Journal: PLoS ONE

Article Title: Centrosome-Kinase Fusions Promote Oncogenic Signaling and Disrupt Centrosome Function in Myeloproliferative Neoplasms

doi: 10.1371/journal.pone.0092641

Figure Lengend Snippet: ( A ) Schematic of normal FGFR dimerization and idFGFR1 dimerization induced with dimerization ligand AP20187. ( B ) Table of idFGFR1 constructs targeted to subcellular domains by the addition of localization tags. ( C ) RPE-1 cells transfected with Myc-idFGFR1, Myc-PACT-idFGFR1, MTS-idFGFR1-Myc, or MYR-idFGFR1-Myc, treated with 10 nM AP20187 for 24 h, fixed, and stained with antibodies against Myc (green) and PY (red). DNA is stained using DAPI (blue). Scale bars: 10 μm; insets: 10× magnification. ( D ) WB analysis of lysates from RPE-1 cells transfected with Myc-idFGFR1, Myc-PACT-idFGFR1, MTS-idFGFR1-Myc, or MYR-idFGFR1-Myc, treated with 10 nM AP20187 or vehicle (100% ethanol) for 24 h in low serum medium, harvested, and probed with antibodies against PY, Myc, and p38 as a loading control. Quantification of PY increase relative to control, normalized by Myc, for each construct is indicated.

Article Snippet: RPE-1 cells were transfected with Lipofectamine LTX (Invitrogen) for 24 h followed by 24 h incubation in DMEM/F12 50/50 medium +0.5% fetal bovine +10 nM AP20187 (ARGENT Regulated Homodimerization Kit Version 2.0; ARIAD Pharmaceuticals, Inc.), or the equivalent volume of 100% ethanol (vehicle).

Techniques: Construct, Transfection, Staining, Control

Journal: PLoS ONE

Article Title: Centrosome-Kinase Fusions Promote Oncogenic Signaling and Disrupt Centrosome Function in Myeloproliferative Neoplasms

doi: 10.1371/journal.pone.0092641

Figure Lengend Snippet: ( A ) WB analysis of lysates from RPE-1 cells transfected with Myc-FOP-FGFR1, Myc-FOP-FGFR1 K259A , Myc-FOP-FGFR1 V74F/E97K , Myc-cFGFR1, Myc-idFGFR1, Myc-PACT-idFGFR1, MTS-idFGFR1-Myc, or MYR-idFGFR1-Myc, treated with 10 nM AP20187 or vehicle (100% ethanol) for 24 h in low serum medium, harvested, and probed with antibodies against phospho-PLCγ (pPLCγ), total PLCγ, Myc, PY, and p38 as a loading control. White boxes indicate quantified regions in PY blot, * marks a non-specific band present in all lanes. ( B ) Graph showing kinase efficiency of each construct with or without AP20187 addition. Kinase efficiency = WB signal intensity of (pPLCγ/PLCγ)/PY. Quantifications were obtained using a Typhoon imaging system and fluorescence-conjugated secondary antibodies. Bars represent mean of 3 independent trials ± SEM. *p<0.05, n.s. p>0.05.

Article Snippet: RPE-1 cells were transfected with Lipofectamine LTX (Invitrogen) for 24 h followed by 24 h incubation in DMEM/F12 50/50 medium +0.5% fetal bovine +10 nM AP20187 (ARGENT Regulated Homodimerization Kit Version 2.0; ARIAD Pharmaceuticals, Inc.), or the equivalent volume of 100% ethanol (vehicle).

Techniques: Transfection, Control, Construct, Imaging, Fluorescence

Journal: Autophagy

Article Title: TBK1 is a signaling hub in coordinating stress-adaptive mechanisms in head and neck cancer progression

doi: 10.1080/15548627.2025.2481661

Figure Lengend Snippet: MUL1 is novel negative regulator of TBK1. (A) TBK1 or MUL1 expression levels were different between tissues from human head and neck cancer (T) and their adjacent non-tumor head and neck (N). Proteins were isolated from tissues of 14 patients with HNC, and p-TBK1, TBK1, or MUL1 expression levels were determined by western blot. (B) Quantification of the p-TBK1:GAPDH, TBK1:GAPDH, and MUL1:GAPDH (14-pair HNC cohort, ** p = 0.0058, **** p < 0.0001 by the Mann-Whitney t test). (C) WT or MUL1 KO FaDu cells were treated with PI+Tg (10 nM bortezomib +100 nM thapsigargin) for the indicated time periods, and subject to western blot. (D) WT or MUL1 KO FaDu cells were treated with 20 μg/ml cycloheximide (CHX) and 10 nM PI +100 nM tg for the indicated times, and subjected to western blot. (E) MUL1 induces TBK1 degradation by the UPS. MYC-His-TBK1 was transfected into MUL1 KO FaDu cells together with or without flag-MUL1. Cells were treated with 10 μM MG132, 10 nM bortezomib, 10 μM lactacystin, or 250 nM epoxomicin for 12 h. (F) TBK1 interacts with MUL1. At 24 h after co-transfection with the indicated plasmids, MUL1 KO FaDu cells were treated with 10 μM MG132 for 8 h before cell harvest and then subjected to Ni-nta affinity-isolation under denaturing conditions. The obtained affinity-isolated samples were subjected to western blot using the indicated antibodies. (G) WT or MUL1 KO FaDu cells were transfected with NC or STING1 siRNA for 24 h, followed by incubation with 10 nM PI +100 nM tg for 30 h. (H) Endogenous p-TBK1 ubiquitination assay. WT or MUL1 KO FaDu cells were treated with 10 nM PI +100 nM tg for the indicated time, followed by ubiquitination assays for analysis with an anti-FK2 antibody. (I) MUL1 induces K48-linked ubiquitination of TBK1. MUL1 KO FaDu cells were transfected MYC-His-TBK1 and flag-MUL1 together with HA-WT ub or ubiquitin mutants (HA-Ub-K48, HA-Ub-K63, HA-Ub K48R , or HA-Ub K63R ). Ubiquitinated TBK1 was identified by Ni-nta affinity-isolation assays for analysis with an anti-ha antibody. (J) MUL1 preferentially induces degradation in active TBK1. Active TBK1 (MYC-His-wt TBK1) or inactive TBK1 (MYC-His-TBK1 S172A ) were co-transfected with flag-MUL1 plasmids (0, 0.25, 0.5, or 1 μg) in MUL1 KO FaDu cells. (K) Activated TBK1 is efficiently ubiquitinated by MUL1. After transfection with plasmids as indicated, MUL1 KO FaDu cells were treated with 10 μM MG132 and then subjected to Ni-nta affinity-isolation ubiquitination assays. (L) Colocalization of EGFP-MUL1 (green), COX4l1 (red), and HSPD1/HSP60 (far red) with or without 20 nM PI +100 nM tg treatment for 12 h in MUL1 KO FaDu cells. (M) Colocalization of EGFP-MUL1 (green), mitochondria (red), and MYC-His-TBK1 (far red) with or without 20 nM PI +100 nM tg treatment for 12 h in MUL1 KO FaDu cells. Mitochondria were labeled with MitoTracker red CMXRos dye. Images were obtained using a nikon N-SIM confocal microscope and overlaid to assess protein localization. Scale bars: 10 μm and 1 μm (inset). (N) The percentages of TBK1 containing colocalization of mitochondria (COX4l1 and HSPD1/HSP60) per field. The data represent the mean ± SD of 25 fields, each of which contains at least 4 cells that meet statistical requirements, from three independent experiments. (O) Quantification of the colocalization of TBK1 and MUL1 signal. Merged images from (M) were analyzed for TBK1 and MUL1 colocalization using NIS Elements software and Pearson’s correlation coefficient. The data represent the mean ± SD of 40 fields, each of which contains at least 4 cells that meet statistical requirements, from three independent experiments. **** p < 0.0001 by unpaired t test.

Article Snippet: The spheroids were then washed with PBS and stained using the LIVE/DEAD TM Viability/Cytotoxicity Kit (Thermo Fisher Scientific, L3224) by incubating with calcein AM and ethidium homodimer-1 (EthD-1) at 37°C for 30 min. A confocal microscope (Nikon A1R, Japan, x20 objective, n = 20) was used for imaging, and z-section imaging was performed.

Techniques: Expressing, Isolation, Western Blot, MANN-WHITNEY, Transfection, Cotransfection, Incubation, Ubiquitin Proteomics, Labeling, Microscopy, Software

Journal: Nature Communications

Article Title: Exercise enhances skeletal muscle regeneration by promoting senescence in fibro-adipogenic progenitors

doi: 10.1038/s41467-020-14734-x

Figure Lengend Snippet: a Cytotoxicity assay shows the LDH release level after stimulation with different concentrations of TNF-α in FAPs isolated from AMI and CIM ( n = 3 per group). b , c Representative image of FITC-Annexin V (green) and ethidium homodimer III (red) after stimulation with 1, 10, and 100 ng/mL of TNF-α, and the quantified data. The number of Annexin V+, apoptotic FAPs increased in a dose-dependent fashion with the addition of TNF-α in AMI, but not in CIM mice ( n = 3 per group). d , e Hierarchical clustering of differentially expressed cytokine–cytokine receptor gene expression was profiled by PrimerArray ® analysis in control, AMI, and CIM mice ( n = 3 per group) ( d ). Genes with higher expression are depicted in magenta, genes with lower expression are depicted in cyan, and genes with no difference are depicted in black ( d ). Scatterplots of gene expression changes in AMI-FAPs ( n = 3) compared with CIM-FAPs ( n = 3) ( e ). f , g Representative images of PDGFRα- and active caspase-3-immunostained triceps surae in control, AMI, and CIM mice ( f ), and quantification of the percentage of active caspase-3-positive FAPs in randomly chosen fields of view ( g ). h Representative confocal images of Bcl-2- and p53-immunostained FAPs isolated from AMI and CIM. i mRNA expression of Cd274 , Pdcd1lg2 , and Cd47 in FAPs isolated from AMI and CIM mice ( n = 3 per group). j Correlation of Cd274 , Pdcd1lg2 , and Cd47 mRNA expression with Cdkn2a mRNA expression in FAPs isolated from AMI and CIM mice. Quantitative data for each specimen are shown in a dot plot. P values were determined by one-way ANOVA adjusted by the Holm method (* P < 0.05, ** P < 0.001). NS, not significant.

Article Snippet: After 2 days culture, a cytotoxicity LDH assay was performed using the Cytotoxicity LDH Assay-WST (Dojindo), and apoptotic and necrotic cell detection assays were performed by staining with FITC-Annexin V, Ethidium Homodimer III, and Hoechst33342 (Apoptotic/Necrotic/Healthy Cells Detection Kit; PromoKine, Germany) according to the manufacturer’s instructions.

Techniques: Cytotoxicity Assay, Isolation, Gene Expression, Control, Expressing

Journal: bioRxiv

Article Title: Comparative analysis of commercially available single-cell RNA sequencing platforms for their performance in complex human tissues

doi: 10.1101/541433

Figure Lengend Snippet: Quality control and data preprocessing. Related to . (A) Cell filtering in the Chromium platform. Cells passing filters are represented on the green regions on each curve. (B) Loess regression curves display the relationships of number of genes detected with different sequencing depth in the C1 96, C1 HT and iCell8 systems. Single-cell data from all experiments on each system are combined. Within each graph, solid line indicates population median and dashed line indicates two standard deviation below population median. Cells passing filters are represented on the green regions on each curve. (C) Relationships of number of genes detected and varying sequencing depth in the Chromium system. Each curve is associated with one Chromium experiment. The endpoint of each curve represents the average sequencing depth for cells passing filter.

Article Snippet: Single cells were stained with 4 μM Ethidium homodimer-1 and 2 μM Calcium AM (ThermoFisher LIVE/DEAD Viability Kit L3224) in the dark at room temperature for 20 min. Stained cells were washed once with PBS + 10% FBS and 10 μl of the cell suspension was loaded onto a C1 96-IFC (Fluidigm 100-5760) or C1 800HT-IFC (Fluidigm 101-4982) at a concentration of 1,000 cells/μl.

Techniques: Sequencing, Standard Deviation

Journal: bioRxiv

Article Title: Comparative analysis of commercially available single-cell RNA sequencing platforms for their performance in complex human tissues

doi: 10.1101/541433

Figure Lengend Snippet: Single-cell RNA-seq can distinguish different pancreatic cell types. See also and . tSNE plots are shown for single-cell data generated on (A) C1 96, (B) C1 HT, (C) iCell8, (D) Chromium V1 and (E) Chromium V2 platforms. Cell type labels are shown on the tSNE plot at the left side of each panel. Marker gene expressions are shown on the six tSNE plots at the right side of each panel.

Article Snippet: Single cells were stained with 4 μM Ethidium homodimer-1 and 2 μM Calcium AM (ThermoFisher LIVE/DEAD Viability Kit L3224) in the dark at room temperature for 20 min. Stained cells were washed once with PBS + 10% FBS and 10 μl of the cell suspension was loaded onto a C1 96-IFC (Fluidigm 100-5760) or C1 800HT-IFC (Fluidigm 101-4982) at a concentration of 1,000 cells/μl.

Techniques: RNA Sequencing Assay, Generated, Marker

Journal: bioRxiv

Article Title: Comparative analysis of commercially available single-cell RNA sequencing platforms for their performance in complex human tissues

doi: 10.1101/541433

Figure Lengend Snippet: Michaelis-Menten (MM) curve illustrates the relationship between expression levels (log10(CPM+1)) and dropout rates. Related to . MM plots for each cell type are shown for (A) C1 96, (B) C1 HT, (C) iCell8, (D) Chromium V1, and (E) Chromium V2 platforms. The MM constant (Km) for each fitting is shown on the upper right corner of each plot.

Article Snippet: Single cells were stained with 4 μM Ethidium homodimer-1 and 2 μM Calcium AM (ThermoFisher LIVE/DEAD Viability Kit L3224) in the dark at room temperature for 20 min. Stained cells were washed once with PBS + 10% FBS and 10 μl of the cell suspension was loaded onto a C1 96-IFC (Fluidigm 100-5760) or C1 800HT-IFC (Fluidigm 101-4982) at a concentration of 1,000 cells/μl.

Techniques: Expressing

Journal: bioRxiv

Article Title: Comparative analysis of commercially available single-cell RNA sequencing platforms for their performance in complex human tissues

doi: 10.1101/541433

Figure Lengend Snippet: tSNE graphs for all the alpha cells in different single-cell platforms. See also . Cluster labels are shown on the left-side tSNE plot in each panel. Donor labels are shown on the right-side tSNE plot in each panel. (A) Alpha cells from different donors distribute in two clusters in the C1 96 system. (B and C) Alpha cell clusters are driven by donors in the (B) C1 HT and (C) Chromium V2 systems.

Article Snippet: Single cells were stained with 4 μM Ethidium homodimer-1 and 2 μM Calcium AM (ThermoFisher LIVE/DEAD Viability Kit L3224) in the dark at room temperature for 20 min. Stained cells were washed once with PBS + 10% FBS and 10 μl of the cell suspension was loaded onto a C1 96-IFC (Fluidigm 100-5760) or C1 800HT-IFC (Fluidigm 101-4982) at a concentration of 1,000 cells/μl.

Techniques: