Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet: Lentiviral splicing reporter and RNA sequencing-based detection of splicing alterations in pAML (A) Schematic of lentiviral dual-fluorescence MAPT splicing reporter. (B) Representative image of Kasumi-1, a pAML cell line, stably transduced with the pCDH-EF1-IRES-Puro MAPT splicing reporter lentiviral vector. Left: confocal fluorescence microscopic image of lentiviral splicing reporter GFP expression (scale bar, 50 μm). Right: confocal fluorescence microscopic image of lentiviral splicing reporter RFP expression (scale bar, 50 μm). (C) FACS analysis of GFP and RFP mean fluorescence intensity (MFI) in the live cell population of CD34 + selected pAML patient samples (n = 4 biological replicates). (D) FACS analysis of GFP MFI in the live cell population of CD34 + selected pAML samples versus CD34 + cord blood (CB) samples (Student’s t test, p = 0.029; n = 4 biological replicates). (E) Whole-transcriptome (RNA-seq)-based quantification of CD44 splice isoforms in pAML (n = 9, blue) versus pNL (n = 3; red) HSCs (Student's t test, p < 0.05). (F) RNA-seq-based quantification of CD47 splice isoform expression in pAML (blue) versus pNL (red) HSCs (Student's t test, CD47-201 p = 0.3766; CD47-202 p = 0.0019; CD47-204 p = 0.0167; CD47-205 p = 0.0134). See also Figure S4 .

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: RNA Sequencing, Fluorescence, Stable Transfection, Transduction, Plasmid Preparation, Expressing

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet: Single-cell proteogenomic sequencing analysis of clonal heterogeneity in pAML Single-cell proteogenomic profiling was performed using the MissionBio Tapestri system to identify clonal somatic mutations and proteomic alterations. Uniform manifold approximation and projection (UMAP) plots (left) are shown from three separate pAML patients and were generated using eight antibody-oligo conjugates (CD3, CD7, CD11b, CD34, CD38, CD45, CD56, and ROR1) and a DNA mutation panel covering 45 myeloid genes with 312 amplicons. (A) pAML 266; correspondence of immunophenotyping and single nucleotide variant (SNV) analysis with ROR1, CD7, CD38, and CD34 cell surface protein expression is shown in a heatmap with each row associated with a single cell (right). (B) pAML 451; correspondence of immunophenotyping and SNV analysis with CD34, CD38, CD7, and ROR1 cell surface protein expression is shown in a heatmap with each row associated with a single cell (right). (C) pAML 678; correspondence of immunophenotyping and SNV analysis with CD7, CD38, CD34, and ROR1 cell surface protein expression is depicted (right). See also Figure S2 .

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: Sequencing, Generated, Mutagenesis, Variant Assay, Expressing

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet: RBFOX2 downregulation and reversion to an embryonic stem cell splicing program in pAML (A) RBFOX2 protein expression levels were quantified in pAML (n = 5 biological replicates) and cord-blood-derived HSCs (n = 3 biological replicates) and hematopoietic progenitor cells (HPCs; pAML, n = 5; pNL, n = 4 biological replicates) by FACS analysis as shown by MFI (Student’s t test HSC [left] p = 0.04; HPC [right] p = 0.06). (B) RNA-seq-based cytoscape network analysis of RBFOX2 and embryonic stem cell (ESC) specific alternative splicing program-related transcripts. Nodes are colored according to log fold change. (C) Volcano plot highlighting RBFOX2 target genes that show differential exon usage in pAML (false discovery rate, FDR < 0.05; percent spliced in, PSI > 0.1). A negative PSI reflects exon inclusion in pAML, while a positive PSI reflects ES in pAML versus pNL cells. (D) Heatmap of exon expression levels after unsupervised clustering of RBFOX2 target genes in pAML HSCs and HPCs (n = 9 and n = 8, respectively; blue) compared with pNL (n = 3 and n = 6, respectively; red). (E) Cytoscape network analysis of differentially spliced RBFOX2 target genes implicated in “Pathways in Cancer” in pAML HPCs. The top 200 ES events in this comparison were used as seeds for network propagation on the STRING high-confidence interactome. Differential expression log fold change is mapped to node color. Triangles depict seed list genes, and circles represent associated genes. See also Figure S5 .

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: Expressing, Derivative Assay, RNA Sequencing, Alternative Splicing, Comparison, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet: Rebecsinib inhibition of pAML LSC survival and self-renewal (A) Colony assay of CD34 + cord blood cells in long-term stromal co-cultures following lentiviral shRNA control (shControl, n = 7 biological replicates) or shRNA knockdown of RBFOX2 (shRBFOX2, n = 7 biological replicates; Student’s t test, p = 0.03). Data represent mean ± SD. (B) Percent colony survival and self-renewal (replating) of CD34 + cord blood samples after treatment with 100 or 1,000 nM of Rebecsinib normalized to vehicle-treated controls. Mean colony percentages ± SD are shown for each treatment condition (n = 7 biological replicates). Statistical analysis was performed by one-way ANOVA (survival [left] 100 nM Rebecsinib shControl versus shRBFOX2 p = 0.003, 1,000 nM Rebecsinib shControl versus shRBFOX2 p < 0.001; self-renewal [right] 100 nM Rebecsinib shControl versus shRBFOX2 p < 0.001, 1,000 nM Rebecsinib shControl versus shRBFOX2 p < 0.001). (C) Survival and self-renewal of CD34 + cells derived from pAML (n = 6 biological replicates) or cord blood (CB) (n = 7 biological replicates) in long-term stromal co-cultures. Data represent mean ± SD for each treatment condition. Statistical analysis was performed by one-way ANOVA (survival [left] 0.1 μM Rebecsinib pAML versus CB p = 0.001, 1 μM Rebecsinib pAML versus CB p = 0.001, 10 μM Rebecsinib pAML versus CB p = 0.001; self-renewal [right] 0.1 μM Rebecsinib pAML versus CB p = 0.001, 1 μM Rebecsinib pAML versus CB p = 0.001, 10 μM Rebecsinib pAML versus CB p = 0.001). (D) Self-renewal of CD34 + pAML (n = 6 biological replicates) versus adult dnAML (n = 9 biological replicates) treated with Rebecsinib. Data represent mean ± SD for each treatment condition. Statistical analysis was performed by one-way ANOVA (pAML versus adult dnAML p < 0.001). (E) Representative fluorescence microscopic imaging analysis of the pAML cell line Kasumi-1 cells stably transduced with the lentiviral pCDH-EF1-IRES-Puro MAPT dual-fluorescence splicing reporter treated with (left) DMSO (scale bar, 200 μm) and (middle) 100 nM Rebecsinib (scale bar, 200 μm). Right: the ratio of RFP/GFP was quantified using Volocity software (Quorum Technologies) following Rebecsinib treatment and normalized to DMSO vehicle controls (n = 1). (F) FACS analysis of RFP/GFP MFI in the live cell population of CD34 + selected pAML patient samples treated with 100 nM Rebecsinib for 72 h (n = 6 biological replicates, Student's t test, p = 0.016). (G) FACS analysis of RFP/GFP MFI in MOLM13 (a leukemia cell line derived from a 20-year-old male) cells stably transduced with the lentiviral pCDH-EF1a-IRES-Puro MAPT dual-fluorescence splicing reporter and treated in vitro with Rebecsinib (100 nM) versus DMSO (n = 3 technical replicates, data represent mean ± SEM, Student's t test, p < 0.0001). (H) MOLM13-MAPT splicing reporter stably transduced cells were engrafted into irradiated NSG-SGM mice, treated with vehicle (n = 3 biological replicates) or Rebecsinib (10 or 20 mg/kg; n = 2, n = 3 technical replicates), and sacrificed 24 h post-treatment. The ratio of RFP/GFP MFI in live, human CD45 + cells in bone marrow, spleen, and peripheral blood was quantified by FACS analysis using a MACS Quant instrument and FlowJo software. Data represent mean ± SEM for each treatment condition. Statistical analyses were performed using unpaired, two-tailed Student’s t test (bone marrow [left] vehicle versus 20 mg/kg p = 0.0013, peripheral blood [middle] vehicle versus 20 mg/kg p = 0.0488, spleen [right] vehicle versus 20 mg/kg p < 0.0001). See also Figure S6 .

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: Inhibition, Colony Assay, shRNA, Control, Knockdown, Derivative Assay, Fluorescence, Imaging, Stable Transfection, Transduction, Software, In Vitro, Irradiation, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Detection and targeting of splicing deregulation in pediatric acute myeloid leukemia stem cells

doi: 10.1016/j.xcrm.2023.100962

Figure Lengend Snippet:

Article Snippet: CD34 Micro-Bead Kit, Ultra-Pure, human , Miltenyi , Cat# 130-100-453.

Techniques: Virus, Clinical Proteomics, Recombinant, Staining, Generated, Software, RNA Binding Assay

Journal: bioRxiv

Article Title: Patterns of Mitochondrial ATP Predict Tissue Folding

doi: 10.1101/2025.08.31.673364

Figure Lengend Snippet: ( a ) Schematic of HH26-stage lung, showing first branch (b1), second branch (b2), third branch (b3), and length of the primary bronchus (L). ( b ) Graph showing L as a function of developmental time. Each smaller dot represents one lung. Shaded region represents the 95% confidence interval of the linear fit. ( c ) Graph showing the relative positions of b1, b2, and b3 as a function of developmental time, normalized to L. Shown are data from 6 lungs for each timepoint. Shaded region represents the 95% confidence interval of the linear fits. ( d ) Volume rendering of epithelium within HH26-stage lung, color-coded to indicate dorsal (gold) and ventral (blue) regions of the epithelium. ( e ) Graph of normalized Tom20 staining intensity in the dorsal (gold) and ventral (blue) epithelium, as a function of distance along the primary bronchus, normalized such that 1 is the mean Tom20 intensity of the lung. ( f ) Graph of Tom20 staining intensity in the mesenchyme, total epithelium, and branching epithelium as a function of developmental stage. Error bars indicate s.e.m. for 3 lungs at the 0-branch stage (116 hr) and 5 lungs at each subsequent stage. (*) indicates p<0.051, (**) p<0.01, (***) p<0.001, ( g ) Fluorescence images of F-actin (green), Tom20 (magenta), and nuclei (blue) in a single branch of a developing lung. Scale bar, 20 μ m. ( h ) Fluorescence images of staining for F-actin (green), Tom20 (magenta), and nuclei (blue) at an uncurved future branch site in the dorsal epithelium. Scale bar, 10 μm. ( i ) Graph showing quantification of normalized Tom20 intensity as a function of position along the apicobasal axis, averaged along the proximal-distal axis of a dorsal branching region. Shaded error bar indicates standard deviation along the proximal-distal axis. Fluorescence images of MitoView633 dye visualizing mitochondrial membrane potential in ( j ) a branch, ( k ) a nascent branch, and ( l ) an uncurved future branch site in the dorsal epithelium. Scale bars, 50, 50, 10 μ m. ( m ) Graph showing quantification of normalized MitoView633 intensity along the apicobasal axis of a branching region. Shaded error bar indicates standard deviation of integrated intensity.

Article Snippet: After blocking, samples were incubated in blocking buffer containing primary antibodies against Tom20 (1:100; D8T4N, rabbit mAb; Cell Signaling 42406S), E-cadherin (1:500; clone 36, mouse mAb; BD 610182), or pMLC (1:50; Ser19, mouse mAb; Cell Signaling 3675S) for 48 hours at 4°C.

Techniques: Staining, Fluorescence, Standard Deviation, Membrane

Journal: bioRxiv

Article Title: Patterns of Mitochondrial ATP Predict Tissue Folding

doi: 10.1101/2025.08.31.673364

Figure Lengend Snippet: ( a ) Schematic of early embryonic chicken lung, indicating sites of apical constriction. ( b ) Projections of confocal images of staining for E-cadherin (orange, left) and Tom20 (color-coded, right) at different stages of development. Scale bar, 200 μm. ( c ) Local intensity of Tom20 within the epithelium, color-coded based on intensity. Scale bar, 25 μm. ( d ) Graph of Tom20 staining intensity in the entire epithelium and mesenchyme, proximal (non-branching) epithelium, medial (branching) epithelium, and adjacent mesenchymal regions. Shown are data from 18 lungs (small circles) across 6 independent replicates (means shown by large circles). Lines match independent replicates between measurements. Box plot shows median, first, and third quartile of data. *, *** indicate p=0.02, p=0.001 respectively. ( e ) Maximum-projection image of staining for Tom20 (magenta) and nuclei (blue), partitioned into four regions: (I) non-branching, (II) branching, (III) nascent branching, and (IV) future branching. Scale bar, 100 μm. ( f ) Graph of normalized Tom20 staining intensity in regions (I-IV) of lungs at different stages of development. Shown are data from 21 lungs across 9 independent replicates. *** indicates p=2e-7, p=9e-5 respectively ( g ) Maximum-intensity projection of staining for Tom20; insets show intensity and apicobasal patterning in single z-planes. Scale bar, 50 μm. ( h ) Maximum-intensity projection of MitoView633 fluorescence. Scale bar, 100 μm. ( i ) Graph of normalized MitoView633 intensity in regions (I-IV) of lungs at different stages of development. Shown are data from 17 lungs. ***, *, * indicate p=0.0003, p=0.01, and p=0.03 respectively.

Article Snippet: After blocking, samples were incubated in blocking buffer containing primary antibodies against Tom20 (1:100; D8T4N, rabbit mAb; Cell Signaling 42406S), E-cadherin (1:500; clone 36, mouse mAb; BD 610182), or pMLC (1:50; Ser19, mouse mAb; Cell Signaling 3675S) for 48 hours at 4°C.

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: Patterns of Mitochondrial ATP Predict Tissue Folding

doi: 10.1101/2025.08.31.673364

Figure Lengend Snippet: ( a ) Timelapse images of ATPRed1 intensity during branch initiation in the embryonic chicken lung. Scale bar, 100 μm. ( b ) Graph showing ATPRed1 intensity at the dorsal side relative to the ventral side (red) and deflection of the epithelium (black) as a function of time, for the inset region shown in (a). Shaded band represents 95% confidence interval of linear fit. ( c ) Graph of normalized ATPRed1 intensity in regions (I-IV; defined in ) of lungs at different stages of development. Shown are data from 17 lungs. **, *, * indicate p=0.001, p=0.04, and p=0.02, respectively. ( d ) Phase-contrast images of lung explants before and 24 hours after culture in the presence of DMF control or blebbistatin (20 μM). Scale bar, 500 μm. ( e ) Maximum intensity projections of ATPRed1 staining immediately after and 3 hours after culture in the presence of DMF control or blebbistatin (20 μM). Scale bar, 100 μm. ( f ) Fold change in ATPRed1 over 2 hours of treatment in dorsal branching tissue. Shown are data from 8 independent control lungs and 7 independent blebbistatin-treated lungs. *, ** indicates p=0.03, and p=0.0004 respectively. ( g ) Top: maximum-intensity projection images of staining for Tom20 (magenta) and F-actin (green) in lungs cultured in the presence of DMF control or blebbistatin. Scale bar, 400 μm. Bottom: insets show pMLC (red) and nuclei (blue) at the region of an initiating branch. Graphs show pMLC from proximal (0) to distal (L) along the outer edge of the epithelium, as indicated by the dotted white line. Data are normalized and averaged over four samples. Shaded region represents standard error of the mean (s.e.m.). ( h ) Schematic of dissections used to divide lungs into proximal (non-branching), medial (branching), and distal (future branching) regions. ( i ) Graph showing oxygen consumption rate of proximal, medial, and distal regions of lungs cultured in the presence of DMF control or blebbistatin. Each dot indicates a replicate, with each replicate containing tissue pooled from 20 lungs. **, *, **, indicate p=0.003, p=0.013 and p=0.002, respectively.

Article Snippet: After blocking, samples were incubated in blocking buffer containing primary antibodies against Tom20 (1:100; D8T4N, rabbit mAb; Cell Signaling 42406S), E-cadherin (1:500; clone 36, mouse mAb; BD 610182), or pMLC (1:50; Ser19, mouse mAb; Cell Signaling 3675S) for 48 hours at 4°C.

Techniques: Control, Staining, Cell Culture

Journal: bioRxiv

Article Title: Patterns of Mitochondrial ATP Predict Tissue Folding

doi: 10.1101/2025.08.31.673364

Figure Lengend Snippet: ( a ) Schematic of two possible connections between mitochondrial energy and apical constriction. Actomyosin-driven apical constriction either precedes (left) or follows (right) mitochondrial ATP production. ( b ) Graph showing oxygen consumption rate of lungs as a function of time before and after treatment with oligomycin (2 μm). ( c ) Phase-contrast images of lung explants before and 24 hours after culture in the presence of DMF control or oligomycin (2 μm). Scale bars, 500 μm. ( d ) Graph showing the mean number of new branches as a function of time in lung explants cultured in the presence of DMF control or oligomycin (2 μm), averaged over 6 independent experiments. Shaded error bar indicates s.e.m. Inset bar graph shows initial and final number of branches. *** indicates p=0.000016. ( e ) Fluorescence images of staining for Tom20 (magenta) and F-actin (green) in lung explants cultured for 12 hours in the presence of DMF control or oligomycin (2 μm). Scale bars, 200 μ m. ( f ) Fluorescence images of staining for Tom20 (magenta), F-actin (green), and DAPI (blue). Insets show: DAPI (blue) and pMLC (red). Scale bars, 20 μ m. ( g ) Graph showing quantification of normalized Tom20 intensity as a function of position along the apicobasal axis of the branching epithelium, manually aligned for 3 independent staining experiments. Shaded error bar indicates s.e.m. at each position.

Article Snippet: After blocking, samples were incubated in blocking buffer containing primary antibodies against Tom20 (1:100; D8T4N, rabbit mAb; Cell Signaling 42406S), E-cadherin (1:500; clone 36, mouse mAb; BD 610182), or pMLC (1:50; Ser19, mouse mAb; Cell Signaling 3675S) for 48 hours at 4°C.

Techniques: Control, Cell Culture, Fluorescence, Staining

Journal: bioRxiv

Article Title: Patterns of Mitochondrial ATP Predict Tissue Folding

doi: 10.1101/2025.08.31.673364

Figure Lengend Snippet: Schematics of ( a ) ventral furrow formation during gastrulation in Drosophila , ( b ) closure of the neural tube during neurulation in chicken, and ( c ) invagination of the lens placode during eye development in mouse embryos. ( d-f ) Confocal images showing staining for Tom20 (magenta) and nuclei (blue) during gastrulation, neurulation, and lens invagination. Insets show z-x sections. Scale bars 50 μ m, 50 μ m, and 20 μ m. ( g-i ) Confocal images showing live imaging of apicobasal mitochondrial patterning, as reported by EYFP-Mito in Drosophila, and ATPRed1 in chicken neural tube and mouse optic cup. Scale bars 10 μ m, 20 μ m, and 10 μ m. ( j ) Graph showing normalized Tom20 intensity in each tissue, revealing enrichment of mitochondria on the apical side. ( k ) Graph showing the ATP/ADP ratio (black) resulting from a mitochondrial density pattern (purple) in a 1D reaction-diffusion model, with Michaelis-Menten kinetics of hydrolysis on the apical side due to actomyosin in the region indicated in red and a baseline rate of hydrolysis throughout the cell. ( l ) Graph showing that the reaction-diffusion model predicts a decrease in myosin phosphorylation rate as the mitochondrial distribution is moved from the apical to the basal side of the cell, plotted for different values of the Michaelis constant K m . ( m ) Graph showing measurements of ATP/ADP ratio in cells undergoing apical constriction in branching epithelium of the chicken lung. Inset: confocal ratiometric image of PercevalHR. Scale bar 30 μ m. ( n ) Cutaway view of initial geometry for a simulation based on mitochondrial patterning of tissue deformation in the mouse optic cup; numerical mesh drawn in green, with applied active stress shown in grayscale. ( o ) Cutaway view of mesh deformed by active stresses, colored by the displacement field. ( p ) Confocal z-slice reconstruction of ATPRed1 signal in the mouse optic cup. Scale bar 50 μ m.

Article Snippet: After blocking, samples were incubated in blocking buffer containing primary antibodies against Tom20 (1:100; D8T4N, rabbit mAb; Cell Signaling 42406S), E-cadherin (1:500; clone 36, mouse mAb; BD 610182), or pMLC (1:50; Ser19, mouse mAb; Cell Signaling 3675S) for 48 hours at 4°C.

Techniques: Staining, Imaging, Diffusion-based Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

doi: 10.1101/2025.05.30.656369

Figure Lengend Snippet: A) Schematic overview of HCR-Proxy. The main part of the in situ workflow is streamlined into tubes, where chemically fixed cells are hybridized with pairs of anti-sense probes towards target RNA. Only the complete probe pair alignment enables signal amplification (HCR) via polymerised DIG-labelled metastable hairpins and therefore recruitment of PL enzyme to catalyse in situ proximity biotinylation (Proxy). B) HCR-FISH / IF micrographs of non-coding RNA Malat1 (yellow, DyLight594-conjugated anti-DIG) in mESCs colocalising with nuclear speckle marker SC-35 (red, SC-35 antibody), merged with DAPI staining (blue). The colocalisation of the signal was calculated using Pearson’s correlation coefficient (PCC) from 13 images (dots represent each cell). C) HCR-Proxy FISH micrographs of Malat1 in mESCs supported with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals. Cells nuclei were visualized with DAPI staining blue). The colocalisation of the signal was calculated using Pearson’s correlation coefficient (PCC) from 10 images (dots represent each cell). D) HCR-Proxy FISH micrographs of Efl1 intronic transcript (red, DyLight594-conjugated anti-DIG) and its proximal vicinity labelled with two different PL enzymes, APEX2 or TurboID (yellow, Alexa647-conjugated streptavidin), merged with DAPI staining (blue). Difference in intensity density was calculated with two-sided Student’s t-test (p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *). E) HCR-Proxy FISH micrographs of Efl1 intronic transcript with signal intensity profiles of RNA FISH (red, DyLight594-conjugated anti-DIG) and HCR-Proxy (yellow, Alexa647-conjugated streptavidin) signals, merged with DAPI staining (blue).

Article Snippet: To visualize RNA labelled with digoxigenin we used DyLight® 594 anti-DIG antibody (goat, Vectorlabs, cat# DI-7594-.5, 1:100 dilution).

Techniques: In Situ, Amplification, Marker, Staining

Journal: bioRxiv

Article Title: HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

doi: 10.1101/2025.05.30.656369

Figure Lengend Snippet: A) Schematic workflow of STED image acquisition platform. In each coarsely sampled confocal image of the tiled panorama scan, nuclei and RNA foci were automatically identified for subsequent super-resolution STED imaging. B) STED microscopy reveals nonspecific biotinylation. STED micrograph and intensity plot of the biotinylation signal (yellow, Alexa647-conjugated streptavidin) beyond RNA FISH boundary (red, DyLight594-conjugated anti-DIG). Reduced intensity of the biotinylation signal in the condensate’s core is barely detected with confocal microscopy. C) Schematic workflow of the fluorescence correlation spectroscopy (FCS) experiment to infer the particle diffusion dynamics (red dots present tracer molecule Alexa 647) sampled in cells’ nucleus, cytoplasm, and extracellular compartment. Autocorrelation curves shifted towards longer lag times indicate slower diffusion. Characteristic diffusion times (DT) shown in panel D were obtained by fitting experimental autocorrelation curves with a 3D diffusion model. D) Quantification of diffusion time for Alexa647 fluorophore from FCS experiment within different cellular compartments for different conditions. Average number of measurements per condition, depicted as dots: extracellular = 30, cytoplasm = 60, nucleus = 60 (two-sided Student’s t-test; p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *). E) Effect of molecular crowding agents on the biotinylation specificity. Top: HCR-Proxy FISH micrographs against Efl1 intronic transcript, where proximity labelling was performed with PBS, 2.5 % BSA, 5 % BSA, 40 % trehalose and Combination of BSA and trehalose (2.5 % BSA & 20 % trehalose). Bottom: Quantification of the difference in radius size between biotinylation (yellow, Alexa647-conjugated streptavidin) and RNA FISH signal (red, DyLight594-conjugated anti-DIG). Approximately 100 images per condition were acquired, depicted as dots (two-sided Student’s t-test; p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *). F) Effect of labelling time extension on the biotinylation specificity. Top: HCR-Proxy FISH micrographs of Efl1 transcript, where proximity labelling was performed under five different labelling times: 1, 3, 5, 15 and 30 minutes, respectively. For this experiment labelling solution Combination 1 was utilized. Bottom: Quantification of the difference (delta) in radius size between biotinylation (yellow, Alexa647-conjugated streptavidin) and RNA FISH signal (red, DyLight594-conjugated anti-DIG). Approximately 100 images per condition were acquired, depicted as dots (two-sided Student’s t-test; p < 0.001 = ***, p < 0.01 = **, p < 0.05 = *).

Article Snippet: To visualize RNA labelled with digoxigenin we used DyLight® 594 anti-DIG antibody (goat, Vectorlabs, cat# DI-7594-.5, 1:100 dilution).

Techniques: Imaging, Microscopy, Confocal Microscopy, Fluorescence, Spectroscopy, Diffusion-based Assay

Journal: bioRxiv

Article Title: HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

doi: 10.1101/2025.05.30.656369

Figure Lengend Snippet: A. HCR-FISH micrographs of pre-rRNA (A’ downstream), Norad and intronic Efl1 sequence (yellow, DyLight594-conjugated anti-DIG) combined with immunofluorescence detection of nucleolar marker NPM1 (red, NPM1 antibody), merged with DAPI (blue). B. UMAP analysis of nucleolar and nuclear targets from benchmarked HCR-Proxy MS experiment. C. Volcano plot for the pre-rRNA-proximal proteome. Proteins marked in red present bona fide nucleolar proteins, meanwhile literature-known nucleolar proteins are labelled (two-sided Student’s t test-based log2 fold-change > 1, fdr- adjusted p-value < 0.05). D. GO term annotation of identified bona fide nucleolar protein interactors.

Article Snippet: To visualize RNA labelled with digoxigenin we used DyLight® 594 anti-DIG antibody (goat, Vectorlabs, cat# DI-7594-.5, 1:100 dilution).

Techniques: Sequencing, Immunofluorescence, Marker

Journal: bioRxiv

Article Title: HCR-Proxy resolves site-specific proximal RNA proteomes at subcompartmental nanoscale resolution

doi: 10.1101/2025.05.30.656369

Figure Lengend Snippet: A. HCR-FISH micrographs of RNA baits targeting sublayers of multiphased nucleolus (A’ upstream, A’ downstream and ITS2) (yellow, DyLight594-conjugated anti-DIG) combined with immunofluorescence detection of FC marker POLR1A, DFC marker FBL and GC marker NPM1 (red), merged with DAPI (blue). B. Radar plot showing the mean difference in amino acid usage in A′ UP, A′ DOWN, and ITS2 proximal proteomes relative to the nuclear background, with 95% confidence intervals. C. Clustermap of mean NARDINI pairwise blockiness Z-scores of eight amino acid residue classes for A′ UP, A′ DOWN and ITS2 proximal proteomes. D. Annotation analysis of RRM domain presence in A′ UP, A′ DOWN, and ITS2 proximal proteomes and nuclear control. Left: percentage of proteins containing at least one RRM domain. Right: percentage of proteins with a given number of RRM domains.

Article Snippet: To visualize RNA labelled with digoxigenin we used DyLight® 594 anti-DIG antibody (goat, Vectorlabs, cat# DI-7594-.5, 1:100 dilution).

Techniques: Immunofluorescence, Marker, Residue, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Biomarker Discovery, Labeling, Single-particle Tracking, Fluorescence Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Diffusion-based Assay, Concentration Assay

Journal: Lab on a chip

Article Title: A human initial lymphatic chip reveals distinct mechanisms of primary lymphatic valve dysfunction in acute and chronic inflammation †

doi: 10.1039/d3lc00486d

Figure Lengend Snippet: An engineered 3D lymphatic vessel model to examine primary lymphatic valve function in vitro. (A) A representation of primary and secondary lymph valves in initial and collecting lymphatic vessels, respectively, in vivo. During the expansion phase, greater hydrostatic pressure in the interstitium surrounding the initial lymphatic vessel causes microvalves to open and fluid to enter (top). During the compression phase, greater pressure in the vessel lumen closes the microvalves and opens the secondary lymph valves, causing fluid to flow downstream (bottom). (B) A schematic of the organotypic 3D lymphatic vessel model (lymphatic vessel-on-chip), with a channel of lymphatic endothelial cells (LECs) embedded in collagen 1 matrix and an acellular channel to induce interstitial fluid flow. (C) A representative bright-field image of engineered rudimentary lymphatic vessels. (D) An engineered lymphatic vessel stained with anti-Prox1 (a lymphatic endothelial marker) antibodies, anti-Ve-cadherin (adherens junctions) antibodies, and DAPI (cell nuclei) to show LEC identification. (E and F) Two settings of experiments to represent primary lymphatic valve function in the initial lymphatic vessels. (E) Upon increase in interstitial fluid pressure, anchoring filaments pull the overlapping junctions apart and allow entry of fluid and immune cells towards the lumen, which can be modeled in vitro by introducing interstitial fluid to the acellular channel (left-side channel) and measuring fluid drainage into the lymphatic vessel (drainage assay). (F) After the initial fluid drainage, the luminal fluid pressure becomes higher than the interstitial fluid pressure. In this condition, primary lymphatic valves close the overlapping junctions and keep the lymph and molecules or immune cells within the lumen, which can be modeled by introducing lymph fluid directly to the lumen of the engineered lymphatic vessel (right-side channel) and measuring diffusion of the fluid to the interstitial space (permeability assay).

Article Snippet: In order to examine the degree of junction disruption between LECs in lymph vessels treated under different inflammatory conditions, the aforementioned fixing, staining, and imaging procedures were used, with the primary antibodies changed to detect VE-cadherin (Santa Cruz Biotechnology, Dallas, TX, 1 : 100) and Prox1 (ReliaTech GmbH, Germany, 1 : 100).

Techniques: In Vitro, In Vivo, Staining, Marker, Diffusion-based Assay, Permeability

Journal: Lab on a chip

Article Title: A human initial lymphatic chip reveals distinct mechanisms of primary lymphatic valve dysfunction in acute and chronic inflammation †

doi: 10.1039/d3lc00486d

Figure Lengend Snippet: Effects of acute and chronic inflammation on lymphatic junctions. (A) Ve-cadherin, prox1, actin (phalloidin), and DAPI staining on control lymphatic vessels with no cytokine treatment. (B) Ve-cadherin, Prox1, actin, and DAPI staining on lymphatic vessels under acute inflammation (10–20 ng ml−1 of TNF-α treatment for 2 hours). (C) Ve-cadherin, Prox1, actin, and DAPI staining on lymphatic vessels under chronic inflammation (10–20 ng ml−1 of TNF-α treatments for 48 hours). Arrows in the enlarged images indicate the presence of junctional disruptions. (D) Quantification of the average number of disruptions per cell across different concentrations and exposure times of TNF-α. *p = 0.0248. (E) Quantification of the actin signal intensity normalized to cell number across different concentrations and exposure times of TNF-α. **p = 0.0049 (control vs. 48 h), **p = 0.0063 (2 h vs. 48 h); (F) quantification of the vessel gap area as a percentage of whole vessel area across different concentrations and exposure times of TNF-α. *p = 0.0244 (control vs. 48 h), *p = 0.0239 (2 h vs. 48 h); (G) quantification of the relative cell junctional area as a percentage of whole vessel area across different concentration and exposure times of TNF-α. *p = 0.0209; one-way ANOVA with Tukey’s HSD tests, n shown as individual data points for each group. Data are expressed as mean ± S.E.M.

Article Snippet: In order to examine the degree of junction disruption between LECs in lymph vessels treated under different inflammatory conditions, the aforementioned fixing, staining, and imaging procedures were used, with the primary antibodies changed to detect VE-cadherin (Santa Cruz Biotechnology, Dallas, TX, 1 : 100) and Prox1 (ReliaTech GmbH, Germany, 1 : 100).

Techniques: Staining, Control, Concentration Assay

Journal: Lab on a chip

Article Title: A human initial lymphatic chip reveals distinct mechanisms of primary lymphatic valve dysfunction in acute and chronic inflammation †

doi: 10.1039/d3lc00486d

Figure Lengend Snippet: Effects of anti-inflammatory dexamethasone treatment on impaired lymphatic drainage and permeability. (A) Quantification of lymphatic drainage (% of total) under acute inflammation and pre-treatment with dexamethasone. **p = 0.0022 (control vs. 2 h), **p = 0.0087 (2 h vs. 2 h + dexamethasone); (B) quantification of area fraction of fibrillin signal normalized by the whole vessel area under acute inflammation and pre-treatment with dexamethasone. *p = 0.0321 (control vs. 2 h), *p = 0.0396 (2 h vs. 2 h + dexamethasone); (C) fibrillin staining on lymphatic vessels under acute inflammation pre-treated with dexamethasone. (D) Quantification of lymphatic permeability under chronic inflammation and pretreatment with dexamethasone. *p = 0.0316; (E) quantification of the vessel gap area as a percentage of the whole vessel area under chronic inflammation and pre-treatment with dexamethasone. *p = 0.0368; (F) Ve-cadherin staining on lymphatic vessels under chronic inflammation pretreated with dexamethasone. One-way ANOVA with Tukey’s HSD tests, n shown as individual data points for each group. Data are expressed as mean ± S.E.M.

Article Snippet: In order to examine the degree of junction disruption between LECs in lymph vessels treated under different inflammatory conditions, the aforementioned fixing, staining, and imaging procedures were used, with the primary antibodies changed to detect VE-cadherin (Santa Cruz Biotechnology, Dallas, TX, 1 : 100) and Prox1 (ReliaTech GmbH, Germany, 1 : 100).

Techniques: Permeability, Control, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Characterization of differentiated PC12 cells and validation of single-molecule labeling. (A,B) Left, Intensity profiles of a single ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) signal. The arrows indicate single-step photobleaching. Right, Histogram showing the intensity value of every spot found in a recording of ATTO 488-labeled GluR2-AMPAR (A) and mGluR1 (B) , superimposed with a single fitted lognormal curve (blue line). (C) Representative trajectories of AMPAR molecules on somas and neurites. Scale bar = 2 μm. (D) The mean square displacement functions and trajectories represent AMPAR molecules with Brownian motion (red) and confined motion (blue). Scale bar = 0.1 μm. (E,F) The cumulative probability functions of D values of AMPAR (E) and mGluR1 (F) on neurites and somas ( n = 510–676 trajectories). *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Biomarker Discovery, Labeling, Single-particle Tracking, Fluorescence Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPAR and mGluR1. (A) Effect of different concentrations of E2 on the diffusion coefficient (D, μm 2 /s) of GluR2-AMPAR (A) and mGluR1 (B) (% of vehicle treatment as the mean ± SEM, n = 425–1145 trajectories per group). (C,D) Line graphs depict changes in D of GluR2-AMPAR (C) and mGluR1 (D) molecules at different time points after the administration of the most effective concentration of E2 (% of vehicle treatment as the mean D ± SEM, n = 117–187 trajectories per time point). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Diffusion-based Assay, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of estrogen receptor modulation on the surface movement of GluR2-AMPAR. (A) Representative PCR gel electrophoresis image depicting the expression of estrogen receptor beta (ERβ) and G protein-coupled estrogen receptor 1 (GPER1) mRNA in dPC12. Estrogen receptor alpha (ERα) mRNA was not detected. (B) Histograms demonstrate the mean D AMPAR as a percentage of vehicle control on somas and neurites in the presence of the estrogen receptor, β (ERβ) agonist diarylpropionitrile (DPN), a GPER1 agonist (G1), G1+DPN together, a GPER1 antagonist (G15) and G15+E2 (with 100 pM of E2 on the somas and 100 nM of E2 on the neurites) (mean ± SEM; n = 215–641 trajectories). *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Gel Electrophoresis, Expressing, Control, Polymerase Chain Reaction

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: The GluR2-AMPAR/GPER1 ratio and molecular distance between GPER1 and GluR2-AMPAR in the membrane. (A) STORM images depicting immunolabeled AMPAR (magenta) and GPER1 (cyan) molecules on dPC12. Dashed lines delineate the borders of the neurites and somas. Scale bar = 2 μm; inset Scale bar = 0.5 μm. (B) The ratio between the number of GPER1 and AMPAR molecules (GPER1/GluR2-AMPAR) on the neurites and somas ( n = 11 somas or neurites). (C1) Photomicrographs depict GPER1 immunoreactivity (visualized with STED microscopy) in dPC12 after 10 min of vehicle (left) or of 100 nM of E2 treatment (right). Scale bar = 2 μm. (C2) One 2 μm 2 (between parallel white bars) and one 10 μm 2 (to the left) areas were selected within each ROI for the membrane and cytoplasmic regions of each cell, respectively. Integrated density was calculated and normalized to the area. Scale bar = 0.5 μm. (D) Dual labeling of plasma membrane and GPER1 molecules defines the membrane regions (approximately 1 μm wide). Scale bar = 0.5 μm. (E) Line graph of the fluorescent intensity calculated from the magnified STED inserts (C2). (F) Integrated density graphs of GPER1 show the effect of vehicle and 100 nM of E2 treatment in the membrane and in the cytoplasm ( n = 15 cells were evaluated in each group). * p < 0.05.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Membrane, Immunolabeling, Microscopy, Labeling, Clinical Proteomics, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: Single-Molecule Imaging Reveals Rapid Estradiol Action on the Surface Movement of AMPA Receptors in Live Neurons

doi: 10.3389/fcell.2021.708715

Figure Lengend Snippet: Effect of E2 on the surface movement of GluR2-AMPA on primary hippocampal neurons. (A) Photomicrograph shows a primary hippocampal neuron labeled with homer-1 (synapse) and β-III tubulin (neuron). Scale bar = 10 μm, insert Scale bar = 2 μm. (B) Dual color STED image of a hippocampal neuron overlayed to differential interference contrast microscopy image depicts live-cell synapse labeling MitoTracker Deep Red (red) and presynaptic protein bassoon (green). Scale bar = 1 μm. (C) Distribution of D values of extrasynaptic and synaptic GluR2-AMPAR under control conditions (median ± IQR, n = 754 extrasynaptic trajectories and n = 104 synaptic trajectories). (D) Effect of E2 (100 pM and 100 nM) on D of extrasynaptic and synaptic GluR2-AMPA with or without chemical LTP (cLTP) induced by glycine/picrotoxin (gly/pic) (% of vehicle treatment as the mean ± SEM; n = 742–928 extrasynaptic trajectories and n = 104–155 synaptic trajectories). (E,F) Effect of vehicle, E2 (100 n, 100 pM) with or without cLTP (gly/pic) on synaptic dwell time (mean ± SEM (s); n = 104–155) (E) and relative surface distribution of synaptic GluR2-AMPAR content (synaptic/total GluR2-AMPA molecule trajectories) (mean ± SEM, n = 8–18 recordings) (F) . * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Before single-molecule imaging, dPC12 were incubated in dRPMI with ATTO 488-labeled antibodies directed against the extracellular N-terminal domain of either rat GluR2 (1:100, Alomone Labs) or rat mGluR1 (1:100, Alomone Labs) at 37°C for 6 min. Specificity of ATTO 488-labeled GluR2-AMPAR antibody has been reported previously in brain sections of GluR2 knockout mice ( ).

Techniques: Labeling, Microscopy, Control, Immunofluorescence

Journal: Molecular cancer therapeutics

Article Title: 3D Collagen vascular tumor-on-a-chip mimetics for dynamic combinatorial drug screening

doi: 10.1158/1535-7163.MCT-20-0880

Figure Lengend Snippet: A. A doxorubicin diffusion profile was captured through confocal microscopy imaging of doxorubicin fluorescent intensity at 1 and 24 h. B. Quantification of doxorubicin diffusion for 1 and 24 h. The grey line was the baseline fluorescence intensity without the presence of doxorubicin. C. 1D simulation of doxorubicin diffusion profile generated by applying the diffusion coefficient of doxorubicin from literature data. The simulation was similar to experimental results for the 1 and 24 h diffusion profiles. D. Viability tests for tumor samples cultured in 96-well plates and in our ECM tumor-on-a-chip device. E. Live/dead staining of tumor samples from Day 0 to Day 6, in 96-well plates and in our device. Scale bar: 200μm.

Article Snippet: Doxorubicin (Selleckchem, in DMSO) was first diluted by DMEM+10%FBS to 100 μM, then injected into the left channel of one blank device (without the presence of tumor samples).

Techniques: Diffusion-based Assay, Confocal Microscopy, Imaging, Fluorescence, Generated, Cell Culture, Staining

Journal: Molecular cancer therapeutics

Article Title: 3D Collagen vascular tumor-on-a-chip mimetics for dynamic combinatorial drug screening

doi: 10.1158/1535-7163.MCT-20-0880

Figure Lengend Snippet: A. 3D devices vs. 2D controls setup. Top: Five tumors were inserted in series into the system. Middle: Simulation of drug diffusion profile for 24, 48, and 72 h. Bottom: In a 2D control, tumors were cultured in 96-well plates and treated directly with constant drug doses equal to the drug dose in the 3D device at 48 h. B. Doxorubicin drug screening results for the device and control. Note that both presented similar trends, yet the absolute drug effect was different. For doxorubicin, 1000 units=100 μM. Experiments were done in triplicates. C. Immunofluorescent staining for DAPI, Ki-67, and caspase 3, and autofluorescence of doxorubicin, in tumor samples treated with doxorubicin. Expression of caspase 3 increased and Ki-67 decreased with higher dosing, which agreed with the live/dead staining results. All images are 700×700μm D. Quantification of immunofluorescent staining intensity of caspase 3 and Ki-67. The lines are polynomial interpolations.

Article Snippet: Doxorubicin (Selleckchem, in DMSO) was first diluted by DMEM+10%FBS to 100 μM, then injected into the left channel of one blank device (without the presence of tumor samples).

Techniques: Diffusion-based Assay, Control, Cell Culture, Drug discovery, Staining, Expressing