Journal: Cell Death Discovery

Article Title: YAP is required for prostate development, regeneration, and prostate stem cell function

doi: 10.1038/s41420-023-01637-1

Figure Lengend Snippet: A The morphology of shYAP and control prostate spheres cultured in Matrigel. B The number of prostate spheres were measured from 3 pairs of sphere samples. The data is presented as mean ± SEM (** P < 0.01). C The diameter of prostate spheres was measured in at least five prostate spheres from three pairs of sphere samples. The data are presented as mean ± SEM (** P < 0.01). D The expression of YAP, CTGF and Cyr61 in prostate spheres. E Notch and Hedgehog signaling pathways were compared by qPCR between shYAP and control prostate spheres. F Notch and Hedgehog signaling pathways in early prostate development were compared via qPCR between scYAPKO and WT in 2-week-old mouse prostates. G Prostate spheres treated 10 days with recombinant Shh or control media.

Article Snippet: In several experiments, 0.25ug/ml recombinant mouse Sonic Hedgehog (MedChemExpress) was used to treat prostate spheres.

Techniques: Control, Cell Culture, Expressing, Protein-Protein interactions, Recombinant

Journal: Journal of Nanobiotechnology

Article Title: Targeting CD39 boosts PD-1 blockade antitumor therapeutic efficacy via strengthening CD8 + TILs function and recruiting B cells in cervical cancer

doi: 10.1186/s12951-025-03500-0

Figure Lengend Snippet: CD39 inhibitor combined with PD-1 blockade improved therapeutic efficacy by promoting CXCL13 release and B cell activation . ( A ) Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of CXCL13 in the serum samples of isotype, αPD-1, CD39i, and combined treatment groups of mice. ( B ) The content of CXCL13 in murine tumors was detected using ELISA. ( C ) Flow cytometry allowed detection of the proportion of CXCR5 + B cells in murine tumors. ( D - E ) Flow cytometry ( D ) and dot plots ( E ) showed the proportion of activated B cell infiltration in murine tumors. ( F ) The dot plot displayed the proportion of GCB cells in tumor B220 + B cells. ( G , H ) Dot plots showed the proportion of dark zone (DZ) ( G ) and light zone (LZ) ( H ) in GCB cells in mouse tumors. ( I , J ) Representative histograms ( I ) and summarized Mean Fluorescence Intensity (MFI) diagram ( J ) showed the expression of BCL6 in GCB cells. ( K ) The dot plot showed the proportion of plasma cells among CD19 + B cells of mouse tumors. ( L ) The dot plot displayed the proportion of memory B cells in mouse tumor B220 + B cells. The values were obtained using the Kruskal–Wallis test. * , P < 0.05; ** , P < 0.01; ***; P < 0.001; ns , not significant . Abbreviations: GCB, geminal center B cells; DZ, dark zone; LZ, light zone

Article Snippet: The sections were stained with a CD19 antibody (27949-1-AP; Proteintech; 1:500), Ki67 antibody(ab16667;Abcam;1:200), TUNEL antibody(G1507; Servicebio; 1:200) followed by a secondary antibody (GB23303; Servicebio; 1:200).

Techniques: Drug discovery, Activation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Expressing, Clinical Proteomics

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Schematic representations of ligand-based conventional and split-design CAR approaches. The extracellular domains of BAFF and APRIL were used as target moieties to generate conventional CAR-T cells, referred to as APRIL CAR and BAFF CAR, respectively. b , d Representative images of cell‒cell conjugates captured at 100× oil objective magnification using a laser scanning confocal microscope (Nikon, A1R). APRIL or 9E10-IgG4m (pre-incubated with Myc-APRIL) CAR-T cells were co-cultured with RPMI8226-GFP cells ( b ), while BAFF or 9E10-IgG4m (pre-incubated with Myc-BAFF) CAR-T cells were co-cultured with IM9-GFP cells ( d ). Fluorescent labels included Hoechst (blue), anti-PKC-θ (red), and GFP (green) and a merged view of all stains. Scale bar = 10 μm. c , e Statistical analysis of the mean fluorescence intensity of PKC-θ at the IS in panels b and d, respectively. In panel c, sample sizes: APRIL CAR, n = 37; 9E10-IgG4m, n = 39. In panel e, BAFF CAR, n = 34; 9E10-IgG4m, n = 44. All n values represent individual cells. P values were determined by paired two-tailed t -tests. f , g Cytotoxicity assays of conventional and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. h , i Inflammatory cytokine release assay. Conventional CAR-T cells or sCAR-T cells along with 1 nM corresponding switches were co-cultured with the specific target cells for 24 h at an E:T ratio of 1:1 in triplicate. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. j , l Schematic representations of ligand-based split-design CAR and FDA-approved CAR, referred to as BCMA CAR ( j ) and CD19 CAR ( l ), respectively. k , m Cytotoxicity assays of FDA-approved CAR-T cells and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. Data in this figure are representative of three independent experiments. Error bars represent mean ± SD. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: Microscopy, Incubation, Cell Culture, Fluorescence, Two Tailed Test, Release Assay

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). b Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). c , d Quantification of the average radiance (p/s/cm /sr) of the luminescence, related to APRIL- ( c ) and BAFF-( d )-based CAR-T-cell therapy. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. e Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Tukey correction were used to assess significance. f , g Survival curves of the mice subjected to the indicated treatments. Survival curves were compared using the log-rank (Mantel‒Cox) test. h Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). i Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). j Quantification of the average radiance (p/s/cm /sr) of the luminescence. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) and CD19/CD22 CAR-T. k Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. l Assessment of the presence of persistent human CD3 + (hCD3 + ) T cells in peripheral blood by flow cytometry over a 3-week follow-up period. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) with BAFF-CAR-T at each time point. m Survival curves of mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. All n represents biological replicates from different mice. Data in this figure are representative of one of two independent experiments. Error bars represent mean ± SEM. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). b Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). c Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. d Quantification of the average radiance (p/s/cm 2 /sr) of the luminescence. Two-way ANOVA multiple comparisons in Sidak correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-APRIL) with BCMA CAR-T. e Survival curves of mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. f Timeline of the in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). g Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). h Evaluation of serum inflammatory cytokine release by ELISA 24 hours after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. i Quantification of the average radiance (p/s/cm 2 /sr) of the luminescence. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) with CD19 CAR-T. j Assessment of the presence of tumor cells (GFP + CD19 + or GFP + CD19 - ) in peripheral blood by flow cytometry on the 18th day of the experiment. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. k Survival curves of the mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. All n represents biological replicates with different mice. Data are in this figure representative of one of two independent experiments. Error bars represent mean ± SEM. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: (A) Representative flow cytometric histograms showing the specificity of dimeric CD20 aptamer WB1/1.CD20.1_3S across CD20-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (B) Bar graph quantifying the mean fluorescence intensities of WB1/1.CD20.1_3S binding, highlighting significant specificity (*p < 0.0001). (C; E) Fluorescence histograms for CD19 homodimeric aptamer WB17/17.CD19.1_3S and WB15/15.CD19.1_3S, respectively, demonstrating selective binding to CD19-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (D; F) Quantification of homodimeric aptamer WB17/17.CD19.1_3S and WB15/15.CD19.1_3S, respectively, binding specificity using mean fluorescence intensity. (G) Fluorescence histograms for CD19 heterodimeric aptamer WB15/17.CD19.1_3S, demonstrating selective binding to CD19-positive cell lines (Raji, Toledo, BJAB, SKLY-16, and Ramos). (H) Quantification of heterodimeric aptamer WB15/17.CD19.1_3S binding specificity using mean fluorescence intensity. (I-L) Confocal microscopy images showing colocalization of bivalent WB1/1.CD20.1_3S (L1-L2: Cy3, RED) with a CD20-specific antibody (J1-J2: APC, GREEN) on Raji cells. DNA random aptamer controls and isotype antibody controls confirmed specificity. The aptamer binds to the cell surface membrane, as shown in zoomed-in views (L1-L2). Panel M-P: Confocal microscopy images of bivalent WB17/17.CD19.1_3S (M1-M2: Cy3, RED) colocalizing with a CD19-specific antibody (N1-N2: APC, GREEN) on Raji cells. The aptamer demonstrates specificity and surface binding with no significant off-target interactions. Zoomed-in views (P1-P2) confirmed aptamer binding to the cell membrane. Scale bars = 7 and 5 μm. Mean fluorescence intensity was calculated using the formula: Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence. The aptamer and random mean fluorescence values correspond to the mean fluorescence observed in their respective histograms. Bar graphs represent mean ± standard deviation from three independent experiments with statistical significance indicated (****p < 0.0001). Data represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Fluorescence, Binding Assay, Confocal Microscopy, Membrane, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Internalization studies of CD19 Antibody and Bivalent CD19 Aptamers in Ramos and Raji Cells. (A–H) Time-course analysis of CD19 antibody and bivalent CD19 aptamer internalization in Ramos cells (A, C, E, and G: CD21-negative) and Raji cells (B, D, F, and H: CD21-positive). (A-B) demonstrate internalization of APC-CD19 antibody in Ramos (A) and Raji (B) cells. In Ramos cells (A), CD19 antibody shows efficient internalization over 48 hours, while in Raji cells (B), CD21 expression inhibits internalization. (C-D) show internalization of bivalent CD19 aptamer WB17/17.CD19.1_3S in Ramos (C) and Raji (D) cells. WB17/17.CD19.1_3S shows robust internalization in Ramos cells (C), but reduced uptake in Raji cells owing to CD21-mediated blocking. (E-F) illustrate internalization of bivalent CD19 aptamer WB15/17.CD19.1_3S in Ramos (E) and Raji (F) cells. The aptamer demonstrates high internalization efficiency in Ramos cells (E), but not CD21-positive Raji cells (F). Panels G and H: Internalization of bivalent CD19 aptamer WB15/15.CD19.1_3S in Ramos (G) and Raji (H) cells. Similar to other bivalent aptamers, WB15/15.CD19.1_3S internalizes efficiently in CD21-negative Ramos cells (G), but CD21 expression in Raji cells (H) significantly blocks its internalization. Data are expressed as the percentage of internalization calculated as . Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Expressing, Blocking Assay, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Colocalization of CD19 and CD21 was confirmed by flow cytometry and confocal microscopy, whereas CD20 does not co-localize with CD21. (A) demonstrates flow cytometry histograms showing CD19 (A1: PE-CD19 mAb, Red) and CD21 (A2: APC-CD21 mAb, Light Blue) fluorescence intensity in Raji cells. (A3) Bi-parametric dot plot confirms colocalization of CD19 and CD21 on the same population of cells. (B) shows bar graph quantifying mean fluorescence intensity of CD19 and CD21, highlighting their robust expression on Raji cells. (C–E) Confocal microscopy images showing CD19 (C: PE-CD19, Red) colocalized with CD21 (D: APC-CD21, Light Blue) on the surface of Raji cells. Arrowheads indicate regions of colocalization (D). Panels F1–F3 illustrate flow cytometry analysis of bivalent CD19 aptamer WB17/17.CD19.1_3S (F1: Pink) binding to Raji cells and its colocalization with CD21 (F2: APC-CD21 mAb, Light Blue). (F3) Dot plot shows overlapping signals, confirming aptamer-CD21 interaction. (G) demonstrates the bar graph of mean fluorescence intensity of bivalent CD19 aptamer (WB17/17.CD19.1_3S) and CD21 antibody. (H–J) present confocal microscopy images showing WB17/17.CD19.1_3S (H: Red) colocalized with CD21 (I: Green) on Raji cells. Arrowheads highlight colocalized regions (J). (K1–K3) Flow cytometry analysis showing CD20 (K1: FITC-CD20 mAb, Green) and CD21 (K2: APC-CD21 mAb, Light Blue) fluorescence intensity in Raji cells. (K3) Dot plot shows no significant overlap between CD20 and CD21, indicating no colocalization. (L) Bar graph showing fluorescence intensity of CD20 and CD21. (M–O) Confocal microscopy images showing no colocalization between CD20 (M: Red) and CD21 (N: Green) on Raji cells (O). (P1–P3) Flow cytometry analysis of bivalent CD20 aptamer WB1/1.CD20.1_3S (P1: Blue) and CD21 (P2: APC-CD21 mAb, Light Blue). (P3) Dot plot confirms the absence of colocalization on the same population of cells. Panel Q: Bar graph of fluorescence intensity for CD20 aptamer and CD21. (R–T) Confocal microscopy images showing that WB1/1.CD20.1_3S (R: Red) and CD21 (S: Green) do not co-localize on Raji cells (T). Scale bars = 5 μm. Mean fluorescence intensity was calculated using the formula: Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence for aptamer. As for antibody it was calculated using the formula: Mean fluorescence intensity=Antibody Mean Fluorescence − Isotype Control Mean Fluorescence. The aptamer/random and antibody/isotype mean fluorescence values corresponds to the mean fluorescence observed in their respective histograms. Data represents mean ± standard deviation from three independent experiments. Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Flow Cytometry, Confocal Microscopy, Fluorescence, Expressing, Binding Assay, Control, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: The expression of CD19, CD20, and CD21, the binding activity of bivalent CD19 aptamers, and the internalization dynamics of CD19 antibody and bivalent aptamers in OCl-LY7 and HBL-1 (DLBCL) cells. Antibody staining for CD19, CD20, and CD21 expression. (C; G) demonstrate flow cytometry histograms showing the expression of CD19 (C2: Red), CD20 (C1: Green), and CD21 (C3: Light Blue) on OCI-Ly7 (C), whereas HBL-1 (G) cells express CD19 (G2: Red), CD20 (G1: Green), and CD21 (G3: Light Blue). Isotype controls (gray) confirm specific binding. (D; H) Bar graphs showing mean fluorescence intensities, confirming robust expression of CD19 and CD20, but the absence of CD21 expression in both cell lines. Binding assay with bivalent CD19 aptamers (A1–A4, E1–E4). Fluorescence intensity histograms of bivalent CD19 aptamers (WB17/17.CD19.1_3S, WB15/17.CD19.1_3S, and WB15/15.CD19.1_3S) compared to random DNA control in OCI-Ly7 (A1–A4) and HBL-1 (E1–E4) cells. (B; F) illustrate bar graphs of mean fluorescence intensities showing high binding specificity of bivalent aptamers compared to controls. Internalization of CD19 antibody with and without Proteinase K (I, K). Flow cytometry histograms (L1) and bar graphs (L2) showing internalization dynamics of CD19 antibody in OCI-Ly7 (I), whereas flow cytometry histograms (K1) and bar graphs (K2) show internalization dynamics of CD19 antibody in HBL-1 (K) cells. Internalization was analyzed at 0 hour and 24 hours in the presence and absence of Proteinase K. Data reveal a significant reduction in surface fluorescence intensity after Proteinase K treatment, confirming internalization. Internalization of bivalent CD19 aptamers under the same conditions (J, L). Fluorescence intensity histograms (J1-J3, L1-L3) and bar graphs (J4 and L4) show the internalization of bivalent CD19 aptamers (WB17/17.CD19.1_3S (J2, L2), WB15/17.CD19.1_3S (J1, L1), and WB15/15.CD19.1_3S (J3, L3)) in OCI-Ly7 (J) and HBL-1 (L) cells. Internalization is measured at 0 hour and 24 hours with and without Proteinase K, demonstrating the effective uptake of bivalent aptamers. Data are expressed as the percentage of internalization calculated as fluorescence intensity was calculated using the formula. Mean fluorescence intensity=Aptamer Mean Fluorescence − Random DNA Mean Fluorescence for aptamer. As for antibody it was calculated using the formula: Mean fluorescence intensity=Antibody Mean Fluorescence − Isotype Control Mean Fluorescence. The aptamer/random and antibody/isotype mean fluorescence values corresponds to the mean fluorescence observed in their respective histograms. Each bar represents mean ± standard deviation from three independent experiments. Data are presented as mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Expressing, Binding Assay, Activity Assay, Staining, Flow Cytometry, Fluorescence, Control, Standard Deviation

Journal: bioRxiv

Article Title: The biochemical function of bivalent aptamer assemblies against B-cell markers CD19 and CD20

doi: 10.1101/2025.01.26.634939

Figure Lengend Snippet: Internalization Assay of CD19 Antibody and Bivalent CD19 Aptamer in Ramos and OCI-Ly7 Cells Visualized by Confocal Microscopy. Internalization of CD19 Antibody in Ramos Cells (A-D). Confocal images showing surface-bound APC-CD19 antibody (A1-A2: Red) at 0 hour with and without Hoechst nuclear staining (A2: Blue). (B1–B2) demonstrate the isotype control at 0 hour. Panels C1-C2 show the internalization of APC-CD19 after 24 hours (C1-C2: Red) with and without Hoechst nuclear staining (C2: Blue). (E) Bar graph quantifying the mean fluorescence intensity of APC-CD19 antibody with and without Proteinase K treatment at 0 and 24 hours. Internalization of Bivalent CD19 Aptamer WB17/17.CD19.1_3S in Ramos Cells (F-I). Initial binding of WB17/17.CD19.1_3S (F1-F2: Green) at 0 hours, as shown with Hoechst-stained nuclei (F2: Blue). (H1–H2) demonstrate internalization of WB17/17.CD19.1_3S at 24 hours with and without Hoechst nuclear staining (H2: Blue). (J) shows the bar graph quantifying the mean fluorescence intensity of WB17/17.CD19.1_3S with and without Proteinase K treatment at 0 and 24 hours. Internalization of CD19 Antibody in OCI-Ly7 Cells (K-N) Surface-bound APC-CD19 antibody at 0 hours (K1-K2). (M1–M2) illustrate reduced surface-bound fluorescence at 24 hours, consistent with internalization. After 24 hours, CD19 antibody is completely uptaken on OCl-Ly7 cells. (O) Bar graph quantifying the mean fluorescence intensity of APC-CD19 antibody with and without Proteinase K treatment at 0 and 24 hours. Internalization of Bivalent CD19 Aptamer WB17/17.CD19.1_3S in OCI-Ly7 Cells (P-S). Confocal images showing WB17/17.CD19.1_3S (P1: Green) binding at 0 hours with and without Hoechst nuclear staining (P2: Blue). (R1–R2) highlight internalized WB17/17.CD19.1_3S at 24 hours with Hoechst nuclear staining (R2). Panel T shows the bar graph quantifying the mean fluorescence intensity of WB17/17.CD19.1_3S with and without Proteinase K treatment at 0 and 24 hours. Scale Bars: 5 μm. Data represent mean ± standard deviation from three independent experiments. Each bar represents mean ± standard deviation from three independent experiments.

Article Snippet: PE-conjugated CD19 anti-human monoclonal antibody (mouse, isotype IgG1, Clone 4G7, Catalog no. PE-65|97, Proteintech), PE-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 12-0198-42, Invitrogen), PE-conjugated CD20 anti-human (mouse, isotype IgG2b, κ, Clone 2H7, Catalog no. 302305, BioLegend), PE Mouse IgG1, κ, isotype control (CloneMOPC-21, Catalog no. 556650, BD Pharmingen), FITC-conjugated CD20 anti-human monoclonal antibody (mouse, isotype IgG2b, Clone 2H7, Catalog no. 35-0209-T100), FITC-conjugated CD19 mouse anti-human (isotype IgG1, κ, Clone SJ25C1, Catalog no. 363008, BioLegend), Monoclonal Rabbit IgG Alexa Fluor 488 (Clone 60024B; Catalog no. IC1051G, R&D Systems, MN), APC Mouse IgG1, κ, Isotype Ctrl (FC) (Clone MOPC-21; Catalog no. 400122), APC-conjugated CD19 anti-human (mouse, isotype IgG1, κ, Clone SJ25C1, Catalog no. 17-0198-42, Invitrogen), APC-conjugated mouse anti-human CD20 (isotype IgG2b, κ, Clone 2H7, Catalog no. 559776, BD Pharmingen), and APC-conjugated CD21 mouse anti-human (isotype IgG2a κ, Clone HB5, Catalog no. 17-0219-42, Invitrogen) were used for routine flow cytometry analysis.

Techniques: Confocal Microscopy, Staining, Control, Fluorescence, Binding Assay, Standard Deviation