Journal: The FASEB Journal

Article Title: Circulating Natural Killer Cells in Patients With Type 2 Diabetes and Peripheral Neuropathy Are Altered in Their Frequency and Exhibit Impaired Functions and Angiogenic Activities

doi: 10.1096/fj.202600263R

Figure Lengend Snippet: Circulating NK cell degranulation and angiogenic capabilities in T2DM patients with neuropathy and controls. Multicolor flow cytometry analysis for circulating NK cell degranulation activity against K562 target cells, showing the frequency of CD107 + circulating NK cells (A) Sample size n = 19–20. Data are shown as mean ± SEM, non‐parametric two‐sample Wilcoxon test, * p < 0.05. Representative dot plots and gating strategies used for the degranulation assay. Singlets were gated on SSC‐A/SSC‐H plot, followed by morphology identification of lymphocytes (SSC‐A/FSC‐A). NK cells were identified as CD3 − CD56 + cells, and CD107a + cells were gated to evaluate their degranulation capabilities. (B) HC: Nondiabetic controls; N: T2DM patients with neuropathy. Graphs of the average expression (2 −ΔCt ) of IFN‐γ (C), CD96 (D), and TIM3 (E) measured by real‐time Q‐PCR of sorted NK cells from N patients and nondiabetic controls. Sample size n = 6–7. EC tube network formation assay on Matrigel. HUVECs exposed to CM of FACS‐sorted NK from healthy control (CM NK‐HC) and T2DM patients with neuropathy (CM NK–N), for 16 h. Representative images (5× magnification) (F) and quantification of the number of segments (G), number of branches (H), total segments length (I), and total branches length (L). Sample size n = 3. Data are presented as mean ± SD, non‐parametric Student t test, * p < 0.05. Spheroid collagen invasion assay. Representative images (5× magnification) (M) of spheroid collagen invasion area on HUVECs exposed for 24 h to CM of FACS‐sorted NK from healthy control and T2DM patients with neuropathy measured as fold change vs. T0. Data are shown as the mean ± SD of 10 different spheroids for HC group and 12 different spheroids for the N group ( n = 10 and 12 biological replicates) (N). Non‐parametric Student t test (*** p < 0.001). The assays were performed on subsets of samples with sufficient PBMCs and sorted NK availability.

Article Snippet: The human erythroid K562 cells (ATCC) were cultured in RPMI 1640 medium (Euroclone) supplemented with 10% FBS (Euroclone), 2 mM L‐glutamine (Euroclone), 100 U/mL penicillin, and 100 μg/mL streptomycin (Euroclone) at 37°C in a 5% CO 2 atmosphere, and used for the NK cell degranulation assay as target cells.

Techniques: Flow Cytometry, Analysis, Activity Assay, Degranulation Assay, Morphology, Expressing, Tube Network Formation Assay, Control, Invasion Assay, Spheroids

Journal: Blood Research

Article Title: Next-generation digital polymerase chain reaction assay for BCR::ABL1 in chronic myeloid leukemia: comprehensive validation for standardized measurable residual disease detection

doi: 10.1007/s44313-026-00188-5

Figure Lengend Snippet: Evaluation of linearity for BCR::ABL1 major fusion transcripts. Linearity was assessed by measuring log 10 -transformed %IS values across a serial dilution range spanning approximately 5 orders of magnitude. A Linearity for the e13a2 transcript was evaluated using KCL-22 cell line dilutions (lot 1: red circles, lot 2: blue circles). B Linearity for the e14a2 transcript was evaluated using K-562 cell line dilutions. The x -axis represents the expected log 10 -transformed %IS values derived from RT-qPCR, and y -axis represents the observed log 10 -transformed %IS values measured using the LQ Suite dPCR system. Solid lines indicate the linear regression fit for each lot. Regression equations and coefficients of determination (R 2 ) are displayed within the plot for each lot, demonstrating strong linear correlation (R 2 > 0.99) across the tested range (e13a2: 10.4301%IS to 0.0006%IS, e14a2: 10.6813%IS to 0.0010%IS). dPCR, digital PCR; %IS, % International Scale; RT-qPCR, real-time quantitative reverse-transcription polymerase chain reaction

Article Snippet: K-562 (ATCC CCL-243), AR230-r (e1a2-transcript CML, ATCC CRL-3346), SUP-B15 (ALL, ATCC CRL-1929), and HL-60 (APL, ATCC CCL-240) were purchased from ATCC; KCL-22 (JCRB1317) was purchased from the Japanese Collection of Research Bioresources Cell Bank; and WHO standard panels were purchased from NIBSC.

Techniques: Transformation Assay, Serial Dilution, Derivative Assay, Quantitative RT-PCR, Digital PCR, Reverse Transcription, Polymerase Chain Reaction

Journal: Science Advances

Article Title: Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition

doi: 10.1126/sciadv.ady0296

Figure Lengend Snippet: ( A ) RT-PCR assays to measure mRNA levels in parental K562; N = 2. Data are normalized means and errors are SEM. ( B ) FACS analysis measuring intracellular phospho-STAT5 (pSTAT5) and phospho-ERK(pERK); N = 3. Data are normalized mean fluorescence intensity (MFI), and errors are SEM. ( C ) Representative Western blot showing BCR::ABL1-p210, untranslocated ABL1, and pSTAT5/pERK expression; N = 3. ( D and E ) Alamar blue assays to assess K562 cell proliferation following BCR::ABL1 targeting with (D) imatinib or (E) Psp Cas13b (time point 0 is 24 hours posttransfection); N = 3. Data are normalized means, and errors are SEM. ( F ) The schematic illustrates the imatinib sensitivity or imatinib resistance of WT BCR::ABL1 and T315I variants, respectively. ( G ) Western blot analysis to examine the suppression of T315I BCR::ABL1-p190 with Psp Cas13b in HEK 293 cells 24 hours posttransfection. A total of 1 μM imatinib treatment for 4 hours was used as a positive control; N = 3. ( H ) Western blot analysis to confirm the imatinib sensitivity or imatinib resistance of parental and TKI-resistant K562 cells, respectively; N = 3. ( I ) Alamar blue assays to assess proliferation of TKI-resistant K562 cells following imatinib treatment; N = 3. Data are normalized means, and errors are SEM. ( J ) The schematic illustrates nucleofection delivery of PspCas13b-mFlame mRNA and crRNA plasmid. ( K ) Western blot analysis to examine the expression levels of Psp Cas13b, BCR::ABL1-p210, and tyrosine phosphorylation 72 hours posttransfection. Imatinib or dimethyl sulfoxide (DMSO) were used as controls (48 hours); N = 3. ( L ) Western blot analysis showing BCR::ABL1-p210 and pSTAT5/pERK; N = 3. ( M ) Alamar blue assays to assess proliferation (time point 0 is 24 hours posttransfection); N = 3. Data are normalized means and errors are SEM. ( N ) FACS plots show K562 cells, parental (top), and TKI-resistant (bottom) 72 hours posttransfection and then stained with annexin V and 7-ADD; N = 3. N is the number of independent biological replicates. Data in (B), (D), (E), (I), and (M) are analyzed by unpaired two-tailed Student’s t test (95% confidence interval).

Article Snippet: Parental K562 (ATCC, CCL-243) cell line was cultured in RPMI 1640 (Thermo Fisher Scientific, 11875083) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, 10100147) and Penicillin-Streptomycin (100 mg/ml; Thermo Fisher Scientific, 151401220).

Techniques: Reverse Transcription Polymerase Chain Reaction, Analysis, Fluorescence, Western Blot, Expressing, Sensitivity, Positive Control, Plasmid Preparation, Phospho-proteomics, Staining, Two Tailed Test, FACS, Proliferation Assay

Journal: Science Advances

Article Title: Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition

doi: 10.1126/sciadv.ady0296

Figure Lengend Snippet: ( A ) Schematic of RNA-seq and mass spectrometry assays. Created in BioRender. W.H. (2026); https://BioRender.com/d25d841 . ( B and C ) Volcano plots of RNA-seq analysis of the transcriptome of parental (top) and T315I (bottom) K562 cells expressing (B) Psp Cas13b or (C) treated with imatinib. Each data point represents a transcript, and transcripts with log 2 FC > 1 (up-regulation; shown in red) or log 2 FC < −1 (down-regulation; shown in blue) are considered significant differentially expressed transcripts (Benjamini-Hochberg adjusted P < 0.05); N = 3. ( D ) Heatmap for 364 common transcripts differentially expressed in Psp Cas13b-treated cell lines (parental or TKI-resistant) and imatinib-treated parental cells. ( E ) Gene Ontology analysis for differentially expressed proteins shown in mass spectrometry data in fig. S7 (B and C). ( F ) Gene Ontology analysis for the 32 common differentially expressed proteins (25 up-regulated and 7 down-regulated) in Psp Cas13b/T crRNA expressing K562 (parental or TKI-resistant) cells and imatinib-treated parental cells shown in the Venn diagram (fig. S7D). The gradient color scheme represents the averaged log 2 FC of each protein’s expression in Psp Cas13b/T crRNA expressing K562 (parental or TKI-resistant) or imatinib-treated parental cells relative to their corresponding control. ( G ) Heatmap for 16 genes differentially expressed in cell lines (parental or TKI-resistant) treated with Psp Cas13b and parental cells treated with imatinib compared to their respective control groups. These genes were confidently detected by both RNA-seq and mass spectrometry across all four comparison groups. ( H ) Gene Ontology analysis for the 16 genes shown in the heatmap (F). LCP2, SMIM1, and FN3K are not shown, as a minimum of two genes in each pathway is required. ( I ) STRING protein-protein interaction network functional enrichment analysis for the 16 genes shown in the heatmap (G). N represents the number of biological replicates. See Dryad for source data.

Article Snippet: Parental K562 (ATCC, CCL-243) cell line was cultured in RPMI 1640 (Thermo Fisher Scientific, 11875083) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, 10100147) and Penicillin-Streptomycin (100 mg/ml; Thermo Fisher Scientific, 151401220).

Techniques: RNA Sequencing, Mass Spectrometry, Analysis, Expressing, Control, Comparison, Functional Assay, Significance Assay

Journal: Science Advances

Article Title: Programmable and selective silencing of oncogenic fusion transcripts by CRISPR-Cas13b via breakpoint sequence recognition

doi: 10.1126/sciadv.ady0296

Figure Lengend Snippet: ( A ) Representative flow cytometry plots showing CD71 and CD235a expression in K562 cells treated with Psp Cas13b (left) or imatinib (right); N = 3. ( B ) Quantification of CD235a relative MFI from (A); N = 3. Data are normalized means, and errors are SEM. Results are analyzed by unpaired Student’s t test (95% confidence interval). ( C ) Western blot analysis of BCR::ABL1 isoforms and pSTAT5 expression in K562 cells overexpressing BCR::ABL1 p190 isoform following nucleofection with a BCR::ABL1 p210–targeting crRNA; N = 3. Data are normalized means, and errors are SEM; results are analyzed by unpaired Student’s t test (95% confidence interval) (uncropped blots are available in the Supplementary Materials). ( D ) Proliferation assays of parental, BCR::ABL1 p190–overexpressing, and empty vector–expressing K562 cells after Psp Cas13b-mediated targeting of BCR::ABL1 p210; N = 3. Data are normalized means, and errors are SEM; results are analyzed by two-way ANOVA with Šídák’s multiple-comparison test (95% confidence interval). ( E ) Quantification of CD235a MFI in BCR::ABL1 p190–overexpressing and empty vector–expressing K562 cells nucleofected with Psp Cas13b and either a targeting (T) or an NT crRNA; N = 3. Data are normalized means, and errors are SEM; results are analyzed by unpaired Student’s t test (95% confidence interval). N is the number of independent biological experiments. See Dryad for source data.

Article Snippet: Parental K562 (ATCC, CCL-243) cell line was cultured in RPMI 1640 (Thermo Fisher Scientific, 11875083) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, 10100147) and Penicillin-Streptomycin (100 mg/ml; Thermo Fisher Scientific, 151401220).

Techniques: Flow Cytometry, Expressing, Western Blot, Analysis, Plasmid Preparation, Comparison