Journal: Frontiers in Microbiology

Article Title: Identification of a small molecule 0390 as a potent antimicrobial agent to combat antibiotic-resistant Escherichia coli

doi: 10.3389/fmicb.2022.1078318

Figure Lengend Snippet: Antimicrobial effects of 0390 against Escherichia coli . (A) Workflow of the in vitro high-throughput screening assay of the MINI Scaffold library, containing 5,033 MINI molecules. The OD value was determined by a microplate spectrophotometer. The structural formula and characteristics of a hit (hit compound), 0390 (6056–0390) were shown. H_acceptor: hydrogen bond acceptor count. H_donor: hydrogen bond donor count. B_rotN: rotatable bond count. LogP: oil- water partition coefficient. (B) Growth of E. coli ATCC 25922 after exposure to different concentrations of 0390 for 18 h. The experiment was repeated three times.

Article Snippet: To identify the antibacterial effects, 5,033 unique MINI Scaffold molecules, the molecules in MINI Scaffold Library (TopScience, L5600), were used to combat against standard strains E. coli ATCC 25922.

Techniques: In Vitro, High Throughput Screening Assay, Screening Assay, Spectrophotometry

Journal: Frontiers in Microbiology

Article Title: Identification of a small molecule 0390 as a potent antimicrobial agent to combat antibiotic-resistant Escherichia coli

doi: 10.3389/fmicb.2022.1078318

Figure Lengend Snippet: Morphologic and microstructural changes of Escherichia coli after being treated with 0390 for 1 h. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of (A) untreated, or (B) 32 μg/ml of 0390 treated E. coli ATCC 25922. Scales for SEM: 5 μm; for TEM: 1 μm.

Article Snippet: To identify the antibacterial effects, 5,033 unique MINI Scaffold molecules, the molecules in MINI Scaffold Library (TopScience, L5600), were used to combat against standard strains E. coli ATCC 25922.

Techniques: Electron Microscopy, Transmission Assay

Journal: Frontiers in Microbiology

Article Title: Identification of a small molecule 0390 as a potent antimicrobial agent to combat antibiotic-resistant Escherichia coli

doi: 10.3389/fmicb.2022.1078318

Figure Lengend Snippet: Combinational bacteriostatic effects between 0390 and SPR741 against Escherichia coli ATCC 25922. (A) Synergistic antimicrobial effects between 0390 and SPR741 by using drug combination assays. (B) Calculation of the fractional inhibitory concentration index (FICI). The red circle indicates the optimal FICI. (C) The time-growth inhibition curves and (D) time-killing curves of E. coli in the presence of 0390 (1/4 × MIC) and SPR741 (1/8 × MIC) alone or in combination. The experiment was repeated three times. (E) Viable cells visualization detected by SYTO 9 and PI staining, with treatment of 0390 (1/4 × MIC) and SPR741 (1/8 × MIC) alone or in combination. Green indicates live cells and red indicates dead cells. Scale bar: 20 μm. (F) Fluorescence intensity analysis of PI-stained cells was carried out by ImageJ software. 100% intensity = the intensity of SYTO9 + the intensity of PI. *** p < 0.001, **** p < 0.0001.

Article Snippet: To identify the antibacterial effects, 5,033 unique MINI Scaffold molecules, the molecules in MINI Scaffold Library (TopScience, L5600), were used to combat against standard strains E. coli ATCC 25922.

Techniques: Concentration Assay, Inhibition, Staining, Fluorescence, Software

Journal: Frontiers in Microbiology

Article Title: Identification of a small molecule 0390 as a potent antimicrobial agent to combat antibiotic-resistant Escherichia coli

doi: 10.3389/fmicb.2022.1078318

Figure Lengend Snippet: Synergistic antimicrobial activity of 0390 and SPR741 combination against Escherichia coli XDR strains. (A) Results of the drug combination assays of four E. coli XDR strains indicate synergism between 0390 and SPR741. (B) Red circles indicate the optimal FICIs. (C) Time-growth curves of the XDR strains treated with 0390 and SPR741 alone or in combination (64 μg/ml 0390 + 8 μg/ml SPR741 for Y0064; 16 μg/ml 0390 + 16 μg/ml SPR741 for Y9592; 16 μg/ml 0390 + 16 μg/ml SPR741 for Y9395; and 16 μg/ml 0390 + 16 μg/ml SPR741 for Y9633, respectively). The experiment was repeated three times.

Article Snippet: To identify the antibacterial effects, 5,033 unique MINI Scaffold molecules, the molecules in MINI Scaffold Library (TopScience, L5600), were used to combat against standard strains E. coli ATCC 25922.

Techniques: Activity Assay

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 1. NPC characterization. A) Phase contrast microscopy of a-synuclein gene triplication (SNCA-Tri), control (Ctrl) and a-synuclein knockdown (SNCA-Tri KD) iPSC-derived NPC lines (Scale bar: 50 mm) shows normal cell morphology. B) Mitochondrial and nuclear morphology of NPCs visualized by fluorescence microscopy using Mitotracker Red CMX Ros (red) and Hoechst 33342 (blue) (Scale bar: 10 mm). C) Stem cell marker expression. Immuno-cytochemistry on fixed NPCs detecting cytoplasmic Nestin expression pattern with secondary Alexa 588 conjugated antibody (orange) by fluorescence microscopy (Scale bar: 100 mm). Insert: Immuno-cytochemistry for the nuclear stem cell marker SOX1, detected by

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Microscopy, Control, Knockdown, Derivative Assay, Fluorescence, Marker, Expressing, Immunocytochemistry

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 2. NPC viability. A) Cell cycle analysis by propidium-iodine (PI) staining and flow cytometry analysis of Ctrl and SNCA-Tri NPCs with staining grouped by cell cycle phase (G0/1, S and G2/M), showing a reduced percentage of SNCA-Tri NPCs in the S phase (n = 3, mean 6 SD, *p = 0.047). B) Survival under nutritional and toxicant stress. NPCs propagated in medium without glucose (NG) untreated or treated with 20 mM rotenone (R) or 20 mM paraquat (PQ). Survival curves (every 12 hours) for the Ctrl, SNCA-Tri and SNCA-Tri KD cell lines after analysis of adherent cell count (ImageJ). Percentage of surviving cells with time (hrs) (n = 3, mean 6 SEM). C) Cell viability assayed by plate reader based high throughput screen (HTS) of NPCs untreated (HG), treated with 20 mM rotenone (HG+R) or without glucose (NG) for 18 hrs. Live cells were stained with 1 mM of the RedOx indicator C12-Resazurin/Alamar Blue for 15 min before analysis. Graphed are endpoint fluorescence units (RFU) normalized to total cellular protein/well (ug protein) (n = 3, mean 6 SEM, *p#0.05). D) Cell viability assayed by flow cytometry evaluation of apoptosis and cell death in live NPCs treated as under A). Cells stained with C12-Resazurin for cell viability and with Sytox-Green. Graphed are percentages of metabolic active NPCs, determined by Resarufin (Ex./Em. 563/587 nm) fluorescence (viable), apoptotic cells (cell membrane asymmetry detected by an Annexin- V Alexa-660 nm conjugated antibody) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, *p = 0.027) or cell death (nuclear fragmentation, detected by Sytox-Green, Ex./Em. 488/530 nm) (n = 3, mean 6 SD, Ctrl/SNCA-Tri: 5.3%/24.4%, **p = 0.004). doi:10.1371/journal.pone.0112413.g002

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Cell Cycle Assay, Staining, Flow Cytometry, Cell Counting, High Throughput Screening Assay, Fluorescence, Membrane

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 3. Mitochondrial membrane potential (MMP) and energy balance. A) Fluorescence microscopy of MMP in live NPCs from patient (SNCA-Tri) and control (Ctrl) loaded with 100 nM TMRM in normal growth medium (HG), medium plus 20 mM Rotenone (HG+R) or with 1 mM of the ionophore CCCP (HG+CCCP) as negative control (Scale bar: 10 mm). B) Plate reader based high throughput screen (HTS) of MMP in live NPCs loaded with 20 mM JC-10 for 45 min. Cells were also treated with medium w/o glucose (NG). Shown are log ratios of reduced (Ex./Em. 540 nm/590 nm) to oxidized JC-10 (Ex./Em. 488 nm/520 nm) normalized to Hoechst 33342 (Log Norm. JC-10 Ratio) after 60 min. (n = 8, mean 6 SEM, Ctrl/SNCA-

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Membrane, Fluorescence, Microscopy, Control, Negative Control, High Throughput Screening Assay

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 4. Protein biosynthesis and proteasome function. A) Mitochondrial protein biosynthesis and protein import. Fluorescent protein expression patterns in confluent adherent NPC cultures (PC: Phase Contrast) transduced with two baculoviral vectors expressing fluorescent proteins targeted to either the peroxisomal (Perox.; Green) or the mitochondrial (Mito.; Red) compartment. Shown are fluorescent protein expression patterns in live confluent Ctrl and SNCA-Tri cell lines grown under normal growth conditions (HG) and evaluated 20 hrs post transduction (Scale bar: 200 mm, 5 mm). B) Time resolved peroxisomal and mitochondrial protein biosynthesis. Fluorescent protein expression patterns as under A), but imaged at 8 and 18 hrs post viral transduction. C) Proteasome activity measured by fluorescence microscopy of adherent NPCs cultured with 20 mM rotenone alone or with 10 mM of the proteasome inhibitor MG132. Depicted are fixed cells stained with 5 mM of the aggresome/ proteasome specific dye Bodipy TMR-AHX3L3VS (red). Hoechst 33342 was used as nuclear counter stain (blue) (Scale bar: 20 mm). D) Proteasome activity measured by flow cytometry evaluation of cells treated and stained as under B). Charted are the aggresome propensity factors (APF) of NPCs calculated from the mean RFU (MRFU) of Bodipy-TMR fluorescence (APF = 1006[MRFU MG132 treated2MRFU untreated]/MRFU MG132 treated (n = 3, mean 6 SD, APF Ctrl/SNCA-Tri: 51/120, *p = 0.041). doi:10.1371/journal.pone.0112413.g004

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Expressing, Transduction, Activity Assay, Fluorescence, Microscopy, Cell Culture, Staining, Flow Cytometry

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 5. Reactive oxygen species (ROS) production. A) Fluorescence microscopy of live adherent NPCs untreated (HG) or treated with 100 mM TBHP (HG+TBHP), loaded with CM-H2DCFDA and imaged under controlled exposure conditions (10 sec fluorescent light exposure before image acquisition). Hoechst 33342 was used as counter stain (Scale bar: 20 mm). B) Plate reader based HTS of ROS levels in adherent NPC in 96- well plates and treated as under A). Relative CM-H2DCFDA fluorescence intensities (RFU) were normalized to Hoechst 33342 (H33342) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.5/1/0.75, HG+R: 0.7/1.3/0.6, NG: 0.4/1.1/0.7, *p#0.046, **p#0.009, ***#0.001). C) ROS production rates by HTS plate reader analysis of CM-H2DCFDA fluorescence development over time (D RFU CM-H2DCFDA/sec + H33342) in cells exposed to TBHP as under A), measured with normal medium (HG) with or without rotenone (R) and in medium without glucose (NG) (n = 12, mean 6 SEM, Ctrl/SNCA-Tri/ SNCA-Tri KD: HG: 22/75/68, HG+R: 177/367/178, NG: 80/353/184, *p#0.010, **p#0.007, ***p#0.001). D) Mitochondrial superoxide production rates assayed by HTS plate reader analysis of the mitochondrial targeted fluorescent superoxide indicator MitoSOX. Depicted are changes in relative fluorescence units normalized to H33342) (D RFU MitoSOX/min + H33342) (n = 4, mean 6 SD, Ctrl/SNCA-Tri/SNCA-Tri KD: HG: 0.28/1.2/0.3, HG+R: 2.1/ 5.5/3.7, NG: 2.3/5.2/0.8,*p#0.038, **p#0.007). doi:10.1371/journal.pone.0112413.g005

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Fluorescence, Microscopy, Staining

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 6. Mitochondrial integrity, MPT opening, and apoptosis. A) Mitochondrial calcein loading by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl2 were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 mM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean 6 SD, Ctrl/SNCA-Tri: 3.4/4.9, *p = 0.039). B) MPT-induced mitochondrial calcein loss in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl2 were assayed 1 hr. after treatment with 4 mM staurosporine under NG conditions. MPT opening results in entry of CoCl2 into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 mm). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 mm). C) HCI automated fluorescence microscopy analysis of MPT in NPCs treated with 4 mM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean 6 SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, *p#0.01). D) Kinetic evaluation of MPT opening and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Negative Control, Fluorescence, Microscopy, Staining, Software

Journal: PloS one

Article Title: Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

doi: 10.1371/journal.pone.0112413

Figure Lengend Snippet: Figure 7. Apoptosis sensitivity and caspase activation. A) Caspase 3 activity in cell lysates from adherent NPCs either left untreated or treated with 20 mM rotenone (R) for 18 hrs and then exposed to 1 uM staurosporine for 120 min before analysis. HTS analysis for caspase 3 activity from cell lysates was by activation of the fluorescent caspase substrate 7-amino-4-methylcoumarin (AMC) (Ex./Em. 340/440 nm) (n = 9, mean 6 SEM, Ctrl/SNCA-Tri/SNCA-Tri KD, HG: 33/69/42, HG+R: 42/129/87, NG: 55/138/85, *p#0.050, **p#0.0035; from three independent experiments). B) Kinetics of caspase 3/7 activity in permeabilized NPCs pretreated as described under B) and assayed 15 min after staurosporine treatment. Changes in caspase 3 activity are depicted as DmM AMC fluorescence/min + mg cellular protein (detected by Bradford protein assay) (n = 9, mean 6 SEM). doi:10.1371/journal.pone.0112413.g007

Article Snippet: High content imaging (HCI) and high throughput screening (HTS) Conventional fluorescence microscopy (Nikon Eclipse Ti, Nikon Planfluor Objectives 10x/.03, 40x/0.75, 60x ELWD/0.7; Chroma 4900 series filtersets: ET-DAPI, -GFP/FITC, -CY3, -mCherry/ Texas Red) was confirmed/validated by high throughput/content screening.

Techniques: Activation Assay, Activity Assay, Fluorescence, Bradford Protein Assay

Journal: Nature chemical biology

Article Title: A small molecule mitigates hearing loss in a mouse model of Usher syndrome III

doi: 10.1038/nchembio.2069

Figure Lengend Snippet: (a) Inhibition of proteasomes by bortezomib increased CLRN1 N48K levels in a D6 cell line stably expressing CLRN1 N48K mRNA. CLRN1 N48K was tagged with an HA epitope that was detected by immunofluorescence microscopy. (b) Cell-containing areas were segmented to measure relative concentrations of CLRN1 N48K . Cells were outlined in the top image and colored in the bottom image. (c) Approximately 50,000 compounds were tested by high-throughput screening for stabilization of CLRN1 N48K , and the measured efficacies of these compounds were normalized to 25 nM bortezomib assayed on the same plate. The top 320 compounds (blue) were selected for further analysis. (d) The top 320 compounds were subjected to the same assay 6 times. Of these, 90 compounds with highest average percentage (%) activities are shown. Among them, 48 compounds (black) were selected for secondary screening but 42 compounds (grey) were eliminated due to unfavorable properties such as autofluorescence, the formation of dye-like structures, or chemical structures unsuitable for further pharmaceutical development , . Data on the y-axis are presented as means ± SEMs (n = 6). Compounds O03, B03, M01, and K01 are labeled. Scale bars = 50 μm.

Article Snippet: RNA (5 μg) was treated with DNase I, and first strand cDNA was synthesized using M-MLV reverse transcriptase (Life Technologies, Grand Island, NY, #28025-013) with an oligo (dT) primer mixture containing 10 μM each of dT12, dT15, and dT18. cDNA was subjected to real-time PCR using the TaqMan Fast Universal Mix system (Life Technologies #4352042) and a probe for human CLRN1 (Life Technologies, Hs00602425_m1).

Techniques: Inhibition, Stable Transfection, Expressing, Immunofluorescence, Microscopy, High Throughput Screening Assay, Labeling

Journal: Nature chemical biology

Article Title: A small molecule mitigates hearing loss in a mouse model of Usher syndrome III

doi: 10.1038/nchembio.2069

Figure Lengend Snippet: (a) Cells were engineered to co-express human CLRN1 N48K fused to Venus fluorescent protein (green) and DsRed-Express-DR (magenta). CLRN1 N48K -Venus and DsRed-Express-DR are both degraded by proteasome (top row). Thus, a proteasome inhibitor will cause both increased Venus fluorescence and DsRed fluorescence (middle row). A molecule specifically stabilizing CLRN1 N48K will cause increased Venus fluorescence but will minimally affect DsRed fluorescence (bottom row). (b) Dual-color assays conducted for 0 nM bortezomib (no treatment), 50 nM bortezomib, 16.8 μM K01, and 16.8 μM O03. O03 specifically increased CLRN1 N48K -Venus as compared to bortezomib and K01 which increased both Venus and DsRed. Scale bar = 50 μm. (c) Fluorescence intensities from Venus and DsRed normalized to intensity values obtained for 50 nM bortezomib. The specific and most significant increase in fluorescence with characteristics of Venus was observed with compounds B03, M01, and O03 (*** P = 1.22 × 10 −5 , ** P = 3.70 ×10 −4 , * P = 2.27 × 10 −3 , respectively, compared to no treatment; two-sided t-test). Results are expressed as means ± SDs (all images are taken in triplicate and 15 different areas from each replicate were picked for quantification). (d) Immunoblotting analysis of human CLRN1 N48K tagged with an HA epitope. Cells were treated with either bortezomib (50 nM), B03 (16.8 and 1.68 μM) or O03 (16.8 μM). CLRN1 N48K expression levels increased dramatically in cells treated with bortezomib and O03 but did not increase in cells treated with B03. Tubulin loading controls are indicated. See also .

Article Snippet: RNA (5 μg) was treated with DNase I, and first strand cDNA was synthesized using M-MLV reverse transcriptase (Life Technologies, Grand Island, NY, #28025-013) with an oligo (dT) primer mixture containing 10 μM each of dT12, dT15, and dT18. cDNA was subjected to real-time PCR using the TaqMan Fast Universal Mix system (Life Technologies #4352042) and a probe for human CLRN1 (Life Technologies, Hs00602425_m1).

Techniques: Fluorescence, Western Blot, Expressing

Journal: Nature chemical biology

Article Title: A small molecule mitigates hearing loss in a mouse model of Usher syndrome III

doi: 10.1038/nchembio.2069

Figure Lengend Snippet: (a) Mouse CLRN1 N48K expressed in NIH/3T3 cells was stabilized by O03 treatment. (b) Quantitative RT-PCR of HEK293 cells stably expressing human CLRN1 N48K . CLRN1 mRNA levels were not affected by either bortezomib or O03 treatment (O03 vs. DMSO, P = 0.9995, two-sided t-test, n=4, means ± SDs). (c) Protein synthesis is not required for the effect of O03. Cells treated with proteasome inhibitors bortezomib and MG132 showed increased levels of CLRN1 N48K as did O03 treatment when compared to DMSO (lanes 1–4, T0). CLRN1 N48K levels remained similar after treatment for an additional 6 h in the presence of 100 μM cycloheximide (CX) (lanes 5–7, T1), indicating that O03 stabilized CLRN1 N48K in the absence of protein synthesis. To confirm that CLRN1 N48K can be effectively degraded in the absence of protein synthesis, cells were treated with bortezomib, MG132, or O03 for 6 h. Compounds then were washed out, and cells were further incubated for 6 h in the presence of CX (lanes 8 – 10, T1). CLRN1 N48K was undetectable in cells after removing the reversible proteasome inhibitor MG132 (compare lanes 3 and 9). Effects of O03 and bortezomib persisted 6 h after the washout. Note: Multiple immunopositive bands were detected by the HA-antibody which recognizes the Ct-tail of CLRN1 N48K (a and c). Based on the analysis of the primary structure , CLRN1 contains a few signal peptide cleavage sites; incomplete cleavage leads to a few differently sized bands. See also .

Article Snippet: RNA (5 μg) was treated with DNase I, and first strand cDNA was synthesized using M-MLV reverse transcriptase (Life Technologies, Grand Island, NY, #28025-013) with an oligo (dT) primer mixture containing 10 μM each of dT12, dT15, and dT18. cDNA was subjected to real-time PCR using the TaqMan Fast Universal Mix system (Life Technologies #4352042) and a probe for human CLRN1 (Life Technologies, Hs00602425_m1).

Techniques: Quantitative RT-PCR, Stable Transfection, Expressing, Incubation

Journal: Nature chemical biology

Article Title: A small molecule mitigates hearing loss in a mouse model of Usher syndrome III

doi: 10.1038/nchembio.2069

Figure Lengend Snippet: (a) Chemical structures of the lead compound (1, O03), the optimized compounds (2, BF981; 3, BF844) and biotinylated compound (4, BF071). BF844, BF981 and BF071 share the same core structure and BF071’s biotin moiety was used for avidin affinity purification. (b) CLRN1 N48K does not bind to the core structure of BF071. BF071 was used for affinity purification (AP) of binding proteins from cell homogenates which contained CLRN1 N48K . As a negative control (Con), target proteins in cell homogenates were blocked with BF981 prior to incubation and affinity purification with BF071. L, whole cell lysates prior to purification; E, the fraction eluted from avidin agarose. (c) BF071 binding proteins were analyzed by SDS-PAGE; protein bands (arrows) at the sizes of ~ 60 and 90 kDa were enriched by BF071 (AP) as compared to the negative control (Con) for which samples were blocked with BF981 prior to affinity purification. Based on quantitative mass spectroscopy, HSP60 was enriched 28 times in position 1 and HSP90 was enriched 10 times in position 2, compared to the control. Positions of the protein molecular mass markers (in kDa) are indicated (lane M). See also .

Article Snippet: RNA (5 μg) was treated with DNase I, and first strand cDNA was synthesized using M-MLV reverse transcriptase (Life Technologies, Grand Island, NY, #28025-013) with an oligo (dT) primer mixture containing 10 μM each of dT12, dT15, and dT18. cDNA was subjected to real-time PCR using the TaqMan Fast Universal Mix system (Life Technologies #4352042) and a probe for human CLRN1 (Life Technologies, Hs00602425_m1).

Techniques: Avidin-Biotin Assay, Affinity Purification, Binding Assay, Negative Control, Incubation, Purification, SDS Page, Mass Spectrometry

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is an epithelial cytokine expressed in alveolar epithelium and airway basal cells in human healthy and asthmatic lungs. (A) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in the LungMAP single-cell human lung atlas. Uniform manifold projection (UMAP) plots show the clustering of 347,970 lung cells (10 single-cell datasets, 148 normal human lung samples from 104 donors: adult, child, and adolescent). Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B and C) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in epithelial cells from human healthy (B) and asthmatic (C) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (B; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (C; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas and downloaded from https://asthma.cellgeni.sanger.ac.uk .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Generated, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Single-cell RNA-seq analysis of IL33 and TSLP expression in human lungs and gating strategy for analysis of mouse lung epithelial cells by flow cytometry. (A and B) Single-cell RNA-seq analysis of IL33 and TSLP expression in epithelial cells from human healthy (A) and asthmatic (B) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (A; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (B; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas , and downloaded from https://asthma.cellgeni.sanger.ac.uk . (C) Gating strategy of Epcam + epithelial cells and CD31 + endothelial cells in the lung of a naïve WT mouse. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with two distinct rat IgG1 isotype controls (rat IgG1 clone eBRG1, D, red; rat IgG1 clone RB40.34, E, red) for the anti-TL1A antibody (rat IgG1, MAB7441, clone 293327). Double staining was performed with antibodies against RAGE (D, green) or IL-33 (E, green). Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Control, Immunohistofluorescence, Staining, Double Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is expressed in mouse alveolar epithelium at steady state. (A) Visualization of Tnfsf15 (TL1A) expressing cells in the LungMAP single-cell mouse lung atlas. UMAP plots show the clustering of 95,658 lung cells (17 samples from late developmental stage to postnatal day 28). The different cell types in the lungs of naïve mice are indicated on the left. Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B) Single-cell RNA-seq analysis of Tnfsf15/TL1A and Il33 gene expression in mouse lung epithelium. UMAP plots show clustering and cell type annotation of 12,536 mouse lung epithelial cells (seven samples from the emergence of the alveolus to postnatal day 28) . The number and percentage of epithelial cells expressing Tnfsf15/TL1A , Il33 , or both are indicated on the right. Results are visualized using ShinyCell and are based upon data obtained by and downloaded from http://www.lungmap.net . (C) Flow cytometry analysis of cell surface TL1A expression on live CD31 + CD45 − endothelial cells and Epcam + CD31 − CD45 − epithelial cells in the lung of a naïve wild type C57BL/6J mouse at steady state. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with antibodies against TL1A (D and E) and RAGE (D) or IL-33 (E) proteins. A tyramide signal amplification (TSA)-based immunofluorescence method was used to detect TL1A-expressing cells in situ. Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Expressing, Generated, RNA Sequencing, Gene Expression, Flow Cytometry, Immunohistofluorescence, Staining, Amplification, Immunofluorescence, In Situ

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: High throughput proteomic analyses of lung ILC2s stimulated ex vivo with IL-33 and/or TL1A. (A) Flow cytometry of cultured lung ILC2s ex vivo. Representative histograms of ST2, CD90.2, Sca-1, CD25, ICOS, KLRG1, and DR3 expression at the surface of cultured ILC2s, 3 days after ILC2 cell isolation from the lung and ex vivo culture in the presence of IL-2. Phenotypic analysis was performed on live Lin – CD45 + cells. (B–D) Large-scale label-free proteomic analyses of mouse lung ILC2s after ex vivo overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plots of IL-33-stimulated ILC2s (B) or TL1A-stimulated ILC2s (C) compared with non-stimulated cells (NS; in culture with IL-2 alone). Volcano plot of IL-33/TL1A-stimulated ILC2s compared to IL-33-stimulated cells (D). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS and IL33 stimulation; n = 3 for TL1A and IL33/TL1A stimulations), using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Representative examples of proteins found modulated in each comparison are shown in color. (E) Flow cytometry of cultured lung ILC2s after 14 h of co-stimulation with IL-33 and TL1A in the presence of IL-2 (ILC2 culture used in ). Intracellular cytokine staining revealed that >99% of ILC2s co-expressed IL-9 and IL-13 intracellularly. Phenotypic analysis was performed on live Lin − CD45 + CD90.2 + cells.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: High Throughput Screening Assay, Ex Vivo, Flow Cytometry, Cell Culture, Expressing, Cell Isolation, Quantitative Proteomics, Comparison, Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A synergizes with IL-33 to induce an IL-9-producing ILC9 phenotype in lung ILC2s. (A and B) Large-scale label-free proteomic analyses of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice and cultured with IL-2 prior to overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plot of IL-33/TL1A-stimulated ILC2s (ILC9 cells) compared with nonstimulated cells (NS; in culture with IL-2 alone) (A). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS; n = 3 for IL33/TL1A stimulation) using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Examples of proteins modulated in both IL-33/TL1A-stimulated ILC2s and IL-33-stimulated ILC2s are shown in blue. Proteins shown in red are representative of molecules specifically modulated in IL-33/TL1A-stimulated ILC2s (A). Heat-map of fold changes of selected proteins in three independent biological replicates (B). (C–K) Analysis of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice , and cultured with IL-2 prior to 14 h stimulation with rIL-2 ± rIL-33 ± rTL1A. Flow cytometry analysis of live Lin − CD45 + cells (C, E, and J), frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) (D and K), and MFI fold change of IL-9 in ILC2s (E), after cytokines treatment and restimulation by PMA, ionomycin, and brefeldin A (4 h, C–E) or brefeldin A (4 h, J and K). Concentration of IL-9 secreted by ILC2s, measured by ELISA (F). Relative STAT5 mRNA expression levels measured by real-time qPCR (G). Samples were normalized to the expression of HPRT and are shown relative to IL-2-stimulated ILC2s. Immunoblot analysis of activated phosphorylated STAT5 (pSTAT5) and α-tubulin (H) or β-actin (I); Arrowheads indicate the migration of the protein of interest; cropped images. Cultured ILC2s were treated with rIL-2 + rIL-33 + rTL1A and increasing doses of a STAT5 inhibitor (STA5i, CAS 285986-31-4) or control vehicle (DMSO) (I–K). Numbers inside outlined areas (C) indicate percent of cells in the relevant gate. Each symbol represents an individual biological replicate (D–G and K). Data are pooled from six (D and E), six to eight (F) or three (G and K) independent experiments, or are representative of six (C and E) or three (H–J) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (D–G and K): ns not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Isolation, Cell Culture, Quantitative Proteomics, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Migration, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s ex vivo. (A) Analysis of cultured lung ILC2s 14 h after ex vivo stimulation by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Flow cytometry analysis of live Lin − CD45 + cells and frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) after cytokine treatment and incubation with brefeldin A (4 h), without restimulation by PMA and ionomycin. Numbers inside outlined area indicate percent of cells in the relevant gate and data are representative of eight independent experiments. (B) Concentration of IL-9 secreted by ILC2s treated with rIL-2 (20 ng/ml) and various concentrations of rIL-33 and rTL1A measured by ELISA. (C and D) MFI of nuclear factor IRF4 (C) and flow cytometry (D) of ILC2s 14 h after ex vivo stimulation of cultured ILC2s by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Numbers inside outlined areas (D) indicate percent of cells in the relevant gate and data are representative of three independent experiments. (E) Immunoblot analysis of JunB and α-tubulin14 h after cytokine stimulation of lung ILC2s; Arrowheads indicate the migration of the protein of interest; cropped image. Data are representative of three independent experiments. (F–H) Relative mRNA expression levels by real time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed relative to IL-2-stimulated ILC2s (F) or relative to HPRT mRNA quantity (G and H). (I and J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-33 + rTL1A ± rIL-2 ± rIL-7 ± rTSLP. Frequency of IL-9 high ILC2s (Lin − CD45 + CD90.2 + cells), after cytokines treatment and re-stimulation by PMA, ionomycin and brefeldin A (4 h, I). Concentration of IL-9 secreted by ILC2s, measured by ELISA (J). (K) Concentration of IL-9 (ELISA) secreted by ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rIL-4 ± rTGF-β. Each symbol represents an individual biological replicates with n = 2–5 independent experiments (A–C and F–K). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (A, C, and F–J) or Dunnett’s (B and K) multiple-comparisons tests: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. In H, all significant P values are annotated with stars, all other comparisons are not significant. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Ex Vivo, Cell Culture, Flow Cytometry, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Migration, Expressing

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A induce phenotypic changes in cultured lung ILC2s at the protein and mRNA levels. (A–J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rTL1A. MFI of the indicated cell surface markers determined by flow cytometry (A, B, D, and E). Relative mRNA expression levels of various genes (C and F–I), including genes characteristic of ILC1s or ILC3s (I), determined by real-time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed as relative to HPRT mRNA quantity. Concentration of IL-5 or IL-13 in cell supernatants, measured by ELISA assay (J). Each symbol represents an individual biological replicate from independent experiments (A–J). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test (B, E, and J) or one-way ANOVA followed by Tukey’s multiple-comparisons test (A, C, D, and F–I): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. In I, all significant P values are annotated with stars, all other comparisons are not significant.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Cell Culture, Ex Vivo, Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A cooperates with IL-33 for induction of IL-9 high ILC2s in vivo. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B) Gating strategy of IL-9 high IL-5 + IL-13 + ILC2s. (C–I) Flow cytometry of IL-5 + IL-13 + ILC2s gated on live ILCs (Lin − CD45 + CD90.2 + cells) (C) and IL-9 high ILC2s gated on live IL-5 + IL-13 + ILC2s (E), frequency of lung IL-5 + IL-13 + ILC2s among live ILCs (D), IL-9 high ILC2s among live IL-5 + IL-13 + ILC2s (F), and IL-9 high IL-13 + ILC2s among live ILCs (G) or IL-9 high ILCs (H), and concentration of IL-9 in BAL fluids (ELISA assay, I) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate and data are representative of two independent experiments (C and E). Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (D) or Dunnett’s (F, G, and I) multiple-comparisons tests: ns, not significant, ** P < 0.01, **** P < 0.0001. (J) Frequency of lung eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells, at day 7 after a single i.n. exposure to rIL-33 or rIL-33 plus rTL1A. Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test: * P < 0.05. (K and L) Multiphoton imaging (K) and intravital microscopy (L) of whole lungs of INFER IL-9 fluorescent reporter mice, with detection of IL-9-eGFP + ILC2s (green) and staining of blood vessels (red) and collagen fibers (blue), 16–18 h after a single i.n. administration of IL-33/TL1A combination (1 μg rIL-33 plus 5 μg rTL1A). To increase the numbers of lung IL-9 high ILC2s accessible to in vivo imaging, the single i.n. exposure to IL-33/TL1A combination was performed after prior expansion of lung ILC2s by repeated i.p. injections of IL-33 (K and L). Multiphoton image (K) is a 3D reconstitution of stitched images (7 × 7 tiles and 181 z-stack). Time-lapse images (L) illustrate the migratory behavior of IL-9-eGFP + ILC2s. Time in h/min/s. Scale bars: K, 300 μm; L, 20 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Imaging, Intravital Microscopy, Staining, In Vivo Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s in vivo. (A) Gating strategy and representative flow cytometry plots of live lung ILCs (live Lin − CD45 + CD90.2 + cells), live lung IL-5 + IL-13 + ILC2s (live IL-5 + IL-13 + ILCs) and live lung IL-9 high ILC2s (live IL-9 high IL-5 + IL-13 + ILC2s) in vivo in wild type (WT) C57BL/6J mouse, 14 h after a single i.n. administration of rIL-33 (1 μg) and rTL1A (5 μg). (B) Verification of the absence of contamination of the IL-5 + IL-13 + ILC2s and IL-9 high ILC2s populations by TCR + cells (T cells and NKT cells) using anti-TCRβ and anti-TCRγδ antibodies. (C) Confirmation of the expression of IL-5 and IL-13 in live Lin − CD3/TCR − NK1.1 − CD45 + CD90.2 + lung ILCs using antibodies against CD3/TCR and NK1.1 with a different fluorescence from the Lin cocktail (CD4, CD19, CD45R, CD11b, CD11c, Ter119, Ly6G, FcεRI). (D and E) Frequency of lung IL-9 high Lin − cells among live CD45 + cells (D), and flow cytometry of IL-9 high IL-13 + ILC2s (live IL-9 high IL-13 + Lin − CD45 + CD90.2 + cells) (E) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate. (F) Frequency of lung IL-9 high Lin − cells among live CD45 + cells of WT mice pretreated with six daily i.p. injections of rIL-33 (days 1–6) prior to one i.n. injection of PBS or rIL-33 and/or rTL1A (day 7). Flow cytometry analyses were performed on day 8. (G) Frequency of IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αIL-2 mAb) or without (Iso, isotype control mAb) IL-2 blockade. (H and I) Analysis of IL-9 and TL1A release in BAL fluids by ELISA at different time points after the third exposure to A. alternata in a chronic exposure model (repeated i.n. administration of 12.5 μg A. alternata at days 0, 3, and 6). Each symbol represents an individual mouse and data are pooled from two (D and G) or three (F, H, and I) independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (G) or one-way ANOVA followed by Dunnett’s multiple-comparison test (D, F, H, and I): * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Expressing, Fluorescence, Injection, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s accumulate around blood vessels after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by multiphoton imaging in the cleared lung of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. 360° rotation of a 3D static representation at a frame rate of 25 fps (500 frames per 20 sec).

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s migrate along collagen fibers after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by lung intravital multiphoton imaging of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A functions as an epithelial alarmin rapidly released after allergen exposure. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B–F) Analysis of TL1A and IL-33 release in BAL fluids after a single allergen exposure. TL1A (B and E), IL-33 (C and F), and LDH (D) levels in BAL fluids were determined by ELISA (B, C, E, and F) or LDH (D) assays, 15 min (B–D) or at different time points (E and F) after a single i.n. administration of A. alternata extract (12.5 μg). Each symbol represents an individual mouse and data are pooled from two independent experiments (B–F). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (B–D) or Dunnett’s (E and F) multiple-comparisons tests: ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G–K) Analysis of TL1A release in cell supernatants after exposure of TL1A-expressing cells to A. alternata or bee venom phospholipase A2 (PLA2). U2OS epithelial cells transfected with a mouse TL1A-Flag expression vector (mTL1A-Flag vector) or control vector were analyzed by indirect immunofluorescence microscopy with anti-mTL1A and anti-Flag antibodies (G). Scale bar, 20 μm. TL1A (H and J) and LDH (I and K) levels in cell supernatants were determined by ELISA (H and J) or LDH cytotoxicity assays (I and K) 15 min after treatment with A. alternata extract ( A. alternata , H and I) or 1 h after treatment with bee venom PLA2 (J and K). NT, not treated. Each symbol represents an individual biological replicate and data are pooled from three independent experiments (H–K). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (treatment versus NT): ** P < 0.01, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Control, Immunofluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A is important for early induction of IL-9 high ILC2s after allergen exposure. (A) Treatment schedule of naïve WT mice. (B) IL-9 mRNA levels in the lungs analyzed by qPCR at different time points after a single allergen exposure. Data are expressed as relative to IL-9 mRNA levels in mice treated with PBS. (C–H) Flow cytometry and frequency of IL-9 high Lin − cells among live CD45 + cells (C and D) and IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) (E and F), flow cytometry (G), and MFI of IRF4 expression in ILC2s (H), in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αTL1A mAb) or without (Iso, isotype control mAb) TL1A blockade. Numbers inside outlined areas indicate the percent of cells in the relevant gate (C, E, and G) and data are representative of two (G) or three (C and E) independent experiments. Each symbol represents an individual mouse and data are pooled from three (D and F) or two (B and H) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (B) or unpaired two-tailed Student’s t tests (D, F, and H): ns, not significant, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: ILC9 cells have an increased capacity to initiate IL-5-dependent allergic airway inflammation. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice by a single i.v. adoptive cell transfer of classical IL-33-activated ILC2s (ILC2) or IL-33/TL1A-activated ILC2s (ILC9). (B–H) Flow cytometry (B and D) and frequency of eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells from BALF (C and F) or lung (E and G), and number of Red5 + ILC2s or ILC9s in total lung of mice (H), at day 7 after a single i.v. adoptive transfer of 5 × 10 5 ILC2s or ILC9s in separate host mice. Adoptively transferred ILC2s and ILC9s were prepared from Rag2 −/− mice ( Il5 +/+ cells) (B–E) or Red5 mice ( Il5 −/− cells) (F–H). Control mice received an intravenous injection of PBS. Red5 + cells indicate the activity of the Il5 promoter. Each symbol represents an individual mouse and data are representative (B and D) or pooled (C and E–H) from two independent experiments. (I–K) Live imaging of ILC2s and ILC9 cells in the lung. Lung intravital microscopy was performed 1–4 h after adoptive transfer of 6 × 10 5 of each cell type in the same host (green, classical IL-33-activated ILC2s-CFSE + ; red, IL-33/TL1A-activated ILC9 cells-CTO + ) (I). Imaging of the migratory behavior of ILC2s and ILC9 cells in the lung (J) and cell quantification from lung intravital microscopy data (K). Time-lapse images, 2 h after adoptive cell transfer (J). A maximum intensity projection of stitched images (2 × 2 tiles and 18 z-stack) is shown (K). Time in h/min/s. Scale bars: J, 20 μm; K, 100 μm. Lung intravital microscopy data are representative (J and K) or analyzed (K) from three adoptive transfer experiments on four mice. Data are expressed as mean (±SEM) with P values determined by paired two-tailed Student’s t test (K) or one-way ANOVA followed by Tukey’s multiple-comparisons test (C and E–H): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Adoptive Transfer Assay, Control, Injection, Activity Assay, Imaging, Intravital Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Adoptively transferred ILC2s and ILC9s are equally recruited to the lung and exhibit an ameboid-like mode of migration. IL-33-activated ILC2s (CFSE/green), IL33/TL1A-activated ILC9s (CTO/red), blood vessels (Evans Blue/dark blue), and collagen fibers (second harmonic generation/light blue) were observed by lung intravital multiphoton imaging 2 h after intravenous adoptive transfer (6 × 10 5 cells). Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Migration, Imaging, Adoptive Transfer Assay

Journal: Journal of Immunology Research

Article Title: Genetic Manipulation of Calcium Release-Activated Calcium Channel 1 Modulates the Multipotency of Human Cartilage-Derived Mesenchymal Stem Cells

doi: 10.1155/2019/7510214

Figure Lengend Snippet: Modulation of Ca 2+ in CRAC-manipulated MSCs. The following experiments were conducted at 7 days after gene transfection of wild-type MSCs, pcDNA3.1-Orai1-transfected MSCs (M1-MSCs), and CRACM1-specific gRNA vector and linear EF1a-GFP-P2A-Puro donor-cotransfected MSCs (KOM1-MSCs). (a) PCR amplification of reverse transcription products produced the expected band following genetic modification. Molecular marker (lane 1); CARCM1 expression (523 bp) in MSCs, M1-MSCs, and KOM1-MSCs (lanes 3, 4, and 5, respectively); and GAPDH expression (214 bp) in MSCs, M1-MSCs, and KOM1-MSCs (lanes 7, 8, and 9, respectively) are shown. (b) CRACM1 mRNA expression in MSCs, M1-MSCs, and KOM1-MSCs (a.u. (arbitrary units); ∗ P < 0.05 and ∗∗∗ P < 0.001). Results are expressed as mean ± SEM ( n = 4). (c) The relative expression of CRACM1 to housekeeping GAPDH in MSCs, M1-MSCs, and KOM1-MSCs using quantitative real-time PCR. Relative fold of CRACM1 expression was achieved using the comparative Ct method (2 -ΔΔCt ) ( ∗∗ P < 0.01 and ∗∗∗ P < 0.001). (d) Time sequential patterns of Ca 2+ imaging in single MSCs, M1-MSCs, and KOM1-MSCs. The imaging period was 200 s without stimulation, followed by 500 s after stimulation. After a 200 s baseline measurement, cells were slowly perfused with TG (0.5 μ M) and then perfused with a CaCl 2 solution (2 mM) at the 400 s time point (scale bar: 10 μ m). (e) Typical Ca 2+ influx patterns of MSCs, M1-MSCs, and KOM1-MSCs shown in (c). (f) Maximum increases in fluorescent intensity values of MSCs, M1-MSCs, and KOM1-MSCs. Quantification was performed using images acquired from 100–120 cells of each group ( ∗ P < 0.05). Results are expressed as mean ± SEM. (g) Initial rate of Ca 2+ influx (in the first 15 s after Ca 2+ addition) into MSCs, M1-MSCs, and KOM1-MSCs. Quantification was performed using images acquired from 100–120 cells of each group ( ∗ P < 0.05 and ∗∗ P < 0.01). Results are expressed as mean ± SEM.

Article Snippet: PcDNA3.1-Orai1 carrying CRACM1 was a gift from Anjana Rao (Addgene plasmid # 21638) [ ], which was transfected into MSCs using Lipofectamine® 3000 Reagent (Thermo Fisher Scientific K.K., Tokyo, Japan), according to the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Amplification, Reverse Transcription, Produced, Modification, Marker, Expressing, Real-time Polymerase Chain Reaction, Imaging

Journal: Journal of Immunology Research

Article Title: Genetic Manipulation of Calcium Release-Activated Calcium Channel 1 Modulates the Multipotency of Human Cartilage-Derived Mesenchymal Stem Cells

doi: 10.1155/2019/7510214

Figure Lengend Snippet: Overexpression of CRACM1 inhibits MSC differentiation to adipocytes. MSCs, M1-MSCs, and KOM1-MSCs were cultured in adipogenic differentiation medium for 7 days. Cells were then stained for mFABP (red) as a marker of adipocytes and counterstained with Hoechst® 33342 for nuclear staining (blue). (a) Typical images of MSCs, M1-MSCs, and KOM1-MSCs observed by fluorescence microscopy (×200; scale bar: 50 μ m). (b) Typical imaging screening panel for quantification of mFABP4 expression. MSCs, M1-MSCs, and KOM1-MSCs were seeded on 8-well plates, and 32 fields were captured in each well using a high-throughput image quantitation system. One of 32 fields is shown. Well number and average intensity are indicated on the image. (c) FABP expression in MSCs, M1-MSCs, and KOM1-MSCs. The average fluorescent intensity was obtained from 256 images for each group ( ∗ P < 0.05). Results are expressed as mean ± SEM. (d) Typical images of lipid droplets analysis. Lipid droplets, which were stained using Oil Red O, present as bright refractive round structures. (×400; scale bar: 25 μ m). (e) Relative positive area of lipid droplet analysis. The red-stained area was segmented from the background, and the relative positive area was quantified. More than four fields per section and an average of five sections from each sample were used for semiquantitative analysis ( ∗∗∗ P < 0.001). Results are expressed as mean ± SEM.

Article Snippet: PcDNA3.1-Orai1 carrying CRACM1 was a gift from Anjana Rao (Addgene plasmid # 21638) [ ], which was transfected into MSCs using Lipofectamine® 3000 Reagent (Thermo Fisher Scientific K.K., Tokyo, Japan), according to the manufacturer's instructions.

Techniques: Over Expression, Cell Culture, Staining, Marker, Fluorescence, Microscopy, Imaging, Expressing, High Throughput Screening Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A) Schematic illustration showing the generation of engineered decoy EVs at the cellular level. Producer cells are genetically modified to express cytokine receptors without the signalling domain fused to an EV sorting domain for efficient display of cytokine receptors on the surface of the secreted EVs (decoy EVs), which can decoy cytokines specifically. B) and C) Schematic illustrations of the evolution of cytokine receptors to facilitate EV surface display and assessment of various designs in a cytokine induced reporter cell system for high throughput screening of TNFR1 and IL6ST decoy EVs. D) and E) List of various TNFR1 or IL6ST sorting domain fusions assessed in the initial screen. F) Engineered decoy EVs displaying TNFR1 purified from HEK293T cells transfected with the constructs encoding the different display constructs (Figure1D) evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. Data were normalized to control cells treated with TNFα only (5 ng/ml). G) Engineered EVs displaying IL6ST purified from HEK293T cells transfected with constructs encoding the different display constructs evaluated for IL6/sIL6R decoy in an in vitro cell assay respondent to IL6/sIL6R induced STAT3 activation. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). F, G , Error bars, s.d. ( n = 3), **** P < 0.0001, *** P < 0.001, ** P < 0.01, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Genetically Modified, High Throughput Screening Assay, Purification, Transfection, Construct, In Vitro, Activation Assay, Control

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A) Schematic illustration showing the evolution of TNFR1 design by addition of trimerization domains to enhance loading and binding efficiency of the EV- displayed decoy receptors. B) and C) Systematic comparison of various TNFR1 designs with multimerization domains. Engineered EVs displaying TNFR1 purified from HEK293T cells transfected with constructs encoding TNFR1 multimerization sorting domain fusion proteins (as listed in ) evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. Data were normalized to control cells treated with TNFα only (5 ng/ml). D) Schematic illustration showing the evolution of IL6ST designs by addition of different multimerization domain to enhance loading and binding efficiency of displayed decoy receptors on EVs. E) and F) Engineered EVs displaying IL6ST purified from HEK293T cells transfected with constructs encoding IL6ST multimerization sorting domain fusion constructs (as listed in ) respectively evaluated for IL6/sIL6R decoy in an in vitro cell assay respondent to IL6/sIL6R induced STAT3 activation. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). B, C, E, F , Error bars, s.d. ( n = 3), **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Binding Assay, Comparison, Purification, Transfection, Construct, In Vitro, Activation Assay, Control

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A) Engineered EVs displaying IL6ST purified from MSC cells stably expressing the optimised IL6STΔ-LZ-NST display construct, evaluated for IL6/sIL6R decoy in an in vitro cell assay respondent to IL6/sIL6R induced STAT3 activation. EVs purified from MSC stably expressing Ctrl construct were used as control. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). B) Engineered EVs displaying TNFR1 purified from MSC cells stably expressing the optimized TNFR1ΔΔ-FDN-NST display construct, evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. EVs purified from MSC stably expressing Ctrl construct were used as control. Data were normalized to control cells treated with TNFα (5 ng/ml) treated cells. C) WB of MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and Ctrl cells and EVs indicating the presence of classical EV markers; ALIX (96 kDa), TSG101 (44 kDa) and absence of Calnexin (67 kDa) in the isolated EVs. The WB results further demonstrate the presence of respective His-tagged decoy receptors; TNFR1ΔΔ-FDN-NST (48 kDa) and IL6STΔ-LZ-NST (94 kDa) both on cells and EVs. D) Transmission electron microscopy of MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and Ctrl-EVs with nanogold labelled antibody staining of respective decoy receptor indicated by white arrows. E) Schematic illustration showing the workflow of the multiplex bead-based flow cytometry assay. Isolated EVs incubated with up to 39 different bead populations coated with different capture antibodies, which are distinguishable by flow cytometry due to their different fluorescence intensities. EVs captured by the different beads are detected with detection antibodies either against PAN (CD63-APC, CD81-APC and CD9-APC), mIL6ST-APC, or hTNFR1-APC. F-H) Characterization of EV surface protein composition by using F) anti-PAN (CD63, CD81 and CD9), G) anti-hTNFR1 and H) anti-mIL6ST detection antibodies in multiplex bead-based assays to confirm marker co-expression on MSC TNFR1ΔΔ-FDN-NST, IL6STΔ-LZ-NST and ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound to respective capture beads and upon using APC labelled detection antibody. A, B , Error bars, s.d. ( n = 3), **** P < 0.0001, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Purification, Stable Transfection, Expressing, Construct, In Vitro, Activation Assay, Control, Isolation, Transmission Assay, Electron Microscopy, Staining, Multiplex Assay, Flow Cytometry, Incubation, Fluorescence, Marker

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: Comparison of A) inhibitory dose response curves and B) calculated IC50 values (95% Confidence interval) for TNFα sequestration by HEK293T TNFR1ΔΔ-FDN-NST EVs and Etanercept. HEK293T NF-kB reporter cells were challenged with 10 ng/ml (1 ng) of TNFα along with increasing doses of either HEK 293T TNFR1ΔΔ-FDN-NST EVs or Etanercept. C) Effect of HEK293T TNFR1ΔΔ-FDN-NST EVs and IL6STΔ-LZ-NST EVs on TNFα levels in conditioned medium determined by ELISA at 6 hours post LPS stimulation of RAW 246.7 macrophages. Data were normalized to control cells treated with LPS only. D) Survival curve of LPS (15 mg/kg) induced systemic inflammation in mice treated with intravenous injection of either 1×10 HEK 293T TNFR1ΔΔ-FDN-NST EVs ( n =3) or 2×10 HEK293T IL6STΔ-LZ-NST EVs ( n =4) or PBS ( n =5) 3 hours post induction. E) Survival curve of LPS (15 mg/kg) induced systemic inflammation in mice treated with intravenous injection of either 1×10 MSC TNFR1ΔΔ-FDN-NST EVs or 1×10 MSC Ctrl EVs or 160 µ g Etanercept 3 hours post LPS induction. F) Percent relative bodyweight to initial bodyweight over time of mice induced with LPS (15 mg/kg). Mice were treated with intravenous injection of either 3.25×10 MSC IL6STΔ-LZ-NST EVs + 3.25×10 MSC TNFR1ΔΔ-FDN-NST EVs ( n =6), 6.5×10 MSC TNFR1ΔΔ-FDN-NST EVs ( n =6), 6.5×10 MSC IL6STΔ-LZ-NST EVs ( n =6), 6.5×10 MSC Ctrl EVs ( n =6), or PBS ( n =6) 3 hours post induction. A , Error bars, S.E.M ( n =3), **** P < 0.0001, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose. F , Error bars, S.E.M ( n = 6), **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective observation time.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Control, Injection

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A ) Description of the treatment protocol for TNFα decoy EVs in EAE. B) Clinical score (EAE-score, see Supplementary table 1) of disease progression over time and C) EAE-score at endpoint (day 16) in mice induced with EAE using MOG 35-55 peptide and treated with subcutaneous (S.C) administration of either 4×10 MSC TNFR1ΔΔ-FDN-NST EVs ( n =5), MSC Ctrl EVs ( n =5), or saline ( n =5) (on day 7, 10 & 13). D) Schematic description of treatment protocol for IL6 decoy EVs in EAE. E) Clinical score of disease progression over time and F ) EAE-score at endpoint (day 16) in mice induced with EAE using MOG 35-55 peptide and treated with intravenous (I.V) administration of either 5×10 MSC IL6STΔ-LZ-NST EVs ( n =5), MSC Ctrl EVs ( n =5), or saline ( n =6) (on day 1,3,5,7,8,9 & 11). G) Schematic description of treatment protocol for IL23 decoy EVs in EAE. H) Clinical score of disease progression over time and I) EAE-score at endpoint (day 16) in mice induced with EAE using MOG 35-55 peptide and treated I.V with either 1×10 HEK293TIL23B-LZ-NST EVs pre symptomatic ( n =5) (on day 5, 7 & 10), 6×10 HEK293TIL23B-LZ-NST EVs post symptomatic ( n =5) (on day 13), or saline ( n =6). C, F, I , Error bars, SEM *** P < 0.001, ** P < 0.01, * P < 0.05 statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response to mock treated animal.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Biomarker Discovery, Saline

Journal: bioRxiv

Article Title: Engineering of extracellular vesicles for display of protein biotherapeutics

doi: 10.1101/2020.06.14.149823

Figure Lengend Snippet: A) Imaging flow cytometry analysis (IFCM) with dot plots and example event images in the double positive (DP) gate of MSC TNFR1ΔΔ-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. PBS + antibodies were used for background adjustment and for determining the gating strategy. B) Percentage of detected events positive for either hTNFR1 or mIL6ST or both in Imaging flow cytometry analysis of MSC TNFR1ΔΔ-FDN-NST and MSC double decoy EVs stained with mIL6ST APC conjugated and hTNFR1 PE conjugated antibody. Percentage values determined from objects/ml in different gates. C) Transmission electron microscopy of double decoy EVs with nanogold labelled antibody staining of hTNFR1 (10 nm) and mIL6ST (5 nm). D) Multiplex EV surface characterization of PAN (CD63, CD81, and CD9) positive, hTNFR1 positive and mIL6ST positive population in MSC double decoy EVs and MSC Ctrl EVs. Data represented as background corrected median APC fluorescence intensity determined by flow cytometry of EVs bound different capture beads and upon using APC labelled detection antibody. E) Engineered EVs displaying TNFR1 purified from MSC cells stably expressing either the optimized TNFR1ΔΔ-FDN-NST display construct or TNFR1ΔΔ-FDN-NST and IL6STΔ-LZ-NST construct, evaluated for TNFα decoy in an in vitro cell assay responsive to TNFα induced NF-κB activation. EVs purified from MSC stably expressing either the IL6STΔ-LZ-NST display construct or Ctrl construct were used as control. Data were normalized to control cells treated with TNFα (5 ng/ml). F) Engineered EVs displaying IL6ST purified from MSC cells stably expressing either the optimized IL6STΔ-LZ-NST display construct or TNFR1ΔΔ-FDN-NST, evaluated for IL6/sIL6R decoy in an in vitro cell assay responsive to IL6/sIL6R induced STAT3 activation. EVs purified from MSC stably expressing either the TNFR1ΔΔ-FDN-NST display construct or Ctrl construct were used as control. Data were normalized to control cells treated with IL6/sIL6R (5 ng/ml). G) Schematic of the treatment protocol for double decoy EVs in TNBS induced colitis. H) Percent change in relative bodyweight to initial weight over the disease course and I) survival curve in mice induced with colitis by intrarectal injection of TNBS and treated I.V with either 3×10 MSC double decoy EVs ( n =13), 3×10 MSC double decoy EVs ( n =15), 10 µ g Tocilizumab and 1 µ g Etanercept ( n =14), or saline (n=15) 24 hours post disease induction. E, F , Error bars, s.d. ( n =3). **** P < 0.0001 statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Ctrl EVs at the respective dose. H , Error bars, SEM. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 statistical significance calculated by two-way ANOVA with Dunnett’s post-test compared with response of Saline treated animal.

Article Snippet: Surface expression of decoy constructs on engineered MSC lines was assessed by using either APC-conjugated rat-anti-mouse gp130 (IL6ST) antibodies (clone FAB4681A, R&D Systems) or AlexaFluor647-conjugated mouse-anti-human CD120a (TNFR1) antibodies (clone H398, Bio-Rad).

Techniques: Imaging, Flow Cytometry, Staining, Transmission Assay, Electron Microscopy, Multiplex Assay, Fluorescence, Purification, Stable Transfection, Expressing, Construct, In Vitro, Activation Assay, Control, Injection, Saline

Journal: Cell reports

Article Title: Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity

doi: 10.1016/j.celrep.2020.108381

Figure Lengend Snippet: (A) FLT3L-derived BMDCs were either left untreated or treated with indicated stimuli. Spider plots depict individual cell trajectories from an origin point (0;0) from four regions of interest. Each trajectory line represents one cell (n = 30–50 cells). Straightness index and mean velocity were calculated (right panels). (B) cDC1s or cDC2s were either left untreated or treated as indicated. The mean fluorescence intensity (MFI) of surface CCR7 (among CD11c + live cells) was measured by flow cytometry. Means and SDs from three replicates are shown, and data are representative of at least three independent experiments. (C) The absolute number of CD45.2 + CFSE + among CD11c + live cells was calculated by flow cytometry. Means and SDs from five mice are shown, and data are representative of at least three independent experiments. (D and E) Hyperactive DCs that migrated to the skin dLN were sorted as CD11c + CD45.2 + CFSE + live cells. Alternatively, resident myeloid cells from the skin dLN were sorted as CD11c + CD45.1 + CFSE neg live cells. (D) Cells were cultured in media for 24 h, and IL-1β and LDH release were measured. Means and SDs from three independent experiments are shown. (E) DCs were stained with the markers indicated and examined by confocal microscopy. Scale bar: 5 μm on representative images (left panel). Quantification of the percent of cells containing ASC specks (right panel). DCx: DC injection. In (C)–(E), BMDCs were either left untreated or treated with the stimuli indicated. Cells were stained with CFSE and injected subcutaneously into CD45.1 mice. At 15–18 h post-DC injection, skin dLNs were dissected. p values of < 0.05 (*), < 0.01 (**), < 0.001 (***), or ≤ 0.0001 (****) are indicated.

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , 130–110–618.

Techniques: Derivative Assay, Fluorescence, Flow Cytometry, Cell Culture, Staining, Confocal Microscopy, Injection

Journal: Cell reports

Article Title: Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity

doi: 10.1016/j.celrep.2020.108381

Figure Lengend Snippet: (A) Mice were injected s.c. on the right flank with OVA either alone or as indicated. Seven or 40 days post-immunization, T cells were isolated from the dLN. (A) The percentage of Teff cells as CD44 low CD62L low , TEM cells as CD44 hi CD62L low , and TCM cells as CD44 hi CD62L hi are represented among CD3 + CD8 + live cells. (B and C) CD8 + T cells were sorted from the dLN 7 days post-immunization, then (B) treated either with PMA plus ionomycin, or co-cultured with B16OVA cells (target cells). CD8 + T cells degranulation was assessed by monitoring the percentage of CD107a + . (C) CD8 + T cells were cultured with BMDC loaded (or not) with a serial dilution of OVA protein starting from 1000 μg/ml. IFNγ secretion was measured by ELISA. Means and SDs of five mice are shown. (D) Seven days post-immunization, the percentage of Teff, TEM, TCM, and T naive cells in the skin dLN was measured by flow cytometry. (E) The percentage of SIINFEKL + among CD8 + live T cells in the dLN (left panel) or in the spleen (right panel) was measured by flow cytometry. (F and G) Total CD8 + T cells were sorted from the dLN and (F) co-cultured with untreated BMDCs loaded (or not) with OVA for 7 days at a ratio of 1:10 (DC: T cell). IFNγ secretion was measured by ELISA. (G) CD8 + T cells were co-cultured with B16OVA cells (target cells) at ratio of 1:3 (effector: target). The percentage of LDH release was measured from B16OVA-CD8 + T cells co-culture and normalized to the LDH released from B16OVA cells or CD8 + T cells cultured separately. Means and SDs from five mice are shown. In (D)–(F), CD45.1 mice were irradiated then reconstituted with mixed BM of the genotypes indicated. Six weeks post-reconstitution, chimera mice were injected with DTx 3 times a week for a total of 9 DTx injections. Chimeric mice were then immunized s.c. on the right flank with OVA with LPS plus PGPC. p values of < 0.05 (*), < 0.01 (**), < 0.001 (***), or ≤ 0.0001 (****) are indicated.

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , 130–110–618.

Techniques: Injection, Isolation, Cell Culture, Serial Dilution, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Co-Culture Assay, Irradiation

Journal: Cell reports

Article Title: Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity

doi: 10.1016/j.celrep.2020.108381

Figure Lengend Snippet: (A) Mice were injected s.c. on the right flank with PBS (unimmunized), with B16OVA cell WTLs alone (“None”), or with LPS, or B16OVA WTLs plus LPS and oxPAPC or PGPC. Fifteen days post-immunization, mice were challenged s.c. on the left upper back with 3 × 10 5 of B16OVA cells. One hundred fifty days later, tumor-free mice were re-challenged s.c. with 5 × 10 5 of B16OVA cells. (A) Tumor growth (left panel) and mice survival (middle panel) was monitored every 2 days. The percentage of tumor-free mice 300 days post-tumor inoculation is indicated (right panel) (n = 8–15 mice per group). (B and C) Tumors were harvested at the endpoint of tumor growth, and (B) the percentages of tumor infiltrating CD3 + CD4 + and CD3 + CD8 + T cells among CD45 + live cells were assessed by flow cytometry. (C) Tumor-infiltrating T cells were sorted then stimulated in the presence of anti-CD3 and anti-CD28 dynabeads. IFNγ release was measured by ELISA (n = 4 mice per group). (D) Mice were either left untreated (unimmunized) or were immunized s.c. on the right flank with B16OVA WTLs plus the stimuli indicated. Fifteen days post-immunization, mice were challenged with 3 × 10 5 B16OVA cells s.c. on the left upper back. The percentage of survival is monitored every 2 days (n = 5 mice per group). (E and F) Mice were either left untreated (unimmunized) or were immunized s.c. on the right flank with B16OVA WTLs (E) or with (F) MC38OVA WTLs or OVA alone or in combination with the treatments indicated. Fifteen days post-immunization, mice were challenged with (E) 3 × 10 5 of viable B16OVA cells or (F) 5 × 10 5 MC38OVA cells s.c. on the left upper back. (E) Ninety days later, tumor-free mice were re-challenged with 5 × 10 5 B16OVA cells s.c. on the back. (F) Fifty days later, tumor-free mice were re-challenged s.c. with 1 × 10 6 MC38OVA cells. Survival was monitored every 2 days (n = 3–5 mice per group). P values of < 0.01 (**) is indicated.

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , 130–110–618.

Techniques: Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity

doi: 10.1016/j.celrep.2020.108381

Figure Lengend Snippet: Mice of the indicated genotypes were inoculated subcutaneously on the left upper back with (A) 5 × 10 5 of MC38OVA cells, (B) 3 × 10 5 B16OVA cells, (C) 3 × 10 5 B16-F10 cells, (D) 3 × 10 5 CT26 cells, or (E and F) 3 × 10 5 LLC1 cells. In (A)–(E), when tumors reached 3–4 mm in size, mice were either left untreated (unimmunized) or were injected s.c. on the right flank with WTLs plus LPS and PGPC with or without neutralizing anti-IL-1β antibodies, anti-CD4, anti-CD8α antibodies, or (F) IL-RA. Mice received two boost injections with WTLs and LPS plus PGPC. In (B)–(E), alternatively, tumor-bearing mice were injected with anti-PD-1 antibody. The percentage of survival is indicated (n = 10–12 mice per group). In (F), LLC1 tumors were harvested at the endpoint of tumor growth. The percentage of CD8 + TILs (left panel) among CD3 + CD45 + live cells and CD69 + CD103 + TRM cells among CD8 + TILs (middle panel) were measured by flow cytometry. CD45 + live TILs were cultured for 48 h on anti-CD28 and anti-CD3 coated plates. IFNγ was measured by ELISA (right panel) (n = 5 mice per group). p values of < 0.05 (*), < 0.01 (**), < 0.001 (***), or ≤ 0.0001 (****) are indicated.

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , 130–110–618.

Techniques: Injection, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity

doi: 10.1016/j.celrep.2020.108381

Figure Lengend Snippet: (A) Zbtb46 DTR mice were s.c. injected with B16OVA cells. Mice were either injected with DTx every other day for four consecutive injections, or mice were injected with PBS. Seven days post-tumor injection, all mice were immunized with B16OVA WTLs plus LPS and PGPC, followed by two boost injections. The percentage of mice survival is indicated (n = 10 mice per group). (B) CD45.1 mice were irradiated then reconstituted with mixed BM from Zbtb46 DTR mice plus either WT or Nlrp3 −/− , Casp1 / 11 −/− , or Ccr7 −/− mice. Six weeks post-reconstitution, mouse chimeras were injected s.c. with B61OVA cells, then all mice received DTx 3 times a week for a total of 12 consecutive injections. Seven days post-tumor inoculation, chimeric mice were immunized with B16OVA WTLs and LPS plus PGPC and received two boost injections. The percentage of mice survival is indicated (n = 5 mice per group). (C and D) WT or Batf3 −/− mice were injected s.c with B16OVA cells. Seven days post-tumor inoculation, mice were either left untreated, or WT and Batf3 −/− mice were immunized with B16OVA WTLs and LPS plus PGPC followed by two boost injections. (C) The percentage of mice survival is indicated (n = 10 mice per group). (D) Twenty-one days post-tumor inoculation, the percentage of OVA-specific CD8 + T cells and CD4 + T cells was assessed using tetramer staining (n = 5 mice per group). (E and F) Batf3 −/− mice were injected s.c on the right flank with B16OVA cells. Seven days post-tumor inoculation, mice were left untreated (no cDC1 injection) or were injected s.c. on the left flank with FLT3-derived naive cDC1s or active cDC1s treated with LPS or with hyperactive cDC1s pretreated with LPS plus PGPC. All cDC1s were loaded with B16OVA WTLs for 1 h prior to their injection. (E) The percentage of mice survival is indicated (n = 5 mice per group). (F) Twenty-one days post-tumor inoculation, OVA-specific CD8 + T cells and CD4 + T cells were assessed using tetramer staining (n = 5 mice per group). p values of < 0.05 (*), < 0.01 (**), or < 0.001 (***) are indicated.

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , 130–110–618.

Techniques: Injection, Irradiation, Staining, Derivative Assay

Journal: Cell reports

Article Title: Inflammasomes within Hyperactive Murine Dendritic Cells Stimulate Long-Lived T Cell-Mediated Anti-tumor Immunity

doi: 10.1016/j.celrep.2020.108381

Figure Lengend Snippet:

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , 130–110–618.

Techniques: Purification, Recombinant, Adjuvant, Enzyme-linked Immunosorbent Assay, CyQUANT Assay, LDH Cytotoxicity Assay, Expressing, High Throughput Screening Assay, RNA Sequencing, Software, Microscopy, Imaging

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: (A) GDF5 gradient surface, 18 × 18 mm, seeded with c-iPSCs visualized using high-throughput confocal IN CELL Analyzer 6000. The bar on the left upper side indicates the extent of the gradient, where the continuous density increase is shown with a marker on the left-hand side, with low GDF5 density at the bottom of the image and high at the top (the damaged lower left corner does not affect the gradient result). (B) Focusing on the budding cell clusters identified at a density range 500–1,500 particles/μm 2 . Two white lines indicate the budding zone. (C–D) Close-up of buds identified in the budding zone. Scale bars 10 μm.

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: High Throughput Screening Assay, Marker

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: GDF5, TGFβ-1, and TGFβ-3 h.d. surfaces, 18 × 18 mm, visualized using high-throughput confocal IN CELL Analyzer 6000. (A) GDF5 surfaces with a density of 400 particles/μm 2 (control). No buds are identified. (B) GDF5 surface with a density in the range of the budding zone, 900 particles/μm 2 . Buds are clearly identified as brighter colored clusters. (C) TGFβ-1 surfaces with a density of 600 particles/μm 2 . Cell-free cavities were formed, a response comparable to that of cells on laminin 521 gradient surfaces (Fig. S2). (D) Evenly distributed cells on TGFβ-1 surfaces with a density of 2,000 particles/μm 2 , (E) TGFβ-3 surfaces with a density of 600 particles/μm 2 , and (F) a density of 1,900 particles/μm 2 .

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: High Throughput Screening Assay, Control

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: Immunostaining for SOX9 in c-iPSCs after five days of differentiation on a GDF5 gradient. (A1–A3) Images from the budding zone were acquired using fluorescence microscopy, 20X objective. Scale bars denote 100 μm. (B1–B3) Images from the budding zone were acquired using a confocal microscope, 40X objective. Scale bars denote 10 μm.

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: Immunostaining, Fluorescence, Microscopy

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: TBX3 are localized in the nuclei and in vesicles in the budding zone. TBX3 expression is visualized in c-iPSCs after five days of differentiation on a GDF5 gradient. (A) Budding zone on a GDF5 gradient. Scale bar 100 μm B1–B3 and C1–C3 show two different budding clusters on the GDF5 gradient. (D1-D3) Close-up of a budding zone. (B–C) Images were acquired with a confocal microscope, 40X objective. Scale bars 20 μm. (D) Images were acquired using a 60X objective. Scale bars 10 μm.

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: Expressing, Microscopy

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: iPSCs on h.d. surfaces immunostained with TBX3 monoclonal (Table S2) antibody; images acquired using confocal microscopy. TBX3 expression is clearly upregulated on the GDF5 h.d. surface with a particle density in the range of the budding zone, as compared with on all other surfaces. (A) GDF5 surface with low particle density. (B) GDF5 budding zone surface. (C) TGFβ-3 surface with the same particle density as (B). (D) TGFβ-3 surface with the same particle density as (B), but with GDF5 (10 ng/ml) added to the differentiation medium. (E) Surface without biomolecules, coated with Coat-1 (DEF-CS 500 Coat-1, Cellartis, Sweden).

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: Confocal Microscopy, Expressing

Journal: Heliyon

Article Title: GDF5 induces TBX3 in a concentration dependent manner - on a gold nanoparticle gradient

doi: 10.1016/j.heliyon.2020.e04133

Figure Lengend Snippet: Histological sections of pellets generated from c-iPSCs (A–C) and chondrocytes (D–E). A1–A2 to E1–E2 were stained with Alcian Blue van Gieson, demonstrating the presence of proteoglycans (blue) and collagen (red/purple). A3–A6 & B3–C3 were immunostained for TBX3, which was clearly upregulated in the areas with higher amounts of GAG. (A1–A6) c-iPSCs pre-differentiated on a GDF5 h.d. surface with low particle density. (B1–B3) c-iPSCs pre-differentiated on a GDF5 h.d. surface with budding zone particle density. (C1–C3) c-iPSCs pre-differentiated on a GDF5 gradient surface. (D1–D2) Chondrocyte pellet pre-differentiated on a GDF5 gradient surface. (E1–E2) Chondrocyte pellet with 5 % human serum in the medium. (A1–E1, A3–C3, A5–A6) 100 μm scale bars. (A2–E2) 50 μm scale bars. (A4) 20 μm scale bars.

Article Snippet: Recombinant human GDF5 (R&D Systems, Bio-Techne, MN, USA), TGFβ-1 (R&D Systems, Bio-Techne, MN, USA), and TGFβ-3 (R&D Systems, Bio-Techne, MN, USA) were biotinylated to facilitate binding to streptavidin.

Techniques: Generated, Staining

Journal: Cytotherapy

Article Title: Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods

doi: 10.1016/j.jcyt.2012.07.001

Figure Lengend Snippet: Monitoring adipogenic progression of MSCs and fibroblasts with the use of q-PCR. MSCs and fibroblasts were cultured in adipogenic medium for 21 days. Expression of (A) PPAR-γ and (B) FABP4 was determined on days 0, 3, 7, 14 and 21. Relative levels of gene expression were normalized to reference gene GAPDH and displayed as fold increase over day 0. Each data point represents the mean of three replicates.

Article Snippet: FABP4 protein was detected with the use of a purified polyclonal goat anti-human FABP4 antibody (IgG, R & D Systems, Abingdon, UK) at 1:20 dilution in PBS.

Techniques: Cell Culture, Expressing, Gene Expression

Journal: Cytotherapy

Article Title: Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods

doi: 10.1016/j.jcyt.2012.07.001

Figure Lengend Snippet: Monitoring adipogenic progression of MSCs with the use of flow cytometry for FABP4 and Nile red. (A) Representative histogram plots for FABP4 staining. (B) The increase in SSC characteristics for the whole population of cells as the time course progresses; error bars represent standard deviation of the mean for three donors tested. (C) Representative histogram plots for Nile red staining. (D) Nile red fluorescence of both populations of gated low- and high-SSC cells, demonstrating a parallel increase throughout the time course. Days 0 and 3 are omitted because of very low frequency of high-SSC early in differentiation. Numbers in the top right corners of histograms represent median fluorescence intensities. FI is fluorescent intensity.

Article Snippet: FABP4 protein was detected with the use of a purified polyclonal goat anti-human FABP4 antibody (IgG, R & D Systems, Abingdon, UK) at 1:20 dilution in PBS.

Techniques: Flow Cytometry, Staining, Standard Deviation, Fluorescence

Journal: Cytotherapy

Article Title: Assay validation for the assessment of adipogenesis of multipotential stromal cells—a direct comparison of four different methods

doi: 10.1016/j.jcyt.2012.07.001

Figure Lengend Snippet: Advantages and disadvantages of different assays for the evaluation of MSC adipogenesis.

Article Snippet: FABP4 protein was detected with the use of a purified polyclonal goat anti-human FABP4 antibody (IgG, R & D Systems, Abingdon, UK) at 1:20 dilution in PBS.

Techniques: Staining, RNA Extraction, Quantitative Proteomics, Biomarker Discovery, TaqMan Assay, Flow Cytometry, Purification, Fluorescence, High Throughput Screening Assay, Microscopy

Journal: Acta Pharmaceutica Sinica. B

Article Title: Functional aptamer evolution-enabled elucidation of a melanoma migration-related bioactive epitope

doi: 10.1016/j.apsb.2025.03.003

Figure Lengend Snippet: Construction of aptamer libraries mapping A375 membrane proteomes. (A) Procedures for library construction involved DNA library modification through click chemistry, positive selection, negative selection, and PCR amplification. Libraries obtained after 7 rounds of selection were utilized for sequencing and functional screening. (B) The molecular formula of the click reaction to convert EdU into IndU. (C) Flow cytometry analysis showing aptamer libraries enrichment on target cell A375. (D) Flow cytometry analysis showing no enrichment on control cell HEK293T. (E) Sequencing results showing libraries enrichment round-by-round. (F) Flow cytometry analysis of top 9 aptamers demonstrating robust binding capacities of libraries for A375.

Article Snippet: The human melanoma cell line A375 and the Human embryonic kidney cell line HEK293T, obtained from the American Type Culture Collection (ATCC), were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA).

Techniques: Membrane, Modification, Selection, Amplification, Sequencing, Functional Assay, Flow Cytometry, Control, Binding Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Functional aptamer evolution-enabled elucidation of a melanoma migration-related bioactive epitope

doi: 10.1016/j.apsb.2025.03.003

Figure Lengend Snippet: High-throughput phenotypic screening to obtain aptamer XH3C with migration-inhibitory function. (A) Schematic illustration of functional aptamer discovery based on the wound healing assay and high-content analysis. (B) The volcano plot showing the normalized migration inhibition rates and P -values of 96 aptamer candidates. P -values between MIR of aptamers and MIR of Library on each plate were calculated by one-way ANOVA using Graphpad software ( P -values: XH3C 0.022, XH3F 0.038, XH3G 0.045, XH4F 0.048, XH7E 0.062, XH11D 0.046). Condition: incubate 1 μmol/L aptamer and library on A375 in blank medium for 36 h after scratching ( n = 3) (C) Representative scratch images of XH3C directly showing migration-inhibitory function. (D) Migration inhibition rate curves of XH3C at various administered drug concentrations after 36 h ( n = 3). (E) Time-normalized blank area curves of XH3C at different concentrations ( n = 3). (F) Binding curves of XH3C to A375 cells ( n = 3). (G) Flow cytometry analysis showing robust binding affinity of XH3C to A375. (H) Flow cytometry analysis showing minimal binding affinity of XH3C to HEK293T.

Article Snippet: The human melanoma cell line A375 and the Human embryonic kidney cell line HEK293T, obtained from the American Type Culture Collection (ATCC), were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA).

Techniques: High Throughput Screening Assay, Migration, Functional Assay, Wound Healing Assay, High Content Screening, Inhibition, Software, Binding Assay, Flow Cytometry

Journal: Acta Pharmaceutica Sinica. B

Article Title: Functional aptamer evolution-enabled elucidation of a melanoma migration-related bioactive epitope

doi: 10.1016/j.apsb.2025.03.003

Figure Lengend Snippet: Identification of CSPG4 as the target of functional aptamer XH3C. (A) Schematic illustration of procedures to identify binding target of XH3C. (B) Analysis of target type through cell membrane proteins digestion by trypsin. (C) Silver-stained SDS-PAGE showing specific band in XH3C group (red rectangle) under two denaturation conditions (room temperature for 30 min and 95 °C for 5 min). Mass spectrometry identified this band as Chondroitin sulfate proteoglycan 4 (CSPG4). Other bands (orange and yellow arrows) were verified as cleavage product of CSPG4 or non-specific binding proteins. (D) Western blotting (WB) of pull-down samples stained with CSPG4 antibody. (E) Typical WB images and (F) quantitative analysis showing that the expression level of CSPG4 was reduced to about 60% by siCSPG4 ( n = 3, ∗∗ P < 0.01; unpaired Student's t test). (G) Flow cytometry analysis of the binding ability of XH3C on CSPG4-knockdown A375 cells. (H) Quantitative analysis of G showing that the binding ability of XH3C was reduced to about 60% after CSPG4 knockdown, the mean fluorescence intensity was calculated by flowjo ( n = 3, ∗∗∗ P < 0.001; unpaired Student's t test). (I) SPR experiments measuring K d value of XH3C and CSPG4 eukaryotic proteins. (J) Structure predictions of CSPG4 and XH3C complex. Structure of CSPG4 was predicted by AlphaFold3.Structure of XH3C was obtained by molecular dynamics simulation. The complex structure was predicted by HDOCK Server. Four interaction protein domains are colored by yellow, wheat, green and light blue. The four IndU sites on XH3C are magentas, while potentially interacting amino acids on the CSPG4 are red. 995S is chondroitin sulfate modification site. Trucation1, 2, 3 and 4 are shown in cyan, gold, blue and gray respectively.

Article Snippet: The human melanoma cell line A375 and the Human embryonic kidney cell line HEK293T, obtained from the American Type Culture Collection (ATCC), were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA).

Techniques: Functional Assay, Binding Assay, Membrane, Staining, SDS Page, Mass Spectrometry, Western Blot, Expressing, Flow Cytometry, Knockdown, Fluorescence, Modification

Journal: Acta Pharmaceutica Sinica. B

Article Title: Functional aptamer evolution-enabled elucidation of a melanoma migration-related bioactive epitope

doi: 10.1016/j.apsb.2025.03.003

Figure Lengend Snippet: XH3C-induced cytoskeletal rearrangement by inhibiting the interaction between the bioactive epitope chondroitin sulfate chain on CSPG4 and ITGA4. (A) Schematic illustration of mechanism of cell migration inhibition by XH3C. (B) Flow cytometry analysis showing the reduced XH3C binding capacity after chondroitin sulfates digestion by chondroitinase ABC (ChABC). (C) Flow cytometry analysis of the membrane ITGA4 content showing less distribution of ITGA4 on membrane after XH3C treatment. (D) Statistical analysis of Rcoloc between CSPG4 and ITGA4, calculated from multiple fields, showing significant difference between the control group and XH3C group ( n = 5 and 6, ∗ P < 0.05; unpaired Student's t test). Rcoloc values between CSPG4 and ITGA4 were calculated by Fiji software. (E) Representative immunofluorescent (IF) images showing attenuated co-localization of ITGA4, CSPG4 and F-actin and more distribution of ITGA4 in cytoplasm in the XH3C group. (F) Confocal microscopy images showing F-actin cytoskeleton in A375 cells in the absence and presence of the XH3C aptamer. Cell Contours were used to measure areas and perimeters per cell to calculate cell circularity. (G) Statistical analysis of cell circularity showing significant difference between the control group and XH3C group. ( n = 17 and 21, ∗∗∗ P < 0.001; unpaired Student's t test). (H) Co-IP (Co-Immunoprecipitation) assay showing the interactions between CSPG4 and ITGA4 in the absence and presence of the XH3C aptamer.

Article Snippet: The human melanoma cell line A375 and the Human embryonic kidney cell line HEK293T, obtained from the American Type Culture Collection (ATCC), were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA).

Techniques: Migration, Inhibition, Flow Cytometry, Binding Assay, Membrane, Control, Software, Confocal Microscopy, Co-Immunoprecipitation Assay