Journal: BMC Biology

Article Title: A CD24 + CD271 + melanoma cancer stem cell possesses hybrid characteristics of its single marker counterparts and promotes invasion and therapeutic resistance

doi: 10.1186/s12915-025-02336-2

Figure Lengend Snippet: Cellular heterogeneity and stem cell attributes differ between melanoma cell lines. A Flow cytometric analysis of the four cell lines for CD44 and EpCAM (left) and CD24 and CD271 (right). The graphs show mean ± SEM of sub-populations in each of 4 quadrants based on ± gating set using isotype controls, as a percentage of the total cells, presented as stacked bars. Representative flow cytometry plots for each cell line are to the left of each graph. B Brightfield images of four melanoma cell lines in culture. C Sphere counts for the four cell lines, for primary spheres and secondary spheres after dissociation and re-plating, in serum-containing FAD medium (top) and serum-free medium (bottom). D Gene expression in FACS sorted CD24 + vs CD24 − sub-populations. For each gene, expression was measured by QPCR and is represented as the expression level in CD24 + cells relative to that in CD24 − cells. For each experiment, the number of biological repeats is indicated next to the graph. P -values were calculated using two-way ANOVA

Article Snippet: Antibodies for cell line staining were CD44-PE (clone G44-26, BD Bioscience), CD24-FITC (clone ML5, BD Bioscience, and clone REA832, Miltenyi Biotec), CD271-PerCP/Cy5.5 and CD271-APC (both clone C40-1457, BD Bioscience), and EpCAM-APC (clone HEA-125, Miltenyi Biotec).

Techniques: Flow Cytometry, Gene Expression, Expressing

Journal: BMC Biology

Article Title: A CD24 + CD271 + melanoma cancer stem cell possesses hybrid characteristics of its single marker counterparts and promotes invasion and therapeutic resistance

doi: 10.1186/s12915-025-02336-2

Figure Lengend Snippet: CD24 and CD271 mark a stem cell sub-population in the CHL-1 melanoma line. A Primary sphere counts for four FACS sorted sub-populations from the 4 quadrants based on ± gating set using isotype controls. Results are presented as paired individual data points (due to considerable baseline variation between repeats), with each of the three marker-positive sub-populations compared individually to the CD24-CD271-sub-population. P-values were calculated using paired ANOVA (due to the variation between repeats). B Colony counts in 2D culture for the four FACS sorted sub-populations, with representative images of crystal violet stained colonies for each sub-population. P-values were calculated using unpaired ANOVA. C Flow cytometric analysis for CD24 and CD271, 7 days after sorting and re-plating the four sub-populations, with accompanying brightfield images of the four sorted sub-populations in culture. The graph shows mean ± SEM of sub-populations in each of 4 quadrants based on ± gating set using isotype controls, as a percentage of the total cells, presented as stacked bars. P -values were calculated using two-way ANOVA. Representative flow cytometry plots for each re-plated sub-population are to the left of the graph. For each experiment, the number of biological repeats is indicated next to the graph

Article Snippet: Antibodies for cell line staining were CD44-PE (clone G44-26, BD Bioscience), CD24-FITC (clone ML5, BD Bioscience, and clone REA832, Miltenyi Biotec), CD271-PerCP/Cy5.5 and CD271-APC (both clone C40-1457, BD Bioscience), and EpCAM-APC (clone HEA-125, Miltenyi Biotec).

Techniques: Marker, Staining, Flow Cytometry

Journal: BMC Biology

Article Title: A CD24 + CD271 + melanoma cancer stem cell possesses hybrid characteristics of its single marker counterparts and promotes invasion and therapeutic resistance

doi: 10.1186/s12915-025-02336-2

Figure Lengend Snippet: The CD24 + CD271 + sub-population in the CHL-1 melanoma line has enhanced ability to migrate and invade. A The number of cells that have migrated to the other side of the membrane in transwell assays, for each of the four FACS sorted sub-populations. B Invasion of the four FACS sorted sub-populations into a collagen gel, when seeded in an inner matrigel gel. Left: Representative images, with each blue diamond representing a cell, projected onto x – y co-ordinates ( z co-ordinates are flattened to zero). Units are pixel numbers in the x – y plane, and the orange star indicates the centre of the inner matrigel gel from which distance was calculated for each invading cell. Cells remaining in the inner gel are excluded from the visualisation. Top right: Average invasion distance for the four FACS sorted sub-populations. Bottom right: A representative brightfield image of cells breaking away from the inner gel and invading into the surrounding collagen. For each experiment, the number of biological repeats is indicated next to the graph. P -values were calculated using unpaired ANOVA

Article Snippet: Antibodies for cell line staining were CD44-PE (clone G44-26, BD Bioscience), CD24-FITC (clone ML5, BD Bioscience, and clone REA832, Miltenyi Biotec), CD271-PerCP/Cy5.5 and CD271-APC (both clone C40-1457, BD Bioscience), and EpCAM-APC (clone HEA-125, Miltenyi Biotec).

Techniques: Membrane

Journal: BMC Biology

Article Title: A CD24 + CD271 + melanoma cancer stem cell possesses hybrid characteristics of its single marker counterparts and promotes invasion and therapeutic resistance

doi: 10.1186/s12915-025-02336-2

Figure Lengend Snippet: The CD24 + CD271 + sub-population is drug resistant. A Surviving cells in the CHL-1 melanoma line, after 3-day treatment with the indicated drugs and a 2-day recovery. B – E Surviving cells in the A375M and CHL-1 melanoma lines, after 3-day treatment with the indicated drugs and a 2-day recovery. The asterisk “*” denotes p < 0.05. In the brightfield images in E , blue arrows indicate cells with altered morphology. F Changes in distribution of sub-populations after generation of dasatinib-resistant CHL-1 (left) and A375M (right) melanoma lines. G Changes in distribution of sub-populations after TGFβ treatment for 6 days on CHL-1 (left) and A375M (right) melanoma lines. For all experiments, flow cytometric analysis for CD24 and CD271 is shown. The CD24/CD271 graphs show mean ± SEM of sub-populations in each of 4 quadrants based on ± gating set using isotype controls, as a percentage of the total cells, presented as stacked bars ( A , F , G ) or side-by-side bars ( D ). P -values were calculated using two-way ANOVA ( A , F , G ) or paired t -test ( C , D ). Representative flow cytometry plots for each condition are to the left of each graph. For each experiment, the number of biological repeats is indicated next to the graph

Article Snippet: Antibodies for cell line staining were CD44-PE (clone G44-26, BD Bioscience), CD24-FITC (clone ML5, BD Bioscience, and clone REA832, Miltenyi Biotec), CD271-PerCP/Cy5.5 and CD271-APC (both clone C40-1457, BD Bioscience), and EpCAM-APC (clone HEA-125, Miltenyi Biotec).

Techniques: Flow Cytometry

Journal: BMC Biology

Article Title: A CD24 + CD271 + melanoma cancer stem cell possesses hybrid characteristics of its single marker counterparts and promotes invasion and therapeutic resistance

doi: 10.1186/s12915-025-02336-2

Figure Lengend Snippet: Pathological assessment of CD24 and CD271 staining in 31 melanoma specimens

Article Snippet: Antibodies for cell line staining were CD44-PE (clone G44-26, BD Bioscience), CD24-FITC (clone ML5, BD Bioscience, and clone REA832, Miltenyi Biotec), CD271-PerCP/Cy5.5 and CD271-APC (both clone C40-1457, BD Bioscience), and EpCAM-APC (clone HEA-125, Miltenyi Biotec).

Techniques: Staining

Journal: BMC Biology

Article Title: A CD24 + CD271 + melanoma cancer stem cell possesses hybrid characteristics of its single marker counterparts and promotes invasion and therapeutic resistance

doi: 10.1186/s12915-025-02336-2

Figure Lengend Snippet: A CD24 + CD271 + sub-population in human melanoma tumour specimens. A H&E of a human melanoma specimen, showing regions of melanoma cells (black arrows). B Isotype control staining. C Melanoma specimen with a region of CD24 + CD271 + staining. Blue—DAPI nuclear stain; Green—CD24; Red—CD271. D Magnification of the region of CD24 + CD271 + staining. Displayed images are image-stitches of multiple fields of view at 40 × magnification

Article Snippet: Antibodies for cell line staining were CD44-PE (clone G44-26, BD Bioscience), CD24-FITC (clone ML5, BD Bioscience, and clone REA832, Miltenyi Biotec), CD271-PerCP/Cy5.5 and CD271-APC (both clone C40-1457, BD Bioscience), and EpCAM-APC (clone HEA-125, Miltenyi Biotec).

Techniques: Control, Staining

Journal: BMC Biology

Article Title: A CD24 + CD271 + melanoma cancer stem cell possesses hybrid characteristics of its single marker counterparts and promotes invasion and therapeutic resistance

doi: 10.1186/s12915-025-02336-2

Figure Lengend Snippet: Scatter plot of CD24 and CD271 (NGFR) expression in scRNAseq of tumour Mel110 from the Broad Institute melanoma immunotherapy dataset [ , ]. Axis scales are gene expression expressed as log2[1 + (TPM/10)], where TPM is transcripts per million. Colour coding represents our designation of cells into the 4 phenotypes based on the bimodal expression. These four designated phenotypes were used to perform the differential expression analysis presented in Additional file 1: Table S1

Article Snippet: Antibodies for cell line staining were CD44-PE (clone G44-26, BD Bioscience), CD24-FITC (clone ML5, BD Bioscience, and clone REA832, Miltenyi Biotec), CD271-PerCP/Cy5.5 and CD271-APC (both clone C40-1457, BD Bioscience), and EpCAM-APC (clone HEA-125, Miltenyi Biotec).

Techniques: Expressing, Gene Expression, Quantitative Proteomics

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Receptor binding and fusogenicity of S proteins of omicron variants (A) Amino Acid sequence alignment of RBDs from different omicron variants. Dashed lines represent residues identical to WT. Residues of RBM are shown in red. (B and C) Cleavage of S proteins from different omicron variants. Plasmids encoding codon-optimized S proteins were transfected into HEK293T cells and the cells were then lysed at 40 h post-transfection. The expression of S proteins was detected by western blot using rabbit polyclonal anti-S2 antibodies. Actin served as the loading control. Experiments were done three times and one representative was shown. Band densities were quantified using ImageLab software to determine the cleaved/FL ratio and normalized to WT. FL S, full-length spike protein. (D) Binding of soluble hACE2 to S proteins of omicron variants. HEK293T cells transiently expressing the indicated S proteins were incubated with biotinylated hACE2 followed by Alexa Fluor 488-conjugated streptavidin and then analyzed by flow cytometry. Experiments were done three times, and one representative was shown. (E and F) Cell-cell fusion mediated by S proteins of omicron variants. HEK293T cells were co-transfected with indicated S protein and eGFP. The cells were detached with trypsin at 48h post-transfection and overlaid on the HEK293 cells expressing hACE2 or Calu3 cells. After 2 h of incubation, five randomly selected images of syncytia were captured using a fluorescent microscope. Fusion areas were quantified using ImageJ software and normalized to WT as fusion efficiency. Experiments were done three times in triplicate and one representative is shown. The statistical difference relative to WT was determined by one-way ANOVA with Dunnett’s multiple testing correction ( n = 3 for (C) and (D); n = 5 for (E) and (F)). The statistical difference between BA.2.86 and JN.1 was determined by a two-tailed t-test ( n = 3 for (C) and (D); n = 5 for (E) and (F)). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Scale bar = 200μm. See also .

Article Snippet: Goat anti-human polyclonal cy5-conjugated antibodies , Bioss Biological Inc (Beijing, China) , Cat#bs-0297G-Cy5.

Techniques: Binding Assay, Sequencing, Transfection, Expressing, Western Blot, Control, Software, Incubation, Flow Cytometry, Microscopy, Two Tailed Test

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Transduction of various omicron S pseudovirions on 293/hACE2 and Calu3 cells (A) S proteins incorporation in pseudovirions. Pseudovirions with different omicron S proteins were pelleted down by centrifugation through 20% sucrose cushion and separated in a 10% SDS-PAGE. Detection of S proteins in pseudovirions was performed by Western blot using rabbit polyclonal anti-S2 antibodies. The p24 served as the loading controls. Experiments were done three times and one representative was shown. (B and C) Entry of pseudovirions of omicron variants. HEK 293/hACE2 and Calu3 cells were transduced with omicron S pseudovirions and lysed at 40 h post-transduction. The transduction efficiencies were determined according to luciferase activities. Experiments were done three times in triplicate and one representative is shown. The statistical difference relative to WT was determined by one-way ANOVA with Dunnett’s multiple testing correction ( n = 3). The statistical difference between BA.2.86 and JN.1 was determined by a two-tailed t-test ( n = 3). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Goat anti-human polyclonal cy5-conjugated antibodies , Bioss Biological Inc (Beijing, China) , Cat#bs-0297G-Cy5.

Techniques: Transduction, Centrifugation, SDS Page, Western Blot, Luciferase, Two Tailed Test

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Receptor binding of omicron variants to various animal ACE2s (A) Relative receptor binding efficiencies of RBDs of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, and JN.1 variants to various animal ACE2s. HEK293T transiently expressing different animal ACE2s were detached with EDTA and incubated with mouse Fc-tagged RBDs of WT, BA.1, XBB.1.16, EG.5.1, BA.2.86, or JN.1, followed by FITC-conjugated polyclonal goat anti-mouse antibodies. The cells were analyzed by flow cytometry. Receptor binding efficiencies were calculated according to WT/hACE2, set as 100%. Experiments were performed three times, and the averages are shown in the heatmap. (B) Statistic analyses of levels of receptor binding between different omicron variant S protein by Wilcoxon test. Significant differences indicated by p < 0.05 are highlighted in green background.

Article Snippet: Goat anti-human polyclonal cy5-conjugated antibodies , Bioss Biological Inc (Beijing, China) , Cat#bs-0297G-Cy5.

Techniques: Binding Assay, Expressing, Incubation, Flow Cytometry, Variant Assay

Journal: iScience

Article Title: Enhanced reverse zoonotic potential and immune evasion by omicron JN.1 variant

doi: 10.1016/j.isci.2025.112824

Figure Lengend Snippet: Effect of L455S mutation on thermal and proteolytic stability on XBB.1.16 and BA.2.86 (A and B) Thermal stability of JN.1 compared to its parental BA.2.86 (left panel) and XBB.1.16–455S to XBB.1.16 (right panel). Pseudovirus of XBB.1.16, XBB.1.16–455S, BA.2.86 and JN.1 were centrifuged through 20% sucrose to remove serum and resuspended in serum-free DMEM, followed by incubation either at 37°C for the indicated time (A) or at the indicated temperature for 2 h (B). The virus suspension was used to transduce 293/hACE2 cells to access their remaining transduction capability. Experiments were performed three times in triplicate, and the representative one was shown. The statistical difference was determined by a multiple t-test ( n = 3). Error bars indicate SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (C) Proteolytic stability of JN.1 compared to its parental BA.2.86 (left panel) and XBB.1.16–455S to XBB.1.16 (right panel). Pseudovirus of XBB.1.16, XBB.1.16–455S, BA.2.86 and JN.1 were resuspended in serum-free DMEM and incubated with soluble hACE2(20 μg) at 37°C for 30 min, followed by incubation with TPCK-treated trypsin. The mixture was immediately boiled with loading buffer containing DTT and separated in a 10% SDS-PAGE. Detection was carried out using rabbit polyclonal anti-SARS-CoV-2 S2 antibodies (1:3000). The numbers below S protein blot are the relative quantification of ratio of S2’/S2 using Image Lab (Bio-Rad). Experiments were performed at least three times, and the representative one was shown.

Article Snippet: Goat anti-human polyclonal cy5-conjugated antibodies , Bioss Biological Inc (Beijing, China) , Cat#bs-0297G-Cy5.

Techniques: Mutagenesis, Incubation, Virus, Suspension, Transduction, SDS Page, Quantitative Proteomics