Journal: International Journal of Molecular Sciences

Article Title: Intraclonal Enrichment of IL-23 Receptor Complex Expression in the Proliferative Fraction of Chronic Lymphocytic Leukemia

doi: 10.3390/ijms27031202

Figure Lengend Snippet: IL-23R and IL-12R expression in ex vivo CLL cells assessed by flow cytometry. FSC is proportional to cell size and SSC reflects intracellular complexity/granularity (each dot represents one cell). Axes in marker plots show fluorescence intensity for the indicated markers. Percentages within gates indicate the fraction of cells relative to the parent population. Lymphocytes were identified by FSC/SSC properties, followed by doublet exclusion using pulse height vs. width plots. Within the single-cell gate, CD19 + /CD5 + CLL cells were selected. This population was further sub-divided into RF and PF based on the differential expression of CD184 and CD5. ( A ) Gating strategy used to identify lymphocytes, single cells, CD19 + CLL cells, and to discriminate proliferative fraction (PF) and resting fraction (RF); ( B ) surface expression of IL-23R, IL-12Rβ1, and IL-12Rβ2 receptor subunits in ex vivo CLL cells, shown for CD19 + PF and RF populations; ( C ) representative flow cytometry plots showing sorting of PF and RF populations from six CLL samples according to the gates indicated; ( D ) IL-12Rβ1 mRNA expression in sorted RF and PF cells, measured by RT-qPCR. Data are shown as mean ± SEM. Statistical significance of the difference is evaluated using the two-sided Wilcoxon signed-rank test. * p < 0.05; ** p < 0.01, ns: not significant. FSC: forward scatter; SSC: side scatter; PE Phycoerythrin; PE-Cy7: Phycoerythrin–Cyanine 7; APC: Allophycocyanin; AF 488: Alexa Fluor 488; PE VIO 770: near-infrared fluorochrome emitting in the ~770–780 nm range.

Article Snippet: The following monoclonal antibodies were used: mouse anti human IL-23R-PE (Cat. #FAB14001P, R&D Systems, Minneapolis, MN, USA), mouse anti human IL-12Rβ1-BB515 (CD212, BD Hori-zon, Cat. #565043, BD Biosciences, San Jose, CA, USA, and mouse anti human IL-12Rβ2-PerCP (Cat. #FAB1959C, R&D Systems).

Techniques: Expressing, Ex Vivo, Flow Cytometry, Marker, Fluorescence, Single Cell, Quantitative Proteomics, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Intraclonal Enrichment of IL-23 Receptor Complex Expression in the Proliferative Fraction of Chronic Lymphocytic Leukemia

doi: 10.3390/ijms27031202

Figure Lengend Snippet: IL-23R and IL-12R expression in CLL cells after incubation with CpG and CpG + IL-15 for 72 h assessed by flow cytometry analyses. ( A ) Representative flow cytometry gating strategy used to identify CLL cells and to assess IL-23R and IL-12R complex expression under the indicated culture conditions (medium alone, CpG, or CpG + IL-15) after 72 h; ( B ) percentage of CLL cells expressing the IL-23R receptor subunit, IL-12Rβ1 receptor subunit, and IL-12Rβ2 receptor subunit following 72 h incubation under the indicated conditions; ( C ) percentage of CLL cells expressing the IL-23R complex and the IL-12R complex following 72 h incubation under the indicated conditions. Statistical significance of the difference is evaluated using the two-sided Wilcoxon signed-rank test. * p < 0.05; **** p < 0.0001. FSC: forward scatter; SSC: side scatter; PE Phycoerythrin; PE-Cy7: Phycoerythrin–Cyanine 7; APC: Allophycocyanin; AF 488: Alexa Fluor 488.

Article Snippet: The following monoclonal antibodies were used: mouse anti human IL-23R-PE (Cat. #FAB14001P, R&D Systems, Minneapolis, MN, USA), mouse anti human IL-12Rβ1-BB515 (CD212, BD Hori-zon, Cat. #565043, BD Biosciences, San Jose, CA, USA, and mouse anti human IL-12Rβ2-PerCP (Cat. #FAB1959C, R&D Systems).

Techniques: Expressing, Incubation, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Intraclonal Enrichment of IL-23 Receptor Complex Expression in the Proliferative Fraction of Chronic Lymphocytic Leukemia

doi: 10.3390/ijms27031202

Figure Lengend Snippet: Differential expression of IL-23R and IL-12R in CLL RF and PF subsets after stimulation with CpG and CpG + IL-15 assessed by flow cytometry analyses. ( A ) Representative flow cytometry gating strategy used to identify CLL cells and to distinguish RF and PF subsets, followed by assessment of IL-23R and IL-12R complex expression after 72 h incubation with CpG or CpG + IL-15; ( B ) percentage of RF and PF CLL cells expressing the IL-23R receptor subunit, IL-12Rβ1 receptor subunit, and IL-12Rβ2 receptor subunit after 72 h incubation under the indicated conditions; ( C ) percentage of RF and PF CLL cells expressing the IL-23R complex and the IL-12R complex after 72 h incubation under the indicated conditions. Each symbol represents an individual CLL sample; horizontal bars indicate mean ± SEM. Statistical significance of the difference is evaluated using the two-sided Wilcoxon signed-rank test. ** p < 0.01; **** p < 0.0001, ns: not significant. FSC: forward scatter; SSC: side scatter; PE Phycoerythrin; PE-Cy7: Phycoerythrin–Cyanine 7; APC: Allophycocyanin; AF 488: Alexa Fluor 488; BV421 = Brilliant Violet 421.

Article Snippet: The following monoclonal antibodies were used: mouse anti human IL-23R-PE (Cat. #FAB14001P, R&D Systems, Minneapolis, MN, USA), mouse anti human IL-12Rβ1-BB515 (CD212, BD Hori-zon, Cat. #565043, BD Biosciences, San Jose, CA, USA, and mouse anti human IL-12Rβ2-PerCP (Cat. #FAB1959C, R&D Systems).

Techniques: Quantitative Proteomics, Flow Cytometry, Expressing, Incubation

Journal: Cancer Medicine

Article Title: Arsenic trioxide rewires mantle cell lymphoma response to bortezomib

doi: 10.1002/cam4.511

Figure Lengend Snippet: ATO combined with BTZ inhibited the proliferation of MCL cells. MCL cell lines, Jeko-1 (A), Granta-519 (B), were treated by BTZ/ATO in fixed ratio (5/1, 7.5/1.5, 10/2, 15/3 [nmol/L]/[ μ mol/L]). After 12 h, cell viability was determined by CCK8. ATO, arsenic trioxide; BTZ, bortezomib; MCL, mantle cell lymphoma.

Article Snippet: Cells were incubated for 12–48 h single or combined of BTZ (Millennium Pharmaceuticals, Cambridge, MA) and ATO (the Pharmacy of Chinese Traditional Medicine in the First Hospital affiliated to Harbin Medical University).

Techniques:

Journal: Cancer Medicine

Article Title: Arsenic trioxide rewires mantle cell lymphoma response to bortezomib

doi: 10.1002/cam4.511

Figure Lengend Snippet: ATO combined with different concentrations of BTZ induced apoptosis of MCL cells. (A) The apoptotic status of MCL cells, 24 h after the Jeko-1 and Granta-519 being treated by 1 μ mol/L ATO, 5 or 7.5 nmol/L BTZ, or combination of the two drugs. (B) The expression of apoptosis-related proteins in Jeko-1 and Granta-519 cells detected by western blot, 12 h after treatment of ATO alone, BTZ alone, or combination of the two drugs. (C) The apoptotic status of Jeko-1 cells, after incubation with the caspase inhibitor, zVAD-FMK, for 1 h, followed by treatment of BTZ and ATO for 24 h. ATO, arsenic trioxide; BTZ, bortezomib; MCL, mantle cell lymphoma.

Article Snippet: Cells were incubated for 12–48 h single or combined of BTZ (Millennium Pharmaceuticals, Cambridge, MA) and ATO (the Pharmacy of Chinese Traditional Medicine in the First Hospital affiliated to Harbin Medical University).

Techniques: Expressing, Western Blot, Incubation

Journal: Cancer Medicine

Article Title: Arsenic trioxide rewires mantle cell lymphoma response to bortezomib

doi: 10.1002/cam4.511

Figure Lengend Snippet: ATO combined with BTZ activated the intrinsic apoptotic pathway of MCL cells. The loss of Δ ψ m (A), Bak conformational changes (B), Bax conformational changes (C) were detected after 24-h treatment of ATO, BTZ, and combination of the two drugs. ATO, arsenic trioxide; BTZ, bortezomib; MCL, mantle cell lymphoma.

Article Snippet: Cells were incubated for 12–48 h single or combined of BTZ (Millennium Pharmaceuticals, Cambridge, MA) and ATO (the Pharmacy of Chinese Traditional Medicine in the First Hospital affiliated to Harbin Medical University).

Techniques:

Journal: Cancer Medicine

Article Title: Arsenic trioxide rewires mantle cell lymphoma response to bortezomib

doi: 10.1002/cam4.511

Figure Lengend Snippet: The effect of ATO combined with BTZ on the Bcl-2 family proteins and the release of Bak. Jeko-1 and Granta-519 cells were treated by ATO, BTZ, or combination of the two drugs for 24 h, after that, (A) the expression of Bcl-2 family proteins was detected. One representative experiment is shown from three independent experiments. (B) The protein levels of Noxa and Mcl-1 were quantified by Quantity One software (Bio-Rad). (C) The ratio between expression levels of Noxa and Mcl-1 was showed. * P < 0.05. (D) The expression levels of Mcl-1, Noxa, Bak, and Bcl-XL proteins in the immunoprecipitated (bound) and nonimmunoprecipitated (unbound) fractions were detected. ATO, arsenic trioxide; BTZ, bortezomib.

Article Snippet: Cells were incubated for 12–48 h single or combined of BTZ (Millennium Pharmaceuticals, Cambridge, MA) and ATO (the Pharmacy of Chinese Traditional Medicine in the First Hospital affiliated to Harbin Medical University).

Techniques: Expressing, Software, Immunoprecipitation

Journal: Cancer Medicine

Article Title: Arsenic trioxide rewires mantle cell lymphoma response to bortezomib

doi: 10.1002/cam4.511

Figure Lengend Snippet: ATO combined with BTZ induced apoptosis in primary cells of MCL patients. (A) The apoptotic status of CD45+CD19+CD5+ and CD45+CD19− cells from BMMCs of patient 1, 2, and 3 were determined after 18-h treatment of 2 μ mol/L ATO, 7.5 or 10 nmol/L BTZ, and combination of the two drugs. (B) The apoptotic status of BMMCs of patient 4 was determined after 18-h treatment of 2 μ mol/L ATO, 7.5 nmol/L BTZ and combination of them. The protein levels of Mcl-1 and Noxa in immunoprecipitated (bound) and nonimmunoprecipitated (unbound) fractions were detected. ATO, arsenic trioxide; BTZ, bortezomib; MCL, mantle cell lymphoma; BMMCs, bone marrow mononuclear cells.

Article Snippet: Cells were incubated for 12–48 h single or combined of BTZ (Millennium Pharmaceuticals, Cambridge, MA) and ATO (the Pharmacy of Chinese Traditional Medicine in the First Hospital affiliated to Harbin Medical University).

Techniques: Immunoprecipitation

Journal: Cancer Medicine

Article Title: Arsenic trioxide rewires mantle cell lymphoma response to bortezomib

doi: 10.1002/cam4.511

Figure Lengend Snippet: The in vivo efficacy of ATO combined with BTZ on MCL cells. (A) The pictures of mice taken at day 0, 9, 15, 19, and 23 after treatment. One representative picture from each group (control, BTZ, ATO, BTZ+ATO) was shown. (B) The volumes of tumor bulk of mice in each group altered over time. * P < 0.05. ATO, arsenic trioxide; BTZ, bortezomib; MCL, mantle cell lymphoma.

Article Snippet: Cells were incubated for 12–48 h single or combined of BTZ (Millennium Pharmaceuticals, Cambridge, MA) and ATO (the Pharmacy of Chinese Traditional Medicine in the First Hospital affiliated to Harbin Medical University).

Techniques: In Vivo

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: The effect of estradiol treatment and loss-of-function mutations in neurodevelopmental disorder (NDD) risk genes is measured across three modalities: (1) single-cell RNAseq in hiPSC-derived NGN2 -induced glutamatergic neurons (iGLUTs), (2) neuronal calcium imaging in iGLUTs, and (3) sensorimotor and arousal behavior in larval zebrafish. Modalities include overlapping but independent gene sets. ASD/NDD target genes encompass myriad functions, including gene expression regulation (pink), neuronal function and communication (green), cellular signaling and protein modification (gray), and cytoskeleton (blue).

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Single Cell, RNA sequencing, Derivative Assay, Imaging, Gene Expression, Modification

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: A. Schematic of experimental design and different effect comparisons. Thirty-six NDD risk genes are targeted using pooled single-cell CRISPR-knockout screening in hiPSC-derived NGN2-glutamatergic neurons (iGLUTs) with and without 24hr exposure to 100nM estradiol. Each gene was targeted with 3-4 gRNAs (i). Various effect comparisons were performed among the treated and untreated groups (ii). Main KO effect measures KO+DMSO vs Non-targeting (NT)+DMSO. Main E2 effect measures NT+E2 vs NT+DMSO. KO effect within E2 measures KO+E2 vs NT+E2. E2 effect within KO measures KO+E2 vs KO+DMSO. E2 and KO joint effect measures KO+E2 vs NT+DMSO. B. Extent of perturbation per target NDD gene (left, dot plot). Circle size indicates negative log 10 (p-value). Stem indicates the log fold change of the knockout +DMSO vs non-targeting control + DMSO. KOs were called based on enrichment of gRNA for target NDD genes and downregulation of target KO gene expression. Number of differentially expressed genes in the main KO effect are shown in the bar plot (FDR <0.05). C . Odds of convergent genes being rescued by E2 compared to nonconvergent genes, tested across NDD KO. Fisher’s exact test, ***p<0.001, **p<0.01, *p<0.05. D. Effect of estradiol reversal on biological pathways across 12 target gene knockouts in iGLUTs. Orange color indicates that the pathway was originally downregulated in main KO effect, and estradiol upregulated the pathway in reversal effect. Blue color indicates that the pathway was originally upregulated in main KO effect, and estradiol downregulated the pathway in reversal effect. Dot size indicates the –log10 of the gene set enrichment significance of E2 effect within KO. E . Effect of estradiol rescue on DEGs across NDD risk genes in iGLUTs. Impact of estradiol on number of main KO DEGs is classified into categories of exacerbated, unchanged, partial rescue, rescue, and over-corrected. For detailed description of thresholds, see Methods. Target genes are ranked from highest to lowest percentage of DEGs that are classified as rescued (nominal p-value <0.05). F-G. Select Gene Ontology terms indicating estradiol reversal among ASH1L ( F ) and SCN2A ( G ) KO in iGLUTs. Dot size indicates the –log10 of the gene set enrichment significance of E2 effect within KO (E2) or main KO effect (KO). Asterisks for Benjamini Hochberg p-adjusted: * <0.05; **<0.01, ***<0.001.

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Single Cell, CRISPR, Knock-Out, Derivative Assay, Control, Gene Expression

Journal: bioRxiv

Article Title: Estradiol modulates neuronal network hyperexcitability in select NDD risk genes

doi: 10.64898/2026.02.18.706588

Figure Lengend Snippet: A. Schematic of experimental workflow to assess impact of estradiol on baseline gene expression in scn1lab -/- larvae. B-C . Comparison of number of Gene Ontology (GO) pathways that are originally upregulated in HOM+DMSO and downregulated by estradiol ( B ) or originally downregulated in HOM+DMSO and upregulated by estradiol ( C ). Selected reversed pathways are highlighted in the bar graphs, with bars indicating -log10(FDR). Asterisk for the p-adjusted values: * <0.05,**<0.01, ***<0.001. D. Schematic of experimental workflow to assess impact of estradiol on baseline brain activity in HOM larvae. Dissected larval brains are stained with phosphorylated ERK (pERK) as a marker of active neurons and total ERK (tERK) as a pan-neuronal marker. Images undergo voxel-by-voxel division of pERK by tERK to obtain an activity ratio. Activity is mapped onto the standard zebrafish brain atlas and quantified based on a 4-region or 149-region atlas. E. Voxel-wise Z score normalized pERK/tERK values representing brain activity differences in scn1lab Δ44/Δ44 + DMSO or estradiol compared to DMSO-treated scn1lab +/+ larvae and the difference (subtraction) between the two conditions. Rescued regions from the 149-region atlas are shown. For specific rescued regions, refer to SI Fig. 7. Images are a representative slice of the axial (top) and sagittal (bottom) view. Scale bar represents Z score (red/yellow, increased in mutant; cyan/purple, decreased in mutant). F. Bar graphs showing regional differences in pERK/tERK brain activity in the rhombencephalon, diencephalon, mesencephalon, and telencephalon. G. Left: Schematic of experimental workflow to assess impact of estradiol on induced seizure activity in scn1lab -/- larvae. Right: Pentylenetetrazole (PTZ)-induced seizure activity, with and without 24 h exposure to 20 𝜇M estradiol, is measured in scn1lab -/- larval zebrafish mutants using video-tracking. Representative activity tracings of control and scn1lab -/- larvae at 5dpf before and after exposure to DMSO vehicle or 20uM estradiol and 10mM PTZ or water. Ribbon shows +/- SEM. H. Average activity 30 min before and after the addition of 10mM PTZ or water to control and scn1lab -/- larvae before and after exposure to DMSO vehicle or 20uM estradiol. scn1lab -/- + E2 + PTZ larvae show significantly decreased activity compared to scn1lab -/- + vehicle + PTZ larvae (Unpaired t-test with Welch’s correction on post values, ****p<0.0001; n=24/group). I. Interaction models assessing genotype-x-estradiol-x-PTZ effect, comparing post- vs pre-PTZ measurements. Dissection of the mutant effect (HOM vs control), estradiol effect (Estradiol vs DMSO), and PTZ effect (PTZ vs water) (i). Three-way interaction model to estimate the PTZ effect among genotype-x-estradiol groups of larvae (ii). J. Left: Schematic of experimental workflow to assess impact of estradiol on neuronal busting activity in CRISPR-engineered SCN2A +/- iGLUTs . Right: Cellular and network bursting activity, with and without exposure to acute 100nM estradiol, is measured in SCN2A +/- NGN2-iGLUTs using multielectrode array. Bar graphs show mean number of cellular bursts and network bursts in controls and SCN2A +/- iGLUTs. Unpaired t-test, cellular bursts *p=0.0415, network bursts SCN2A +/- spontaneous vs SCN2A +/- E2 *p=0.0127, SCN2A +/- spontaneous vs WT spontaneous *p=0.0450.

Article Snippet: H1-iCas9 hESCs were transduced with a lentivirus expressing doxycycline-inducible NGN2 (pLVX-UbC-rtTA-Ngn2:2A, Addgene #127288) modified to express blasticidin resistance in place of EGFP.

Techniques: Gene Expression, Comparison, Activity Assay, Staining, Marker, Mutagenesis, Control, Dissection, CRISPR