Journal: Nature Communications

Article Title: CTCF controls three-dimensional enhancer network underlying the inflammatory response of bone marrow-derived dendritic cells

doi: 10.1038/s41467-023-36948-5

Figure Lengend Snippet: a Western blotting performed with the indicated antibodies. The data are representative of three independent experiments with similar results. b Relative signal intensities of proteins in a were measured using ImageJ software. Error bars represent mean ± standard error of the mean (s.e.m). Significance was calculated using an unpaired two-tailed t -test using n = 3 independent samples. c Histogram showing the average tag density of STAT5 ChIP-seq peaks called for WT and KO BMDCs. d Heatmap of ChIP-seq signals with ±2 kb of unique STAT5 peaks comparing STAT5 enrichment (fold change > 1.3; FDR < 0.05) between WT and KO BMDCs. The data are representative of two independent experiments with similar results. e A pie chart depicting the numbers of genes, assigned to “lost in KO” STAT5 peaks and categorized by the overlap feature of the STAT5 peaks with promoters, distal enhancers, or both. f RNA-seq MA plot for the genes assigned to “lost in KO” STAT5 peaks. The number of genes exhibiting >1.3-fold decreases in WT (red) or KO (blue) BMDCs with a false discovery rate <0.05 has been indicated. g Enrichment of pathway terms on the “Lost in KO” STAT5 target genes whose RNA expression was downregulated in KO. Significance was calculated by one-sided Fisher’s Exact test. h Changes in RNA expression of the genes associated with the pathway term of “Regulation of NF-κB activity”. Error bars represent mean ± standard error of the mean (s.e.m.). n = 3 biologically independent samples. i Snapshots displaying virtual 4C plots, ChIP-seq signal tracks, and significant loops (from top to bottom) at the Trim25 (left) and Irak2 (right) loci. Virtual 4C plots (V4C) shows normalized H3K27ac HiChIP loop strength (represented as -Log10( Q )) with the TSSs of Trim25 (left) and Irak2 (right) genes as the viewpoint. IGV browser shows ChIP-seq signal tracks for STAT5, CTCF, SMC1, H3K27me3, H3K4me3, H3K4me1, and H3K27ac. Arcs show significant interactions with –Log10( Q ) ≥ 5. Only the loops interacting with the viewpoint have been displayed. Source data are provided as a Source Data file.

Article Snippet: After blocking with 5% skim milk, the membrane was incubated with primary antibodies with 1:1000 dilution: β-actin (sc-47778) and α-tubulin (sc32293) from Santa Cruz; CTCF (2899) p-JAK2 (3771), JAK2 (3230) p-STAT5 (9351), STAT5 (94205), p-IKKα/β (2078), IKKα (2682), p-IκBα (2859), IκBα (9242), and RelA (8242) from Cell Signaling Technology; ALDH1A2 (ab156019) and Lamin B1 (ab133741) from Abcam, followed by incubation with horseradish peroxidase-conjugated secondary antibody with 1:2000 dilution: HPR-linked anti-Rabbit IgG (7074), and HPR-linked anti-Mouse IgG (7076) from Cell Signaling Technology.

Techniques: Western Blot, Software, Two Tailed Test, ChIP-sequencing, RNA Sequencing Assay, RNA Expression, Activity Assay, HiChIP

Journal: Nature Communications

Article Title: CTCF controls three-dimensional enhancer network underlying the inflammatory response of bone marrow-derived dendritic cells

doi: 10.1038/s41467-023-36948-5

Figure Lengend Snippet: a Western blotting performed with the indicated antibodies. The data are representative of three independent experiments with similar results. b Relative signal intensities of proteins in a were measured using ImageJ software. Error bars represent mean ± standard error of the mean (s.e.m). Significance was calculated using an unpaired two-tailed t -test using n = 3 independent samples. c Histogram showing the average tag density of STAT5 ChIP-seq peaks called for WT and KO BMDCs. d Heatmap of ChIP-seq signals with ±2 kb of unique STAT5 peaks comparing STAT5 enrichment (fold change > 1.3; FDR < 0.05) between WT and KO BMDCs. The data are representative of two independent experiments with similar results. e A pie chart depicting the numbers of genes, assigned to “lost in KO” STAT5 peaks and categorized by the overlap feature of the STAT5 peaks with promoters, distal enhancers, or both. f RNA-seq MA plot for the genes assigned to “lost in KO” STAT5 peaks. The number of genes exhibiting >1.3-fold decreases in WT (red) or KO (blue) BMDCs with a false discovery rate <0.05 has been indicated. g Enrichment of pathway terms on the “Lost in KO” STAT5 target genes whose RNA expression was downregulated in KO. Significance was calculated by one-sided Fisher’s Exact test. h Changes in RNA expression of the genes associated with the pathway term of “Regulation of NF-κB activity”. Error bars represent mean ± standard error of the mean (s.e.m.). n = 3 biologically independent samples. i Snapshots displaying virtual 4C plots, ChIP-seq signal tracks, and significant loops (from top to bottom) at the Trim25 (left) and Irak2 (right) loci. Virtual 4C plots (V4C) shows normalized H3K27ac HiChIP loop strength (represented as -Log10( Q )) with the TSSs of Trim25 (left) and Irak2 (right) genes as the viewpoint. IGV browser shows ChIP-seq signal tracks for STAT5, CTCF, SMC1, H3K27me3, H3K4me3, H3K4me1, and H3K27ac. Arcs show significant interactions with –Log10( Q ) ≥ 5. Only the loops interacting with the viewpoint have been displayed. Source data are provided as a Source Data file.

Article Snippet: After blocking with 5% skim milk, the membrane was incubated with primary antibodies with 1:1000 dilution: β-actin (sc-47778) and α-tubulin (sc32293) from Santa Cruz; CTCF (2899) p-JAK2 (3771), JAK2 (3230) p-STAT5 (9351), STAT5 (94205), p-IKKα/β (2078), IKKα (2682), p-IκBα (2859), IκBα (9242), and RelA (8242) from Cell Signaling Technology; ALDH1A2 (ab156019) and Lamin B1 (ab133741) from Abcam, followed by incubation with horseradish peroxidase-conjugated secondary antibody with 1:2000 dilution: HPR-linked anti-Rabbit IgG (7074), and HPR-linked anti-Mouse IgG (7076) from Cell Signaling Technology.

Techniques: Western Blot, Software, Two Tailed Test, ChIP-sequencing, RNA Sequencing Assay, RNA Expression, Activity Assay, HiChIP

Journal: Cell Communication and Signaling : CCS

Article Title: Induction of DNMT1-dependent demethylation of SHP-1 by the natural flavonoid compound Baicalein overcame Imatinib-resistance in CML CD34 + cells

doi: 10.1186/s12964-023-01049-9

Figure Lengend Snippet: JAK2/STAT5 signaling and downstream protein were observed in CML CD34 + cells in BM microenvironment. BM microenvironment caused a BCR/ABL-independent activation of JAK2/STAT5 in the presence of IM in CML CD34 + cells. K562 CD34 + cells and primary CD34 + CML cells were cultured with or without hBMSCs for 12 h and then treated with various concentrations of IM (0, 0.125, 0.25, 0.5 μM) for 36 h, respectively. A , B Western blotting showed that activated BCR/ABL kinase was presented by staining for the expression of p-BCR/ABL and p-CrkL. STAT5 activation was shown by p-STAT5 Tyr694 -specific antibodies. GAPDH was regarded as loading control. The percentages of proteins p-BCR/ABL, p-CrkL and p-STAT5 Tyr694 were observed by Western blot analysis in CML CD34 + cells. C , D The expressions of p-JAK2 Tyr1007/1008 , JAK2, XIAP, Mcl-1, CyclinD2, Survivin and Bcl-2 were determined using Western blot in CML CD34 + cells, respectively. E , F After IM treatment, the expression of pro-apoptotic proteins caspase3, cleaved-caspase 3 and Bax were determined by Western blot in K562 CD34 + cells and primary CD34 + CML cells. * p < 0.05, ** p < 0.01, *** p < 0.001 the group treated with IM in monolayer culture versus the group treated with IM in co-culture system

Article Snippet: Antibodies for Western blot were used in our research: p-JAK2 Tyr1007/1008 , p-STAT5 Tyr694 , JAK2, STAT5, BCR-ABL, CrkL, Survivin, Bcl-2, Mcl-1, XIAP, cleaved-caspase 3, bax, caspase 3, GAPDH, DNMT1 were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Cell Culture, Western Blot, Staining, Expressing, Co-Culture Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Induction of DNMT1-dependent demethylation of SHP-1 by the natural flavonoid compound Baicalein overcame Imatinib-resistance in CML CD34 + cells

doi: 10.1186/s12964-023-01049-9

Figure Lengend Snippet: GM-CSF/DNMT1 mediated activation of JAK2/STAT5 signaling contributed to resistance in BM microenvironment. A Cytokine array. Different cytokines (80) were screened for differential expression in monolayer culture (up membrane) versus co-culture model in K562 CD34 + cells (low membrane). Different cytokines are spotted in duplicate on each membrane. Darker spots indicate higher expression. Positive controls (4 dots) are shown in the top left corners. Cytokines with increased expression are indicated by bold-lined squares in co-culture system versus thin-lined squares in monolayer culture. B The survival rate of K562 CD34 + cells after treatment with IM for 36 h in monolayer culture and GM-CSF was evaluated comparing with the control treatment (no GM-CSF) appling MTT assay. *** p < 0.001 versus control group. Data points of triplicate experiments are depicted. C Reversal of microenvironment-mediated IM resistance of K562 CD34 + cells by addition of increasing concentrations of neutralizing anti–human GM-CSF antibodies as indicated. * p < 0.05, ** p < 0.01 versus control group. Data points of 3 independent experiments are depicted. D , E K562 CD34 + cells or primary CD34 + CML cells were treated with IM and the raise of GM-CSF dose as indicated in BM microenvironment or in monolayer culture. After treatment for 24 h, cell lysates were acquired for western blot with the indicated antibodies. GAPDH was regarded as loading control. F , G Supplementation of anti-human GM-CSF antibodies in co-culture system, DNMT1 expression was examined in K562 CD34 + cells or primary CD34 + CML cells. H , I K562 CD34 + cells or primary CD34 + CML cells were treated with IM, then GM-CSF or α- rhGM-CSF antibodies was added into BM microenvironment or monolayer culture, respectively. The activity of DNMT1 was detected by ELISA. * p < 0.05, ** p < 0.01 versus CML CD34 + cells treated with IM in monolayer culture. # p < 0.05, ## p < 0.01 versus CML CD34 + cells treated with IM in co-culture system

Article Snippet: Antibodies for Western blot were used in our research: p-JAK2 Tyr1007/1008 , p-STAT5 Tyr694 , JAK2, STAT5, BCR-ABL, CrkL, Survivin, Bcl-2, Mcl-1, XIAP, cleaved-caspase 3, bax, caspase 3, GAPDH, DNMT1 were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Co-Culture Assay, MTT Assay, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Induction of DNMT1-dependent demethylation of SHP-1 by the natural flavonoid compound Baicalein overcame Imatinib-resistance in CML CD34 + cells

doi: 10.1186/s12964-023-01049-9

Figure Lengend Snippet: The effect of Baicalein on IM resistance of CML CD34 + cells within microenvironment. A Effects of Baicalein on proliferation of K562 CD34 + cells were measured by MTT analysis. The cells were treated with various concentrations of Baicalein for 36 h in the supernatant of hBMSCs. B , C K562 CD34 + cells and primary CML CD34 + cells were exposed to 0.5 μM IM within various concentrations of Baicalein for 36 h in co-culture model, respectively. JAK2/STAT5 signaling pathway was determined by Western blot in treated CML CD34 + cells in co-culture model. D Nuclear translocalization of p-STAT5 Tyr694 (green) was acquired by confocal microscopy after staining the indicated antibody, when K562 CD34 + cells treated with or without 20 μM Baicalein in 0.5 μM IM-induced co-culture model. Scale bar, 10 μm. E K562 CD34 + cells and primary CD34 + CML cells were cultured with or without hBMSCs for 12 h and then treated with or without 0.5 μM IM within various concentrations of Baicalein for 36 h, respectively. Apoptosis was assessed by Annexin V-PI double staining after treatment in co-culture model. F , G K562 CD34 + cells and primary CML CD34 + cells were cultured with or without hBMSCs for 12 h and then treated with the combination of 0.5 μM IM and Baicalein for 36 h in co-culture model, respectively. STAT5 DNA binding activity was assessed by EMSA. H Soft-sugar-colony forming experiment was performed to ascertain Baicalein reversal effect after treatment with or without 0.5 μM IM or the combination

Article Snippet: Antibodies for Western blot were used in our research: p-JAK2 Tyr1007/1008 , p-STAT5 Tyr694 , JAK2, STAT5, BCR-ABL, CrkL, Survivin, Bcl-2, Mcl-1, XIAP, cleaved-caspase 3, bax, caspase 3, GAPDH, DNMT1 were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Co-Culture Assay, Western Blot, Confocal Microscopy, Staining, Cell Culture, Double Staining, Binding Assay, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Induction of DNMT1-dependent demethylation of SHP-1 by the natural flavonoid compound Baicalein overcame Imatinib-resistance in CML CD34 + cells

doi: 10.1186/s12964-023-01049-9

Figure Lengend Snippet: The effect of Baicalein on DNMT1-mediated SHP-1 within BM microenvironment. A , B Effects of Baicalein on GM-CSF secretion in K562 CD34 + cells and primary CD34 + CML cells were detected by ELISA assay, respectively. C , D Effects of Baicalein on DNMT1 and SHP-1 expression were determined in both CML CD34 + cells. ** p < 0.01, *** p < 0.001 versus control group (without Baicalein treatment). E , F The expressions of SHP-1 were determined, after treatment with 10 μM DNMT1 inhibitor(decitabine) in CML CD34 + cells. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control group, Data points of 3 independent experiments are depicted. G pCMV6-Entry shp-1 and pCMV6-Entry vector were transfected into CML CD34 + cells. Subsequently, apoptosis was measured by Annexin V-PI double staining assay after treatment with IM in co-culture model. H , I After transfection, the growth inhibition effect of IM on CD34 + subpopulation in K562 cells or primary CML cells with or without the supernatant of hBMSCs was detected by MTT. J , K Silence of SHP-1 by SHP-1shRNA reversed the effects of Baicalein on p-JAK2 Tyr1007/1008 and p-STAT5 Tyr694 (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: Antibodies for Western blot were used in our research: p-JAK2 Tyr1007/1008 , p-STAT5 Tyr694 , JAK2, STAT5, BCR-ABL, CrkL, Survivin, Bcl-2, Mcl-1, XIAP, cleaved-caspase 3, bax, caspase 3, GAPDH, DNMT1 were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Plasmid Preparation, Transfection, Double Staining, Co-Culture Assay, Inhibition

Journal: Cell Communication and Signaling : CCS

Article Title: Induction of DNMT1-dependent demethylation of SHP-1 by the natural flavonoid compound Baicalein overcame Imatinib-resistance in CML CD34 + cells

doi: 10.1186/s12964-023-01049-9

Figure Lengend Snippet: The effect of combination of IM and Baicalein on the leukemogenic activity engrafted with CML CD34 + cells in vivo. A K562 CD34 + cells (2 × 10 6 cells per mouse) were transplanted into NOD/SCID mice, and then engraftments were randomly divided into four groups respectively. The animals engrafted with CD34 + cells were treated with or without IM (200 mg/kg), combination with or without Baicalein (20 mg/kg) (four groups, 5 mice/per group: media control(DMSO); Baicalein; IM; Baicalein + IM). Mice were performed with death after 6 weeks, and bone marrow contents were obtained. B Flow cytometry graphs showed that the levels of human CML CD45 + cells were regenerated in the BM of mice engrafted with K562 CD34 + cells treated with different drugs at 6 weeks. C Images of spleen were obtained from mice in each group. ( D ) Weight of spleen were examined and analyzed, ** p < 0.01, *** p < 0.001 versus control group (DMSO); ## p < 0.01 versus IM group. E The serial sections of spleens were examined for H&E staining. F The levels of human CML CD34 + , CD33 + , and CD19 + cells regenerated in the BM of mice engrafted with K562 CD34 + cells treated with different drugs were measured at 6 weeks. G The expressions of BCR-ABL mRNA obtained from selected CD45 + cells in BM were measured by qRT-PCR and normalized to GAPDH mRNA levels. H GM-CSF secretion in the supernatant of BM was evaluated. I The effect of Baicalein on JAK2/STAT5 signaling pathway was observed by Western blot from BM CML cells of mice. All data from independent experiments are presented as means ± SD. Significance values: ** p < 0.01,*** p < 0.001 versus control group; ## p < 0.01 versus IM group

Article Snippet: Antibodies for Western blot were used in our research: p-JAK2 Tyr1007/1008 , p-STAT5 Tyr694 , JAK2, STAT5, BCR-ABL, CrkL, Survivin, Bcl-2, Mcl-1, XIAP, cleaved-caspase 3, bax, caspase 3, GAPDH, DNMT1 were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, In Vivo, Flow Cytometry, Staining, Quantitative RT-PCR, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Induction of DNMT1-dependent demethylation of SHP-1 by the natural flavonoid compound Baicalein overcame Imatinib-resistance in CML CD34 + cells

doi: 10.1186/s12964-023-01049-9

Figure Lengend Snippet: Schematic diagram illustrated that Baicalein overcame IM resistance by reducing DNMT1-mediated methylation of SHP-1 and inducing an inhibition of JAK2/STAT5 signaling in CML CD34 + cells

Article Snippet: Antibodies for Western blot were used in our research: p-JAK2 Tyr1007/1008 , p-STAT5 Tyr694 , JAK2, STAT5, BCR-ABL, CrkL, Survivin, Bcl-2, Mcl-1, XIAP, cleaved-caspase 3, bax, caspase 3, GAPDH, DNMT1 were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Methylation, Inhibition

Journal: Communications Biology

Article Title: ANKLE1 cleaves mitochondrial DNA and contributes to cancer risk by promoting apoptosis resistance and metabolic dysregulation

doi: 10.1038/s42003-023-04611-w

Figure Lengend Snippet: a Gene Set Enrichment Analysis (GSEA) of RNA-seq results for ANKLE1 overexpressing cells versus control cells identifies hallmarks of STAT1 activation, mitochondria degradation, DNA damage, and Epithelial to Mesenchymal transition. b Gene ontology analysis for differentially expressed genes (Fig. ) after 3-days post-transfection of ANKLE1 cells versus control cells show enrichment for STAT1 activation and mitochondria degradation. c ANKLE1 cuts nuclear DNA in non-apoptotic cells, as shown by DNA Pulse Field Gel Electrophoresis (PFGE) from non-apoptotic cells that overexpress ANKLE1 (24 h post-transfection). d ANKLE1 leads to STAT1 activation after 24 h of ANKLE1-transfection, as shown by western blots probed for P-STAT1, STAT1, P-STAT3,P-STAT5, P-IκB and α-Actin of three independent replicates per condition. The mean and standard deviations for the control and ANKLE1 are shown below the blot. The p -value for differential phosphorylation as measured by a two-tailed T-test is 0.008.

Article Snippet: The following primary antibodies were used: Anti-Phospho-Stat1 (Tyr701) (58D6) antibody (Cell Signaling Technology 14994), Anti-Stat1 (D1K9Y) antibody (Cell Signaling Technology 80916), Anti-Phospho-Stat3 (Tyr705) (D3A7) antibody (Cell Signaling Technology 9145), Anti-Phospho-Stat5 (Tyr694) (D47E7) antibody (Cell Signaling Technology 4322), Anti-Phospho-IκBα (Ser32) (14D4) antibody (Cell Signaling 2859), Anti-Cleaved PARP (Asp214) (D64E10) antibody (Cell Signaling Technology 5625) and Anti-β-actin (AC-74) antibody (GenWay Biotech Inc. GWB-A0AC74).

Techniques: RNA Sequencing Assay, Activation Assay, Transfection, Nucleic Acid Electrophoresis, Western Blot, Two Tailed Test

Journal: Cancer Research Communications

Article Title: S100A8/S100A9 Promote Progression of Multiple Myeloma via Expansion of Megakaryocytes

doi: 10.1158/2767-9764.CRC-22-0368

Figure Lengend Snippet: Mechanism of S100A9 effect on MK differentiation. A, BM cells were isolated from WT, TLR4 KO, or RAGE KO mice and cultured in the presence of TPO with or without S100A9. Number of MKs was determined on day 5 of culture. B, BM cells from WT or TLR4 KO mice were kept in serum-free media with or without S100A9 for 2 hours. Activation of STAT5 was evaluated by Western blotting. C, WT BM cells were cultured in the presence of supernatants collected from BM cells from WT mice (WT-WT) or BM cells from S100A9 KO mice (KO-WT) without addition of TPO with or without 10 μmol/L TQ. Activation of STAT5 was evaluated by Western blotting. Experiment was repeated three times with similar results; results of two of them are shown. D, BM cells from WT or STAT5 KO mice were cultured in the presence of TPO with or without S100A9 protein for 5 days. Absolute number and proportion of MK were determined. Individual values, mean, and SEM values are shown. Statistics: ANOVA with Tukey multiple comparisons test.

Article Snippet: The following primary antibodies were used: anti-His-tag (RRID: AB_2744546), anti-S100A9 (RRID: AB_2734726), anti-phospho-STAT5 (Tyr694) (RRID: AB_10544692), anti-STAT5 (RRID: AB_2737403; Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnology, RRID: AB_626632).

Techniques: Isolation, Cell Culture, Activation Assay, Western Blot

Journal: Cancer Research Communications

Article Title: S100A8/S100A9 Promote Progression of Multiple Myeloma via Expansion of Megakaryocytes

doi: 10.1158/2767-9764.CRC-22-0368

Figure Lengend Snippet: Mechanism of S100A9 effect on MK differentiation. A, BM cells were isolated from WT, TLR4 KO, or RAGE KO mice and cultured in the presence of TPO with or without S100A9. Number of MKs was determined on day 5 of culture. B, BM cells from WT or TLR4 KO mice were kept in serum-free media with or without S100A9 for 2 hours. Activation of STAT5 was evaluated by Western blotting. C, WT BM cells were cultured in the presence of supernatants collected from BM cells from WT mice (WT-WT) or BM cells from S100A9 KO mice (KO-WT) without addition of TPO with or without 10 μmol/L TQ. Activation of STAT5 was evaluated by Western blotting. Experiment was repeated three times with similar results; results of two of them are shown. D, BM cells from WT or STAT5 KO mice were cultured in the presence of TPO with or without S100A9 protein for 5 days. Absolute number and proportion of MK were determined. Individual values, mean, and SEM values are shown. Statistics: ANOVA with Tukey multiple comparisons test.

Article Snippet: The following primary antibodies were used: anti-His-tag (RRID: AB_2744546), anti-S100A9 (RRID: AB_2734726), anti-phospho-STAT5 (Tyr694) (RRID: AB_10544692), anti-STAT5 (RRID: AB_2737403; Cell Signaling Technology), anti-β-actin (Santa Cruz Biotechnology, RRID: AB_626632).

Techniques: Isolation, Cell Culture, Activation Assay, Western Blot