Journal: RNA nanomed

Article Title: CD133-Guided RNA Nanoparticle Delivery of FTO siRNA Impairs Leukemia Resistance to Tyrosine Kinase Inhibitor Therapy

doi: 10.59566/isrnn.2025.0201e

Figure Lengend Snippet: Expression and regulation of LSC markers in naïve, larger clones and TKI resistant leukemia cells. (A) Quantification of CD25+ cells in K562 cells treated with 0.3 or 1 μM of nilotinib for 48 hours, as measured by flow cytometry. (B) Left: Graphs representing colony numbers of parental and nilotinib-resistant K562 cells cultured in drug-free medium for 72 hours; Right: Flow cytometry analysis depicting the percentage of CD25+ cells in large vs small clones. (C) Graphs showing quantification of CD25+, CD44+ and CD133+ cells measured in parental vs NIR or IMR K562 cells, as measured by flow cytometry. (D) qPCR measuring indicated LSC marker expression in parental vs resistant Kasumi-1, MV4–11 and K562 cells. (E) Western blot and quantification of KIT (CD117) and EZH2 protein expression in parental, NIR-resistant, and IMR-resistant K562 cells. Band intensity was quantified and normalized to a loading control (β-actin). (F-G) qPCR measuring changes in the indicated stem cell markers in FTO knockdown (F) or FTO inhibitor CS2-treated (G) K562 cells resistant to nilotinib. (H) m 6 A immunoprecipitation (IP) was performed in mRNA from K562 resistant cells. The eluted RNA was converted to cDNA and transcript levels of KIT, CD25 and CD44 were measured by qPCR. Data are representative of three independent experiments. Par, parental; NIR, nilotinib resistance; IMR, imatinib resistance; Kas, Kasumi-1; shctrl, scramble control; * P <0.05, ** P <0.01, *** P <0.001; ns, not statistically significant.

Article Snippet: To measure the percentage of LSCs, different TKI resistant and parental cells were stained with antibodies against CD44 (Miltenyi Biotec, 130-113-334), CD25 (Miltenyi Biotec, 170-081-060) or CD133 (Miltenyi Biotec, 170-081-001).

Techniques: Expressing, Clone Assay, Flow Cytometry, Cell Culture, Marker, Western Blot, Control, Knockdown, Immunoprecipitation

Journal: bioRxiv

Article Title: Hypoimmunogenic hPSC-derived cardiac organoids for immune evasion and heart repair

doi: 10.1101/2025.04.09.648007

Figure Lengend Snippet: a-d, Bar charts of flow cytometry data showing expression of HLA-A2, HLA-BC, and HLA-E as percent positive expression in ( a ) hPSC-CMs, ( b ) hPSC-cFbs, ( c ) hPSC-ECs, and ( d ) hPSC-PCs by wild-type hPSC-derived cells (red) and UDET hPSC-derived cells (blue). e , Timeline of T cell cytotoxicity assay to measure T cell-mediated lysis of target cells. f-i , Percent lysis as quantified from LDH assay following exposure of activated CD8+ T cells to wild-type (red) and UDET (blue)-derived ( f ) hPSC-CMs, ( g ) hPSC-cFbs, ( h ) hPSC-ECs, and ( i ) hPSC-PCs. j , Timeline of NK cytotoxicity assay to measure NK cell-mediated lysis of target cells. k-n , Percent lysis as quantified from LDH assay following exposure of NK cells to wild-type (red) and UDET (blue)-derived ( k ) hPSC-CMs, ( l ) hPSC-cFbs, ( m ) hPSC-ECs, and ( n ) hPSC-PCs. Data shown from one donor representative of 2 biological replicates with 3-5 technical replicates. nd=no difference, *=p<0.05.

Article Snippet: Pre-conjugated antibodies used: B2M-APC (1:100; Biolegend, 316312, clone 2M2), HLA-A/B/C-PE (1:40; Biolegend, 311406, clone W6/32), HLA-E-APC (1:40; Biolegend, 342606, clone 3D12), HLA-G-APC (1:100; Biolegend, 335909, clone 87G), HLA-E-APC (1:50; Invitrogen, 17995342, clone 3D12HLA-E), HLA-BC-APC (1:50; Invitrogen, 17593542, clone B1.23.2), HLA-A2-PE-Cy7, clone BB7.2), CD25-APC (1:50; R&D Systems, FAB1020A, clone 24212), CD45-BV421 (1:50; Biolegend, 304032, clone HI30), CD3-BV605 (1:50; Biolegend, 344836, clone SK7), CD8-APC (1:50; Biolegend, 344722, clone SK1), CD4-PE (1:50; Biolegend, 980804, clone SK3).

Techniques: Flow Cytometry, Expressing, Derivative Assay, Cytotoxicity Assay, Lysis, Lactate Dehydrogenase Assay

Journal: bioRxiv

Article Title: SLAMF6 deficiency augments tumor killing and skews towards an effector phenotype revealing it as a novel T cell checkpoint

doi: 10.1101/824946

Figure Lengend Snippet: A , SLAMF6 and Vβ13 expression in Pmel-1 or Pmel-1 x SLAMF6 KO splenocytes measured by flow cytometry. B , Percent CD8, CD4, and CD19 cells in spleens from Pmel-1 or Pmel-1 x SLAMF6 KO untreated mice. C , Pmel-1, and Pmel-1 x SLAMF6 KO CD8 untreated splenocytes were stained with anti-CD44 and anti-CD62L. One representative experiment is shown. D , Percent CD8 cells in Pmel-1 or Pmel-1 x SLAMF6 KO splenocytes after 7 days of in vitro activation with gp100 25-33 peptide and IL-2 (30 IU/ml). E , Flow cytometry for activation markers (CD25, CD69, CD137) in Pmel-1 or Pmel-1 x SLAMF6 KO splenocytes after 3 days of in vitro activation, as in (D). Median fluorescence intensity (MFI) is shown. F , Expression of PD-1 in Pmel-1 or Pmel-1 x SLAMF6 KO CD8 T cells after 7 days of in vitro activation, as in (D). Median fluorescence intensity (MFI) is shown. G, H , After 7 days of activation, Pmel-1 and Pmel-1 x SLAMF6 KO CD8 T cells were stained with anti-CD44 and anti-CD62L. CD8 subpopulations were defined for each mouse strain. G , One representative experiment and H , summary of subpopulations identified by flow cytometry in 5 experiments is shown. EM, effector memory, CM, central memory. Student t-test. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Article Snippet: Anti-IFN-γ (XMG1.2), anti-CD8 (53-6.7), anti-GZMB (NGZN), anti-CD4 (GK1.5), and anti-CD25 (PC61.5) were from Biogems, Westlake Village, CA, USA.

Techniques: Expressing, Flow Cytometry, Staining, In Vitro, Activation Assay, Fluorescence

Journal: bioRxiv

Article Title: Role of Lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1101/2024.05.14.593747

Figure Lengend Snippet: (A) Subcutaneous inoculation (s.c.) with 10 5 p.f.u. of VACV was administered to both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells, as well as CD25 + Foxp3 + Treg cells, was conducted in popliteal lymph nodes (pLN) at 6 and 14 days post-infection. The percentage of Th1 cells, CD8 CTL T cells, and Treg cells was assessed. The data represent means ± SEM from at least 8 mice per group, obtained from 3 independently conducted experiments, and were analyzed using unpaired Student’s t-test between genotypes at each time point. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant). (B) Intraperitoneal inoculation (i.p.) with 10 6 p.f.u. of VACV was performed on both WT and LysM-Lmna -/- mice. Flow cytometry analysis of IFNγ-producing CD4 and CD8 T cells was conducted in mesenteric lymph nodes (mLN) 6 days post-infection. The percentage of Th1 and CD8 CTL cells was determined. Data represent means ± SEM of at least 6 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is indicated by asterisks (*P < 0.05; **P < 0.01). (C) Infection with 10 5 p.f.u. of VACV through skin scarification (s.s.) in the tail was performed on WT and LysM-Lmna -/- mice. Flow cytometry analysis of the percentages of IFNγ-producing CD4 and CD8 T cells was conducted in extracted inguinal lymph nodes (iLN) at 6 and 14 days post-infection. (D) Viral load measurement in the tail skin was performed at 6 and 14 days post-infection. The data represent means ± SEM of at least 4 mice per group, derived from 2 independently conducted experiments, and were analyzed by unpaired Student’s t-test. Statistical significance is denoted by asterisks (*P < 0.05; **P < 0.01; ns, not significant).

Article Snippet: Antibodies against: CD4 (Clone RM4-5, GK1.5) -v450, -APC and PE; CD25 (PC61.5) -APC; CD69 (H1.2F3)-FITC; CD28 (37.51); CD3 (145-2C11); CD45.1 (A20)-Pe-Cy7, -v450, -FITC; CD45.2 (104) -v450, -redFluor710; CD11b (M1/70)-v450; CD86 GL-1 PE; IFNγ XMG1.2 FITC, -APC; Foxp3 (3G3)-FITC; MHC-II (I-A/I-E, M5/115.15.2)-APC; GR1 (RB6-8C5)-Biotin; CD80 (16-10A1)-Biotin; B220 (RA3-6B2)-Biotin; F4/80 (BM8.1)-Biotin; CD11b (M1/70)-Biotin; CD19 (1D3)-Biotin; CD25 (PC61.5)-Biotin from Tonbo.

Techniques: Flow Cytometry, Infection, Derivative Assay

Journal: bioRxiv

Article Title: Role of Lamin A/C on dendritic cell function in antiviral immunity

doi: 10.1101/2024.05.14.593747

Figure Lengend Snippet: Flow cytometry was employed to assess the percentage of activated CD4 T cells, specifically gated on CD25 + or CD69 + cells; Th1-differentiated cells, specifically gated on IFNγ+ cells; and proliferating CellTrace Violet + T cells. Maturation of GM-CSF-derived WT- and LysM-Lmna -/- -BMDCs was undertaken with (A) sonicated VACV extract, (B) Pam3CSK4, or (C) lipopolysaccharide (LPS), and, in all conditions, pulsed with OVA323–339 cognate OT-IIp. (D) WT- or Lmna -/- -BMDCs, matured with LPS and pulsed with the OVA 323–339 cognate OT-II peptide, were s.c. inoculated into CD45.1 OT-II recipient mice. Six days later, pLNs were extracted, and IFNγ-producing CD4 T cells were analyzed by flow cytometry. The data represent means ± SEM and are presented from a representative experiment out of 3 (A and B, n=3) or out of 4 (C, n=4) or out of 2 (D, n=4). Activation data were analyzed using One Way ANOVA and Bonferroni post-test, and proliferation and Th1 differentiation were assessed by unpaired Student’s t-test. Statistical significance was denoted by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant).

Article Snippet: Antibodies against: CD4 (Clone RM4-5, GK1.5) -v450, -APC and PE; CD25 (PC61.5) -APC; CD69 (H1.2F3)-FITC; CD28 (37.51); CD3 (145-2C11); CD45.1 (A20)-Pe-Cy7, -v450, -FITC; CD45.2 (104) -v450, -redFluor710; CD11b (M1/70)-v450; CD86 GL-1 PE; IFNγ XMG1.2 FITC, -APC; Foxp3 (3G3)-FITC; MHC-II (I-A/I-E, M5/115.15.2)-APC; GR1 (RB6-8C5)-Biotin; CD80 (16-10A1)-Biotin; B220 (RA3-6B2)-Biotin; F4/80 (BM8.1)-Biotin; CD11b (M1/70)-Biotin; CD19 (1D3)-Biotin; CD25 (PC61.5)-Biotin from Tonbo.

Techniques: Flow Cytometry, Derivative Assay, Sonication, Activation Assay