Journal: BMC Pulmonary Medicine

Article Title: β-elemene inhibits tumor-promoting in small cell lung cancer by affecting M2 macrophages and TGF-β

doi: 10.1186/s12890-025-03533-z

Figure Lengend Snippet: The Effect of M2 Macrophages on NCI-H209 Cells. ( A ) PMA-induced differentiation of THP-1 into M0 macrophages. ( B and C ) IL-4 and IL-13 induce the differentiation of M0 macrophages into M2 macrophages. ( D ) CCK8 assay to detect the effect of M2 macrophages on the viability of NCI-H209 cells. ( E ) Flow cytometry to analyze the cell cycle distribution of NCI-H209 cells treated with M2 macrophages. ( F ) Flow cytometry to analyze the cell apoptosis of NCI-H209 cells treated with M2 macrophages. ( G ) Transwell assays to examine the effect of M2 macrophages on the migration of NCI-H209 cells. Scale bar = 100 μm. ( H ) ELISA assay to detect the effect of M2 macrophages on TGF-β expression. ** p < 0.01, *** p < 0.001, ns” stands for “no significant difference

Article Snippet: Subsequently, the cells were incubated with THP-1 cell-specific culture medium (CM-0233, Procell, Wuhan, China) containing IL-4 (20 ng/mL) (HY-P700130AF, MCE, Shanghai, China) and IL-13 (5 ng/mL) (HY-P70568, MCE, Shanghai, China) for 48 h to polarize them into M2 macrophages.

Techniques: CCK-8 Assay, Flow Cytometry, Migration, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: IL12B is upregulated in CCR2-deficient hearts and activates autophagy through PI3K/Akt/mTOR pathway inhibition. (A) Western blot results of IL12A and IL12B protein in heart from WT and CCR2 -/- mouse (n = 6). (B) ELISA quantification of IL12A and IL12B levels in heart lysates from WT and CCR2 -/- mouse (n = 6). (C) Western blot results of IL12B protein in BMDMs differentiated from WT and CCR2 -/- mouse (n = 3 independent experiments). (D) Immunofluorescence co-staining of IL12B (green) and the macrophage marker CD68 (red) in heart sections from WT and CCR2 -/- mouse. Scale bar: 5 μm. (E) Western blot results of autophagy markers (LC3B-II and P62) in H9C2 cells treated with recombinant IL12B protein (n = 3 independent experiments). (F) Quantitative PCR analysis of autophagy-related genes Atg12, Atg5, Atg7, Atg3, and Gabarapl1 in H9C2 cells following vehicle or IL12B treatment (2 ng/mL) (n = 3 independent experiments). (G) Western blot results of autophagy-related signaling pathway in H9C2 treated with vehicle or IL12B (2 ng/mL) (n = 3 independent experiments). (H) Western blot results of pPI3K, PI3K and autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in H9C2 treated with vehicle, IL12B (2 ng/mL) alone, or in combination with the PI3K activator 740 Y-P (3 μM) (n = 3 independent experiments).

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Marker, Recombinant, Real-time Polymerase Chain Reaction

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: IL12B promotes cardiomyocyte autophagy in vitro . (A) Cell viability assay of H9C2 treated with DOX (1 μM) in the presence or absence of recombinant IL12B proteins (2 ng/mL) (n = 3 independent experiments). (B) Western blot analysis of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in the same groups (n = 3 independent experiments). (C) Immunofluorescence staining and quantitative analysis of LC3B puncta in the same groups (n = 3 independent experiments). Scale bar: 20 μm. (D) Tandem fluorescence microscopy of H9C2 cells expressing the mRFP-GFP-LC3 reporter. Yellow puncta represent autophagosomes, and red puncta represent autolysosomes. Scale bar: 10 μm (n = 3 independent experiments). (E) Transmission electron microscopy of H9C2. Arrows indicate autolysosomes. Scale bar: 2 μm, 0.5 μm (n = 3 independent experiments).

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: In Vitro, Viability Assay, Recombinant, Western Blot, Immunofluorescence, Staining, Fluorescence, Microscopy, Expressing, Transmission Assay, Electron Microscopy

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: Recombinant IL12B administration protects against DIC through autophagy activation in vivo . (A) Experimental design for in vivo IL12B administration. (B) Representative M-mode echocardiographic tracings of mouse from the vehicle, DOX alone, and DOX + IL12B groups. Scale bar: 0.1 s, 2 mm. (C, D) Quantitative analysis of EF and FS in the indicated groups (n = 6). (E–G) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (H) Western blot analysis of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from the same groups (n = 6). (I) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (J) Immunofluorescence staining and quantitative analysis of LC3B puncta in cardiomyocytes from the same groups. Scale bar: 50 μm (n = 6). (K) Transmission electron microscopy analysis of autolysosome density from the same groups. Arrows indicate autolysosomes. Scale bars: 1 μm and 0.5 μm (n = 6).

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: Recombinant, Activation Assay, In Vivo, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: IL12B neutralization abolishes CCR2 deficiency-mediated cardioprotection in vivo . (A) Experimental design of IL12B neutralization in CCR2 -/- mouse. (B) Representative M-mode echocardiographic tracings of CCR2 -/- mouse treated with vehicle, DOX alone, or DOX + anti-IL12B Neu. Scale bar: 0.1 s, 2 mm. (C, D) Quantitative results of EF and FS in the indicated groups (n = 6). (E–G) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (H) Western blot results of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from CCR2 -/- mouse treated with vehicle, DOX alone, or DOX + anti-IL12B Neu (n = 6). (I) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (J) Immunofluorescence staining and quantitative analysis of LC3B puncta in cardiomyocytes from the same groups. Scale bar: 50 μm (n = 6). (K) Transmission electron microscopy analysis of autolysosome density in cardiomyocytes from the same groups. Arrows indicate autolysosomes. Scale bars: 1 μm and 0.5 μm (n = 6).

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: Neutralization, In Vivo, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy

Journal: Theranostics

Article Title: CCR2 deficiency protects against doxorubicin-induced cardiac dysfunction through enhanced IL12B-dependent autophagy

doi: 10.7150/thno.131005

Figure Lengend Snippet: Pharmacological CCR2 inhibition protects against DIC without compromising the antitumor efficacy of doxorubicin in 4T1 breast cancer model. (A) Cell viability assay of 4T1 breast cancer cells treated with DOX or in combination with CCR2 antagonist4 (n = 3 independent experiments). (B, C) Tumor growth curves and representative tumor images at the study endpoint in a syngeneic 4T1 breast cancer model. Scale bar: 1 cm (n = 6). (D) Representative M-mode echocardiographic tracings of tumor bearing mouse treated with vehicle, DOX alone, CCR2 antagonist4 alone or DOX + CCR2 antagonist4. Scale bar: 0.1 s, 2 mm. (E, F) Quantitative results of EF and FS in the same groups (n = 6). (G–I) The levels of cardiac injury markers in serum, including LDH-L, CK-MB, and cTnI (n = 6). (J) Western blot results of autophagy markers (LC3B-II, ATG12, GABARAPL1, and P62) in heart tissues from the same groups (n = 6). (K) Quantitative PCR analysis of autophagy-related gene (Atg12, Atg5, Atg7, Atg3, and Gabarapl1) in heart tissues from the same groups (n = 6). (L) Working model. DOX administration triggers cardiomyocyte dysfunction, ultimately leading to heart failure. In contrast, loss of function of CCR2 initiates a protective cascade. Specifically, CCR2 deficiency increases the secretion of IL12B. This cytokine directly targets cardiomyocytes, suppressing PI3K/Akt/mTOR signaling to trigger protective autophagy, which clears damaged organelles and toxic proteins, thus sustaining cellular homeostasis and cardiac function.

Article Snippet: For the rescue or inhibitor experiments, 3-methyladenine (MCE HY-19312, 15 mg/kg), rapamycin (MCE HY-10219, 5 mg/kg), recombinant IL12B protein (MCE HY-P700100AF, 3 μg/kg), anti-IL12B neutralizing antibody (Proteintech Group 69006-1-Ig, 5 μg/kg), CCR2 antagonist4 (MCE HY-108323, 10 mg/kg), recombinant Cxcl12 protein (MCE HY-P700219AF, 3 μg/kg), anti-Ifnar2 neutralizing antibody (Thermo Fisher 213851, 5 μg/kg), Cxcr2 inhibitor SB225002 (MCE HY-16711, 2 mg/kg), and CCR1 inhibitor BX471 (MCE HY-12080, 4 mg/kg) were injected as indicated.

Techniques: Inhibition, Viability Assay, Western Blot, Real-time Polymerase Chain Reaction

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