C) HLFs were treated 5 ng/mL TGF-β1 for 48 h, and analyzed for LAMA1 (A) or PXDN (B) mRNA or PXDN protein expression (C). (D – G) BMDM from C57BL6/NJ mice were treated 5 ng/mL TGF-β1 for 48 h, and analyzed for Pxdn (D) , Lama1 (E) , or Lamb1 (F) mRNA, or Laminin α/β1 protein expression by immunofluorescence imaging (G). Scale bar: 50 μm. Values are the mean of at least three independent biological replicates ± SEM. Differences among groups were evaluated by Student's T-test.

Article Snippet: HLF (ATCC PCS-201-013) were cultured in fibroblast basal medium (ATCC PCS-201-030) with fibroblast growth kit-low serum (ATCC PCS-201-041) at 37 °C in a 5% CO 2 atmosphere.

Techniques: Expressing, Derivative Assay, Immunofluorescence, Imaging

Journal: Scientific Reports

Article Title: Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling

doi: 10.1038/s41598-018-21073-x

Figure Lengend Snippet: Hypoxia induces pulmonary fibroblast proliferation. Normal human pulmonary fibroblasts (HPFs) and LL29 IPF fibroblasts were exposed to normoxia (21% O 2 ) or hypoxia (5% or 1% O 2 ) for 2–6 days. ( A ) Bright field imaging. Scale bar: 100 µm. ( B ) Cell count. ( C , D ) Cell proliferation by BrdU assay. HPF and LL29 cells were incubated with BrdU for 3 hrs. Data were normalized to normoxia. The absorbance for BrdU at normoxia was 0.08 ± 0.002 (day 3) and 0.09 ± 0.009 (day 6) compared to 0.09 ± 0.004 (day 3) and 0.10 ± 0.006 (day 6) for HPF and LL29 cells, respectively. ( E , F ) Cell viability by MTT assay. The absorbance for MTT at normoxia was 0.23 ± 0.0796 (day 3) and 0.30 ± 0.091 (day 6) compared to 0.07 ± 0.016 (day 3) and 0.28 ± 0.097 (day 6) for HPF and LL29 cells, respectively. ( G ) Additional normal human pulmonary fibroblasts [HPF (with F12K medium), CCD-13Lu, CCD-19Lu, HPF153 and HLF154] and IPF fibroblasts (LL97A, IPF12 and IPF14) were subjected to normoxia and hypoxia (1% O 2 ) for 3 days, and cell proliferation assessed by BrdU assay. Cells were incubated with BrdU for 12 hrs. The absorbance values for BrdU at normoxia were 0.15 ± 0.006 (HPF with F12K medium), 0.09 ± 0.004 (CCD-13Lu), 0.18 ± 0.008 (CCD-19Lu), 0.28 ± 0.003 (HLF153), 0.14 ± 0.008 (HLF154), 0.11 ± 0.004 (LL97A), 0.05 ± 0.003 (IPF12) and 0.10 ± 0.01 (IPF14); values represent means ± SE. *p < 0.05, **p < 0.01, ***p < 0.001 vs. normoxia. n = 3 independent experiments.

Article Snippet: IPF fibroblasts LL29 and LL97A were isolated from a 26-year-old female Caucasian patient and a 48-year-old male Caucasian patient, respectively, and were purchased from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Imaging, Cell Counting, BrdU Staining, Incubation, MTT Assay

Journal: Journal of medicinal chemistry

Article Title: Discovery of 6-Phenylhexanamide Derivatives as Potent Stereoselective Mitofusin Activators for the Treatment of Mitochondrial Diseases

doi: 10.1021/acs.jmedchem.0c00366

Figure Lengend Snippet: Diastereomer-selective correction of mitochondrial fragmentation and depolarization in Mfn knockout cells for 13. A and B. Dose-response curves for 13 stereoisomers increasing mitochondrial aspect ratio in cells expressing only Mfn2 (A) or only Mfn1 (B). Prototype mitofusin activator 2 data are shown for comparison. C. Effects of compounds on mitochondrial inner membrane polarization in Mfn1 knockout (KO; top) and Mfn2 KO (bottom) cells. D. Representative confocal imaging of mitochondrial morphology and polarization status in Mfn2 null MEFs treated with different compounds (1 μM, 24 h). Green mitochondria are depolarized and have damaged respiratory function. Scale bars are 10 μm. MT Green (mitotracker green), TMRE (tetramethylrhodamine ethyl ester, red) and nuclear Hoescht (blue) were used for staining. Data are means ± SEM of three independent experiments.

Article Snippet: Functional evaluation of mitofusin agonist fusogenicity was performed in MFN1- or MFN2-deficient MEFs purchased from ATCC (Cat #: CRL-2992, CRL-2993) and cultured at 37°C, 5% CO 2 -95% air in Dulbecco’s minimal essential medium (DMEM) containing glucose (4.5 g/l) with 10% (v/v) fetal bovine serum (FBS; Gibco, 26140–079), 1x nonessential amino-acids (Gibco, 11130051), 2 mM L-glutamine (Corning, 34717007), and 1% (v/v) penicillin/streptomycin (Gibco, 15140–122).

Techniques: Knock-Out, Expressing, Imaging, Staining

Journal: bioRxiv

Article Title: Enhancing Drug Delivery with Supramolecular Amphiphilic Macrocycle Nanoparticles: Selective Targeting of CDK4/6 Inhibitor Palbociclib to Melanoma

doi: 10.1101/2023.11.21.567974

Figure Lengend Snippet: Confocal microscopy imaging of NR-MC1 and NR-MC2 NPs internalization in both M14 melanoma and HDFn fibroblast cell lines. The scale bar is 20 µm. Images in each row (left to right): CellMask (green) for staining cell membrane, NR-loaded MC NPs (red), overlapping (green-red), and bright field image (green-red).

Article Snippet: We also obtained RAW 264.7 mouse macrophages, U-87 MG, and adipose stromal cells, human dermal fibroblasts, neonatal (HDFn), human melanoma (Mel29.1) cells from the American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy, Imaging, Staining, Membrane

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet: Quantitative Temporal Analysis of VACV Infection (A) Schematic of experimental workflow for each of three biological replicates. Cells were infected at MOI of 5 or mock infected ( Figure S1 A). Additionally, one mock and one infected sample were treated for 6 h with the viral DNA replication inhibitor cytosine arabinoside (AraC). (B) Hierarchical cluster analysis of all proteins quantified. An enlargement of three subclusters is shown (right panel), including multiple proteins that were substantially up- or downregulated. (C) Scatterplot of all proteins quantified at 18 h of infection. For all analyses in this manuscript, a mean fold change at each time point was calculated by averaging fold changes from each of the biological replicates in which the protein was quantified. For the purposes of comparison, the 18-h mock sample from each replicate was used, because the 0-, 6-, and 18-h mock samples behaved extremely similarly ( Figure S1 E). To perform a comprehensive analysis, “sensitive” criteria were employed, examining proteins down- or upregulated >2-fold on average across all replicates in which the protein was quantified ( A). Sensitive criteria were used in each analysis apart from where indicated. Data from “stringent” criteria that examined only proteins quantified in all three replicates with an average fold change > 2 and p < 0.05 are shown in C. For proteins quantified in all three replicates, a Benjamini-Hochberg-corrected two-tailed t test was used to estimate p values. Only proteins quantified in all three replicates are shown in this scatterplot. (D) Example of a previously unreported target of VACV infection. Data are represented as mean ± SEM; ∗ p < 0.05 (see ). Immunoblot of HFFF-TERTs infected with VACV (MOI = 5) confirmed rapid HDAC5 downregulation.

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Infection, Comparison, Two Tailed Test, Western Blot

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet: VACV Protein C6 Downregulates HDAC5 (A) HDAC5, but not HDAC1, is proteasomally degraded during VACV infection. Bar charts and statistics were generated as described in Figure 5 . (B) Viral class centroid profiles compared to an inverted profile of HDAC5, which had additionally been scaled from 0 to 1. (C) Profile of HDAC5 scaled as in (B), and Tp2-class VACV proteins with known roles in regulation of IFN or ISGs. (D) C6 targets HDAC5. HFFF-TERTs were infected in biological triplicate with WT VACV or vΔC6 (lacking gene C6L ) ( Unterholzner et al., 2011 ) (MOI = 5 and 12 h). The scatterplot shows all proteins quantified. A Benjamini-Hochberg-corrected two-tailed t test was used to estimate p values. (E) Representative immunoblot demonstrating that C6 specifically targets HDAC5. By comparison, HDAC1 was unmodified (MOI = 5). Quantitation of all three replicate immunoblots are shown (right panel). Data are represented as mean ± SEM, ∗ p < 0.05 from a two-tailed t test. (F) Representative immunoblot demonstrating rescue of HDAC5 expression by inhibition of the proteasome (MOI = 5). MG132 was added 2 h after VACV infection. Quantitation of all three replicate immunoblots is shown (right panel). Data are represented as mean ± SEM, ∗∗ p < 0.00005 from a two-tailed t test. (G) Immunoblot demonstrating that inducible expression of C6 is sufficient for HDAC5 degradation. HEK293T cells inducibly expressing C6 with an N-terminal Myc or C-terminal TAP tag were either treated or untreated with 100 ng/ml doxycycline overnight then lysates immunoblotted.

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Infection, Generated, Two Tailed Test, Western Blot, Comparison, Quantitation Assay, Expressing, Inhibition

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet:

Article Snippet: Primary human fetal foreskin fibroblast cells immortalized with human telomerase (HFFF-TERTs, male), BS-C-1 (African green monkey cell line, ATCC CCL-26), HeLa (human cervical ATCC CCL-2,+ female) parental and HDAC5 −/− derivative cell lines, HEK293T (human embryo kidney epithelial cell line, ATCC CRL-11268, female) parental and HDAC5 −/− derivative cell lines and human bone osteosarcoma epithelial (U2OS, ATCC HTB-96, female) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS: 10% v/v), and penicillin/streptomycin at 37°C in 5% CO 2 .

Techniques: Virus, Western Blot, Derivative Assay, Subcloning, Recombinant, Protease Inhibitor, Electron Microscopy, Bicinchoninic Acid Protein Assay, TA Cloning, Sequencing, Mass Spectrometry, CRISPR, Disruption, Plasmid Preparation, Software, Quantitative Proteomics, Imaging, Flow Cytometry, Fluorescence, Microscopy

Journal: European journal of nuclear medicine and molecular imaging

Article Title: Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy.

doi: 10.1007/s00259-022-05842-5

Figure Lengend Snippet: Fig. 3 Correlation of FAP expression analysis by immunohistochem- istry and autoradiography using 111In-FAP-2286. Representative images of patient and mouse xenograft tumors are shown by autora- diography (left, 500 µm) and immunohistochemistry (right, 100 μm) (A). FAP levels by autoradiography demonstrate correlation to immu- nohistochemistry in patient cholangiocarcinoma and sarcoma tumor sections (Pearson correlation coefficient r = 0.79, P = 0.002) (B)

Article Snippet: In vitro assays Surface plasmon resonance assay (SPR) The binding kinetics of FAP-2286 to antibody-immobilized human FAP (Sino Biological) or mouse FAP (R&D Systems) was measured by SPR and calculated using singlecycle kinetic measurements.

Techniques: Expressing, Autoradiography, Immunohistochemistry

Journal: European journal of nuclear medicine and molecular imaging

Article Title: Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy.

doi: 10.1007/s00259-022-05842-5

Figure Lengend Snippet: Fig. 4 Imaging of HEK-FAP tumor-bearing mice with 111In- FAP-2286. SPECT images of one representative mouse at 5 different timepoints are shown (A). Quantification of 111In- FAP-2286 uptake as mean ± SD %ID/g (n = 9) in tumor, liver, kidney, and blood pool surro- gate at various timepoints after injection (B)

Article Snippet: In vitro assays Surface plasmon resonance assay (SPR) The binding kinetics of FAP-2286 to antibody-immobilized human FAP (Sino Biological) or mouse FAP (R&D Systems) was measured by SPR and calculated using singlecycle kinetic measurements.

Techniques: Imaging, Single Photon Emission Computed Tomography, Injection

Journal: PLoS ONE

Article Title: Cross-linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model

doi: 10.1371/journal.pone.0241091

Figure Lengend Snippet: a) Workflow schematics: skinfold chambers were installed on NSG mice (day -2). 48 hours later (day 0): WSU DLCL2 (blue), unstained CT26 cells, and CD2 + T cells (pink) freshly purified from human PBMCs or from HSC-NSG mice were injected intra-dermally in the skinfold chamber together with labeled CD20-TCB (0.5 mg/kg) or with suitable vehicle. Cells were imaged 2 hours post treatment by MP-IVM. Adapted from https://smart.servier.com/ . b) 3D representative rendering of MP-IVM imaging on skinfold chamber of HSC-NSG-NSG mice showing localization of therapy (white) at the contact site between WSU DLCL2 cells (blue) and T cells (pink), 2 hours post treatment. c) MP-IVM analysis of T cells tracks in the skin fold chamber of PBMC-NSG (top) vs HSC-NSG-NSG mice (bottom), +/- CD20-TCB. T cell tracks are plotted according to their displacement in the X and Y axes. Total number of tracks for each plot is: Top left: Vehicle n = 330. Top right: CD20-TCB n = 759. Bottom left: Vehicle n = 741. Bottom right: CD20-TCB n = 185. d-e) Quantification of (d) Track Speed (μm/min) and (e) Track displacement (μm) of T cells in PBMC-NSG or HSC-NSG-NSG mice, +/- CD20-TCB. Shown in yellow are mean values +/- s.d. Unpaired t-test; ****p<0.0001; n.s.: not significant.

Article Snippet: CT26 cells ( Mus Musculus colon carcinoma) were originally obtained from ATCC and after expansion deposited in the Roche Glycart internal cell bank, and were cultured in RPMI containing 10% FCS and 1% Glutamax.

Techniques: Purification, Injection, Labeling, Imaging

Journal: PLoS ONE

Article Title: Cross-linking of T cell to B cell lymphoma by the T cell bispecific antibody CD20-TCB induces IFNγ/CXCL10-dependent peripheral T cell recruitment in humanized murine model

doi: 10.1371/journal.pone.0241091

Figure Lengend Snippet: a-c) Top: Representative histological staining of WSU DLCL2 tumors 24h post second treatment (0.5 mg/kg CD20-TCB or suitable vehicle i.v.). Bottom: Quantification of total number of cells/mm 2 from histological images of vehicle vs CD20-TCB treatment. Whole slide scans quantification of 4 μm FFPE sections with the software (a) Definiens; (b-c) Halo. Statistical analysis: Unpaired 2-tailed t-test with Welch’s correction. *p<0.05, **p<0.005 (a) Red: CD3 staining, brown: CD31 staining. Quantification: Number of CD3 + cells b) red: Ki67, yellow: CD3, blue: DAPI. Quantification: Number of CD3 + Ki67 + cells c) Red: CXCR3, yellow: CD3, Blue: DAPI. Quantification of CD3 + CXCR3 + T cells. d) Percentage of proliferating CD8 + T cells, as assessed by CFSE dilution, freshly purified from PBMCs. Proliferation has been evaluated at 24h, 48h and 72h post CD20-TCB treatment, at the indicated doses, in the presence of WSU DLCL2 cells as target. n = 3 per group, mean and s.d. are shown. One-way Anova, *p<0.05, **p<0.005, ****p<0.0001. e) Workflow schematics: Skinfold chamber were installed on NSG mice. 48h later, WSU DLCL2 (Blue), unstained CT26 cells, and CD2 + T cells freshly purified from HSC-NSG spleens (pink) were injected intra-dermally in the skinfold chamber, together with 0.25 mg/kg of CD20-TCB or with suitable vehicle. Concomitantly, freshly purified CD2 + T cells from HSC-NSG spleens (orange) were injected i.v. to allow visualization of peripheral blood T cells. Cells were imaged 72h post treatment by MP-IVM. f) Representative MP-IVM imaging of the tumors. Blue: WSU DLCL2 cells; Pink: Resident T cells; Orange: Recruited T cells. Images were acquired 72h post intradermal treatment with 0.25 mg/kg CD20-TCB or suitable vehicle. Adapted from https://smart.servier.com/ g) Quantification of peripheral T cells (number/mm 2 ) 72h post treatment. Mean +/- s.d. are shown. Unpaired 2-tailed t-test with Welch’s correction. **p<0.005. h) In the context of the skinfold chamber model, increasing number of T cells (Resident) were co-injected with the tumor and 0.25 mg/kg of CD20-TCB intradermally, while 2.5*10 6 T cells were injected intravenously (Peripheral). 72h post treatment, peripheral blood T cells were counted for each tumor from 5 representative fields. 4 tumors per group were analyzed. Shown is the count of peripheral T cells/mm 2 , Mean +/- s.d. per group. Statistical analysis: One-way Anova. **** p<0.0001. i) 3 hours in vitro chemotaxis assay of T cells toward preconditioned medium derived from WSU DLCL2 co-culture with CD3/CD28 pre-activated T cells. Pre-activated CD8 T cells have been plated with WSU DLCL2 cells at decreasing T cells: Tumor cells ratios, in the presence of 200 ng/ml of CD20-TCB. 24h later the supernatant has been collected and transferred to the bottom chamber of a 24-Transwell plate. In the top chamber 100.000 pre activated T cells, labeled with CFSE, have been seeded and let to migrate for 3 hours. Migration has been evaluated by counting total amount of CFSE positive migrated cells in the bottom chamber, by flow cytometry at constant volume and acquisition speed. Mean fold change and +/- s.d. are shown. n = 5, from two independent experiments 2-way Anova; **p<0.005.

Article Snippet: CT26 cells ( Mus Musculus colon carcinoma) were originally obtained from ATCC and after expansion deposited in the Roche Glycart internal cell bank, and were cultured in RPMI containing 10% FCS and 1% Glutamax.

Techniques: Staining, Software, Purification, Injection, Imaging, In Vitro, Chemotaxis Assay, Derivative Assay, Co-Culture Assay, Labeling, Migration, Flow Cytometry