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: bioRxiv

Article Title: DNA replication stress redistributes the histone methyltransferase G9a and activates lineage-specific gene expression

doi: 10.64898/2026.10.06.757090

Figure Lengend Snippet: (A) Representative immunoblot analysis of G9a, WIZ, BCL11A, H3K9me2 and H3K9me3 in HUDEP-2 whole cell extract, soluble, and chromatin-bound fractions from cells treated with vehicle (Veh), hydroxyurea (HU), or aphidicolin (APH) as indicated. Tubulin serves as a loading control for the soluble fraction and total H3 as a loading control for the chromatin-bound fraction, while Ser345-phosphorylated CHEK1 (p-Chk1) is a marker of DNA replication stress. (B–C) Quantification of immunoblot signals from (A) along with other replicates (n = 3, blots not shown) for (B) whole cell extract and (C) soluble and chromatin-bound fractions as indicated. Signal intensities are normalized to indicated loading controls. Statistical significance was determined via two-way ANOVA and p-values > 0.05 are indicated as ns (not significant). (D) Representative immunoblot of FLAG and G9a signal in parental HUDEP-2 cells, HUDEP-2 cells transduced with a FLAG-G9a overexpression construct containing a puromycin resistance gene prior to selection, and similarly transduced HUDEP-2 cells selected with puromycin for 72 hr (hereafter referred to as G9a OE cells). β-actin serves as a loading control. (E) Quantification of the representative immunoblot shown in (D). FLAG and G9a signal is normalized to their corresponding β-actin loading control. (F) EHMT2 (G9a) RNA transcript levels in parental HUDEP-2 cells and G9a OE HUDEP-2 cells (n=3). p-value was calculated using Student’s t-test. (G) Bar plot comparing the increase in the percent of mCherry+ cells following treatment of parental and G9a OE HUDEP-2 cells with either HU or aphidicolin (APH) as indicated (n=3). p-values were calculated using Student’s t-test. (H) Genome tracks for H3K9ac, H3K9me2, and H3K9me3 ChIP-seq signal in K562 cells, data derived from previously published work . Highlighted regions denote globin gene clusters and the locus control region (LCR) as indicated. (I) EHMT2 (G9a) RNA transcript levels measured by qRT-PCR in parental K562 cells and K562 G9a OE cells as indicated. qRT-PCR signal is normalized to PPIB and RPLP0 . P-values were calculated using Student’s t-test (n=3). (J) HBG1/2 RNA transcript levels measured by qRT-PCR in parental K562 cells and G9a OE K562 cells with and without HU treatment. qRT-PCR signal is normalized to PPIB and RPLP0. P-values were calculated using Student’s t-test (n=3).

Article Snippet: K562 human chronic myelogenous leukemia cells (ATCC, CCL-243) were cultured in RPMI 1640 media (Corning, 15-040-CV) supplemented with 10% fetal bovine serum (Gibco, A52567-01) and 4mM L-glutamine (Cytiva, SH30034.01).

Techniques: Western Blot, Analysis, Control, Marker, Transduction, Over Expression, Construct, Selection, ChIP-sequencing, Derivative Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: DNA replication stress redistributes the histone methyltransferase G9a and activates lineage-specific gene expression

doi: 10.64898/2026.10.06.757090

Figure Lengend Snippet: (A) Schematic of EdU labeling and compound treatment for a subset of assays shown in this figure. Cells were labeled with EdU under three conditions: Vehicle (Veh), with 4 hr vehicle treatment followed by 15 min EdU pulse, Hydroxyurea (HU), with 15 min EdU pulse followed by 4 hr hydroxyurea (HU) treatment at a dose that fully arrests fork progression, and Post replication DNA (post-rep DNA), with a 15 min EdU pulse followed by 4 hr thymidine chase. Timelines of treatment are shown (left) as well as position of EdU labeling indicated in green on replication forks (right). (B) Representative streptavidin-HRP immunoblots for EdU-labeled DNA pulled down by H3K9me2 or IgG click-CUT&RUN in HUDEP-2 cells treated with Veh or HU as described in (A). The indicated amount of DNA was loaded in each lane. Equal DNA loading was assessed using a pan DNA stain (SYBR-Gold). (C) Representative immunofluorescence images of foci per nucleus for G9a, WIZ, H3K9me2, or H3K9me3 in proximity to nascent DNA (labeled by EdU) using a proximity ligation assay (PLA). PLA signals (red) are shown for each condition described in (A) as indicated, and nuclei are counterstained with DAPI (blue). Cells in S phase, determined by the presence of EdU-labeled DNA, are represented by white dashed circles. Representative selection of EdU-positive and EdU-negative signal is shown in Figure S8A. (D) Quantification of PLA foci per nucleus for G9a, WIZ, H3K9me2, and H3K9me3 from the PLA shown in (C). Data are presented as box-and-whisker plots showing median and interquartile range. Statistical significance was calculated using a Wilcoxon test with Benjamini-Hochberg p-value correction (**p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant). The number of nuclei assessed for each condition is indicated. (E) Schematic of EdU labeling and dWIZ-2/HU treatment for subset of assays shown in this figure. Cells were labeled with EdU and treated with dWIZ-2 under two conditions: dWIZ-2 only, with 4 hr dWIZ-2 treatment followed by 15 min EdU pulse, and dWIZ-2 + HU, with 4 hr dWIZ-2 treatment followed by a 15 min EdU pulse followed by 4 hr HU. Timelines of treatment are shown with dWIZ-2 shown in blue (left) as well as position of EdU labeling indicated in green on replication forks (right). (F) Representative immunofluorescence images of PLA signals (red) for G9a or H3K9me2 in proximity to EdU for vehicle-treated, HU, post-rep DNA, dWIZ-2 only, and dWIZ-2 + HU conditions as described in (A) and (E). Nuclei are counterstained with DAPI (blue). Cells in S phase are represented by white dashed circles. (G) Quantification of PLA foci for G9a and H3K9me2 in proximity to EdU in cells treated with vehicle or HU with or without degradation of WIZ through dWIZ-2 pretreatment as shown in (E). Data are presented as box-and-whisker plots. Statistical significance was calculated using a Wilcoxon test with Benjamini-Hochberg p-value correction (**p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant). The number of nuclei assessed for each condition is indicated. (H) Representative immunoblot of 3% pulldown input (left) and proteins associated with nascent DNA identified by iPOND (isolation of proteins on nascent DNA) (right) in K562 cells treated with vehicle or HU, or following a post-replicative chase (see (A)). In iPOND, EdU incorporated into nascent DNA is conjugated to biotin azide by click chemistry , enabling affinity purification of nascent DNA and associated proteins. The (-) azide sample, in which biotin azide was omitted from the click reaction, serves as a negative control for nonspecific protein recovery during purification. (I) Quantification of H3K9me2 iPOND signal from (H), normalized to H3 iPOND signal and the input H3K9me2/H3 ratio, at vehicle and HU-treated nascent DNA and at post-replication DNA (n=3). (J) Representative immunoblot of 3% pulldown input (left) and iPOND fractions (right) from Veh, HU, post-rep DNA, dWIZ-2 only, and dWIZ-2 + HU (see (A) and (E)) from K562 cells. (K) Quantification of H3K9me2 iPOND signal from (J), normalized to H3 iPOND signal and the input H3K9me2/H3 ratio.

Article Snippet: K562 human chronic myelogenous leukemia cells (ATCC, CCL-243) were cultured in RPMI 1640 media (Corning, 15-040-CV) supplemented with 10% fetal bovine serum (Gibco, A52567-01) and 4mM L-glutamine (Cytiva, SH30034.01).

Techniques: Labeling, Western Blot, Staining, Immunofluorescence, Proximity Ligation Assay, Selection, Whisker Assay, Isolation, Affinity Purification, Negative Control, Purification