Journal: STAR Protocols

Article Title: Protocol for studying the immune microenvironment of human hepatocellular carcinoma by Cell DIVE multiplex immunofluorescence imaging

doi: 10.1016/j.xpro.2025.103946

Figure Lengend Snippet: Example of segmentation with Stardist model (A) Original image used for segmentation showing DAPI-labeling of nuclei. (B) Overlay of DAPI-staining and result of segmentation in red (step 22-c). The “Cell display” parameter selected here is “nuclei only” (step 22-d). Scale bar represents 200 μm.

Article Snippet: Mounting medium: DAPI Fluoromount-G , SouthernBiotech , Cat#010-20.

Techniques: Labeling, Staining

Journal: STAR Protocols

Article Title: Protocol for studying the immune microenvironment of human hepatocellular carcinoma by Cell DIVE multiplex immunofluorescence imaging

doi: 10.1016/j.xpro.2025.103946

Figure Lengend Snippet: Example of application of the classification model step 25 (A) mIF image showing CD3 (pink), CD31 (red) and CD68 (green) and DAPI (gray) staining. (B) Objects created by the segmentation and assigned to Lymphocytes CD3+ (pink), Cell endoth. CD31+ (red) or Macrophages CD68+ (green) classes by the classification model on image A. Scale bar represents 200 μm.

Article Snippet: Mounting medium: DAPI Fluoromount-G , SouthernBiotech , Cat#010-20.

Techniques: Staining

Journal: STAR Protocols

Article Title: Protocol for studying the immune microenvironment of human hepatocellular carcinoma by Cell DIVE multiplex immunofluorescence imaging

doi: 10.1016/j.xpro.2025.103946

Figure Lengend Snippet: Example of final mIF image of human hepatocellular carcinoma whole slide For visualization purposes, only 5 markers are displayed: WGA (yellow), CD4 (orange), CD20 (green), CD31 (red), CD68 (blue) and DAPI (gray). Scale bar represents 2 mm.

Article Snippet: Mounting medium: DAPI Fluoromount-G , SouthernBiotech , Cat#010-20.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting autophagy and plasminogen activator inhibitor-1 increases survival and remodels the tumor microenvironment in glioblastoma

doi: 10.1186/s13046-025-03473-w

Figure Lengend Snippet: PAI-1 expression correlates with poor GBM prognosis and is increased in tumors treated with lucanthone. ( A ) Kaplan-Meier survival curve of GBM patients with tumors of all subtypes that express low or high levels of PAI-1 (SERPINE1) (Gliovis/TCGA_GBM dataset). ( B ) Kaplan-Meier survival curve of GBM patients with tumors of the proneural subtype that express low or high levels of PAI-1 (SERPINE1) (Gliovis/TCGA_GBM dataset). ( C ) mRNA expression of PAI-1 (SERPINE1) in GBM compared to non-tumor tissue, unpaired t-test, p-value = 0.00011 (Gliovis/TCGA_GBM dataset using the HG-U133A platform). ( D ) Correlation plots of PAI-1 expression vs. expression of autophagy markers p62 (SQSTM1) and Cathepsin D (CTSD) in GBM tumors, labeled with respective Pearson correlation coefficients (Gliovis). ( E ) Cartoon of in vivo mouse GBM model detailing implantation of GL261 cells and treatment course. ( F ) Representative confocal images of tumors treated with saline control or lucanthone for two weeks and stained with PAI-1, Cathepsin D (CATD), and DAPI nuclear stain. Colocalization of PAI-1 and CATD are shown in the reslice and orthogonal views. Scale bars measure 10 μm. ( G ) Quantification of the signal integrated density divided by the number of DAPI + nuclei, i.e. per cell, shown in F. Each dot represents one animal, average of 3–5 images per animal, n = 11 animals per group, unpaired t-tests

Article Snippet: Cells were then washed 3x with 0.3% TX-100 in PBS, mounted onto glass slides using DAPI Fluoromount counterstain (Southern Biotech 0100 − 20), and imaged under the Leica SP8-x confocal microscopy system, with white light and argon lasers.

Techniques: Expressing, Labeling, In Vivo, Saline, Control, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting autophagy and plasminogen activator inhibitor-1 increases survival and remodels the tumor microenvironment in glioblastoma

doi: 10.1186/s13046-025-03473-w

Figure Lengend Snippet: Lucanthone increases autophagy markers and intracellular PAI-1 both within and outside lysosomal machinery. ( A ) Representative western blot of PAI-1 and LC3 expression in GL261 cells treated with DMSO control or 10 µM lucanthone for 24 h. GAPDH was used as loading control. Numbers next to blots are kDa based on protein ladder. ( B ) Quantifications of PAI-1 and LC3-II signal intensity, normalized to loading control (LC). Data presented as fold change of ctrl (DMSO). Each dot represents one independent experiment, unpaired t-tests. ( C ) Representative images of GL261 cells treated with DMSO control or 10 µM lucanthone for 24 h in regular media, stained with PAI-1, Cathepsin D (CATD), and DAPI. Colocalization of PAI-1 and CATD are shown in orthogonal views. Scale bars measure 10 μm. ( D ) Quantification of C. Each dot represents one independent experiment, average of 3–5 images per experiment, unpaired t-tests

Article Snippet: Cells were then washed 3x with 0.3% TX-100 in PBS, mounted onto glass slides using DAPI Fluoromount counterstain (Southern Biotech 0100 − 20), and imaged under the Leica SP8-x confocal microscopy system, with white light and argon lasers.

Techniques: Western Blot, Expressing, Control, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting autophagy and plasminogen activator inhibitor-1 increases survival and remodels the tumor microenvironment in glioblastoma

doi: 10.1186/s13046-025-03473-w

Figure Lengend Snippet: PAI-1 and autophagy inhibition decrease glioma cell proliferation. ( A, B ). MTT assay measuring percentage of live GL261 cells treated with DMSO control, 1.25 and 2.5 µM MDI-2268, 5 and 10 µM lucanthone, or the combination, respectively, for 72 h in regular media (10% serum) relative to the average of control. n = 3 independent experiments, one-way ANOVA. ( C ) HSA synergy analysis of the two agents using Combenefit software. ( D, E ) Cell viability dose-response curves for MDI-2268 and lucanthone, respectively, in GL261 cells, and their EC50 values (% change = % change in relative live cells) plotted using Combenefit. Each data point is the mean (marked with an X) and error bar for 3 independent experiment values. ( F ) Representative images of GL261 cells treated with DMSO control, 2.5 µM MDI-2268, 10 µM lucanthone, or the combination for 48 h in regular media (10% serum), and stained with Ki67, cleaved Caspase 3, and DAPI. Scale bars measure 10 μm. ( G ) Quantification of F, each dot represents one independent experiment, average of 3–5 images per experiment, one-way ANOVA

Article Snippet: Cells were then washed 3x with 0.3% TX-100 in PBS, mounted onto glass slides using DAPI Fluoromount counterstain (Southern Biotech 0100 − 20), and imaged under the Leica SP8-x confocal microscopy system, with white light and argon lasers.

Techniques: Inhibition, MTT Assay, Control, Software, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting autophagy and plasminogen activator inhibitor-1 increases survival and remodels the tumor microenvironment in glioblastoma

doi: 10.1186/s13046-025-03473-w

Figure Lengend Snippet: Lucanthone and combination treatments increase autophagy markers and intracellular PAI-1 both within and outside lysosomal machinery. ( A ) Representative western blot of PAI-1, p62, CATD, and LC3 expression in GL261 cells treated with DMSO control, 2.5 µM MDI-2268, 10 µM lucanthone, or the combination, for 24 h. Vinculin was used as loading control. Numbers next to blots are kDa based on protein ladder. ( B ) Quantification of A, normalized to loading control (LC). Data presented as fold change of DMSO control. Each dot represents one independent experiment, one-way ANOVA. ( C ) Representative images and orthogonal views of GL261 cells treated with the above conditions, stained with PAI-1, CATD, and DAPI. Scale bars measure 10 μm. ( D ) Quantification of C. Data presented as fold change of DMSO control. Each dot represents one independent experiment, average of 3–5 images per experiment, one-way ANOVA

Article Snippet: Cells were then washed 3x with 0.3% TX-100 in PBS, mounted onto glass slides using DAPI Fluoromount counterstain (Southern Biotech 0100 − 20), and imaged under the Leica SP8-x confocal microscopy system, with white light and argon lasers.

Techniques: Western Blot, Expressing, Control, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting autophagy and plasminogen activator inhibitor-1 increases survival and remodels the tumor microenvironment in glioblastoma

doi: 10.1186/s13046-025-03473-w

Figure Lengend Snippet: Lucanthone abrogates extracellular active PAI-1. ( A-C ) Active PAI-1 ELISA on conditioned media of GL261 cells treated with DMSO control, either 1.25 or 2.5 µM MDI-2268, 5 or 10 µM lucanthone, or the combination, for 12, 24, or 72 h. Gray boxed plots are zoomed-in views of the y-axis of the depicted conditions. Data presented as fold change of the average of control (DMSO), which was measured in ng/mL of active PAI-1. Each dot represents one independent experiment, one-way ANOVAs, or unpaired t-test for boxed plot in A. ( D ) Total PAI-1 ELISA on culture media of GL261 cells treated with the labeled conditions. Data presented as fold change of the average of control (DMSO). Each dot represents one independent experiment, one-way ANOVA. ( E-H ) Active or total PAI-1 ELISA on conditioned media of GL261 cells treated with DMSO control, 3 µM mitoxantrone, or 3 µM mitoxantrone + 10 µM lucanthone, for 1–24 h. Data presented as fold change of the average of control (DMSO). Each dot represents one independent experiment, one-way ANOVA. ( I, J ) Representative images of GL261 cells treated with control (DMSO), 3 µM mitoxantrone, 10 µM lucanthone, or the combination, at 1 and 24 h, stained with PAI-1, CATD, and DAPI. Quantifications of PAI-1 signal in select conditions shown. Each dot represents one independent experiment, one-way ANOVA in I, one-way ANOVA or unpaired t-tests in J. Scale bars measure 10 μm

Article Snippet: Cells were then washed 3x with 0.3% TX-100 in PBS, mounted onto glass slides using DAPI Fluoromount counterstain (Southern Biotech 0100 − 20), and imaged under the Leica SP8-x confocal microscopy system, with white light and argon lasers.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Labeling, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting autophagy and plasminogen activator inhibitor-1 increases survival and remodels the tumor microenvironment in glioblastoma

doi: 10.1186/s13046-025-03473-w

Figure Lengend Snippet: PAI-1 inhibition enhanced cytotoxic T-cell infiltration, while lucanthone reduced vascularization. ( A ) Representative images of tumors at day 21 in the 4 conditions stained with CD8a, CD31, and DAPI. Scale bars measure 20 μm. ( B-E ) Quantifications of A. Each dot represents one animal, average of 3–5 images per animal, one-way ANOVA

Article Snippet: Cells were then washed 3x with 0.3% TX-100 in PBS, mounted onto glass slides using DAPI Fluoromount counterstain (Southern Biotech 0100 − 20), and imaged under the Leica SP8-x confocal microscopy system, with white light and argon lasers.

Techniques: Inhibition, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting autophagy and plasminogen activator inhibitor-1 increases survival and remodels the tumor microenvironment in glioblastoma

doi: 10.1186/s13046-025-03473-w

Figure Lengend Snippet: MDI-2268 and lucanthone treatments decreased Arginase 1 and CD206 expression in vivo. ( A ) Representative images of tumors at day 21 in the 4 conditions stained with Arginase 1, CD206, and DAPI. Scale bars measure 20 μm. ( B-G ) Quantifications of A. Each dot represents one animal, average of 3–5 images per animal, one-way ANOVA

Article Snippet: Cells were then washed 3x with 0.3% TX-100 in PBS, mounted onto glass slides using DAPI Fluoromount counterstain (Southern Biotech 0100 − 20), and imaged under the Leica SP8-x confocal microscopy system, with white light and argon lasers.

Techniques: Expressing, In Vivo, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting autophagy and plasminogen activator inhibitor-1 increases survival and remodels the tumor microenvironment in glioblastoma

doi: 10.1186/s13046-025-03473-w

Figure Lengend Snippet: Inhibition of both autophagy and PAI-1 activated immune-stimulatory myeloid cells. ( A ) Representative images of tumors at day 21 in the 4 conditions stained with Iba1, iNOS, and DAPI. Scale bars measure 50 μm. ( B-G ) Quantifications of A. Each dot represents one animal, average of 3–5 images per animal, one-way ANOVA

Article Snippet: Cells were then washed 3x with 0.3% TX-100 in PBS, mounted onto glass slides using DAPI Fluoromount counterstain (Southern Biotech 0100 − 20), and imaged under the Leica SP8-x confocal microscopy system, with white light and argon lasers.

Techniques: Inhibition, Staining