primary antibodies against ace  (R&D Systems)

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of anti-tumor activity of iMac with induced ACE expression, in vitro. a Proliferation of melanoma (SK-MEL-28), Triple Negative Breast Cancer (TNBC: HCC1806), and FP chemotherapy resistant HNSCC (FaDu/FP-R) cell lines cultured alone or indirectly co-cultured with iMac ( ± Dox) using 3.0 µm pore polyester membrane inserts for 5 days. Total and dead cell counts were determined via trypan blue exclusion on days 1, 3, and 5. b , c Quantification of iNOS and interleukin-12 (IL-12) production in Cnt-iMac and ACE-iMac treated with LPS (500 ng/ml, 12 h), ± Dox. d ROS production in ACE-iMac following LPS stimulation (1 µg/ml, 30 min) ±Dox, assessed by DCFDA (2’,7’-dichlorodihydrofluorescein diacetate) staining. Representative histograms (left) and mean fluorescence intensity (MFI) quantification (right) are shown. Nitric Oxide (NO) production in ACE-iMac stimulated with LPS (75 ng/ml) for 24 h ( e ), and/or conditioned with SK-MEL-28 supernatant for 24 h ( f ) ± Dox. g Perforin and IFN-γ levels in human peripheral blood NK and Tc cells, respectively, after co-culture with Cnt-iMac or ACE-iMac pretreated with LPS (24 h), followed by melanoma conditioning (24 h). To induce ACE expression, myeloid progenitors were differentiated in the presence of Dox and maintained at 1 µg/ml throughout all in vitro assays. Experiments were performed in triplicates across three independent replicates. Statistical analyses included one-way ANOVA ( a ), two-way ANOVA with Bonferroni correction ( b , c , g ), and two-sided unpaired Student’s t -test ( d – f ). Data are presented as means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Activity Assay, Expressing, In Vitro, Cell Culture, Membrane, Staining, Fluorescence, Co-Culture Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Analysis of anti-tumor macrophage activation in the TME post-iMac treatment. Tumors were harvested and single-cell suspensions were prepared for flow cytometry analysis as detailed in the methods section (refer to Fig. ). a Gating strategy for distinguishing M1 and M2 macrophages using flow cytometry based on surface marker immunostaining. b Assessment of CD86 + (M1) and CD206 + (M2) expression in tumor associated macrophages (TAMs) in SK-MEL-28-derived xenografts (±Dox). The bar plot presents FACS quantification of M1 and M2 populations ( n = 5/group). c Assessment of arginase expression in TAMs. d ACE expression in TAMs. e – g Measurement of proinflammatory and anti-tumor markers IFN-γ, IL-12, and iNOS (left: representative histogram; right: mean fluorescence intensity) in TAMs in melanoma xenografts ( n = 5/group). Statistical analysis was performed using a two-sided unpaired Student’s t -test for comparisons in ( b , d , g ), and one-way ANOVA with Bonferroni’s correction for multiple comparisons in ( c , e , f ). Data are presented as means ± SEM. * p < 0.05 and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Activation Assay, Single Cell, Flow Cytometry, Marker, Immunostaining, Expressing, Derivative Assay, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: The effect of ACE-iMac treatment on human immune cells in a humanized mouse tumor model. a Growth of SK-MEL-28 tumors in humanized BLT-NSG mice; comparing tumor volumes between PBS, iMac and ACE-iMac treated groups. b Representative images of tumors are shown (see Supplementary Fig. for additional images). c Final tumor volumes on Day 28 post-implantation. d Tumor weight on Day 28 post-implantation. Flow cytometry analysis of anti-tumor activation of human CD8 T cells in the TME, measured by intracellular IFN-γ ( e ) and perforin ( f ) in tumor-infiltrating CD3 + CD8 + T cells at sacrifice on Day 28 post-implantation. g Flow cytometry analysis of anti-tumor activation of NK cells, measured by intracellular IFN-γ in tumor-infiltrating CD45 + CD56 + NK cells in the TME on Day 28. The flow cytometry gating strategy for identifying human immune cells is provided in Supplementary Fig. . One-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SEM ( n = 9–10). * p < 0.05, ** p < 0.01, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Flow Cytometry, Activation Assay

Journal: Annals of Hematology

Article Title: Chidamide and venetoclax synergistically regulate the Wnt/β-catenin pathway by MYCN/DKK3 in B-ALL

doi: 10.1007/s00277-024-06110-2

Figure Lengend Snippet: Chidamide and venetoclax synergistically induce cell cycle arrest, promote apoptosis, inhibit the expression of HDACs and the Wnt/β-catenin signaling pathway in B-ALL cells. Western blots showing the effects of chidamide, venetoclax, and their combination on the expression of BCL-X L ( A ), MCL1 ( B ), Cyclin D1 ( C ), BCL2 ( D ), BAX ( E ) Ace-H3K18 ( F ), Ace-H4K8 ( G ), β-catenin (nuclear and cytoplasmic) ( H , I ) and p-GSK3β, GSK3β ( J ) proteins in Nalm6 and SupB15 cells. GAPDH served as the internal reference. ns P > 0.05; * P < 0.05; ** P < 0.01 *** P < 0.001; **** P < 0.0001. # P < 0.05; ## P < 0.01 ### P < 0.001; #### P < 0.0001. Control: B-ALL cell lines without chidamide or venetoclax

Article Snippet: Primary antibodies against MYCN, DKK3, Ace-H3K18, and Ace-H4K8 were purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China), and antibodies against cyclin D1, BCL2, BCL-X L , MCL1, BAX, β-catenin, p-GSK3β, GSK3β, and GAPDH were purchased from Wanlei Biotechnology Co., Ltd. (Shenyang, China).

Techniques: Expressing, Western Blot, Control

Journal: Annals of Hematology

Article Title: Chidamide and venetoclax synergistically regulate the Wnt/β-catenin pathway by MYCN/DKK3 in B-ALL

doi: 10.1007/s00277-024-06110-2

Figure Lengend Snippet: Chidamide combined with venetoclax inhibits the expression of MYCN and increases the expression of DKK3 by inhibiting the activity of HDAC and BCL2, inhibiting both the Wnt/β-catenin signaling pathway and B-ALL cell proliferation in vivo. Western blots showing the effects of chidamide, venetoclax, and their combination on the expression of MYCN ( A ), DKK3 ( B ), BCL2 ( C ), BCL-X L ( D ), MCL1 ( E ), Ace-H3K18 ( F ), Ace-H4K8 ( G ), β-catenin (nuclear and cytoplasmic) ( H , I ) and p-GSK3β, GSK3β ( J ) proteins in Nalm6 xenograft mouse model cells. GAPDH served as the internal reference. ns P > 0.05; * P < 0.05; ** P < 0.01 *** P < 0.001; **** P < 0.0001. # P < 0.05; ## P < 0.01 ### P < 0.001; #### P < 0.0001. Control: B-ALL cell lines without chidamide or venetoclax

Article Snippet: Primary antibodies against MYCN, DKK3, Ace-H3K18, and Ace-H4K8 were purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China), and antibodies against cyclin D1, BCL2, BCL-X L , MCL1, BAX, β-catenin, p-GSK3β, GSK3β, and GAPDH were purchased from Wanlei Biotechnology Co., Ltd. (Shenyang, China).

Techniques: Expressing, Activity Assay, In Vivo, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: A RT-qPCR analysis of Sirt6, Acox1, Lpl, Pparα, Cpt1a, and Cyp4a14 levels in the liver tissues of mice. B The protein levels of Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1, Lpl, Cpt1a, and Cyp4a14 in livers were determined by Western blotting. C Primary hepatocytes were isolated from FoxA1-LKO mice and infected with Ad-LacZ or Ad-FoxA1 before treatment with palmitic acid (PAL) (0.4 mM). Lipid droplet formation was evaluated by BODIPY staining (scale bar = 100 µm). D , E RT-qPCR and Western blotting analyzed Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1, Lpl, Cpt1a and Cyp4a14 expression in the primary hepatocytes. F , G Primary hepatocytes were isolated from FoxA1 flox/flox mice and infected with Ad-shLacZ or Ad-shFoxA1, followed by treatment with PAL. Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1, Lpl, Cpt1a, and Cyp4a14 expression levels were measured by RT-qPCR and Western blotting. H , I Primary hepatocytes from FoxA1 flox/flox mice were infected with Ad-FoxA1, Ad-shSirt6, or a combination of them, and then treated with PAL. Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1, Lpl, Cpt1a, and Cyp4a14 expression levels were detected by RT-qPCR and Western blotting. Data was repeated at least 3 times. * p < 0.05, ** p < 0.01, and *** p < 0.001. For A-G, Student’s t test was performed. For H-I, one-way ANOVA followed by Tukey’s multiple comparison test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Quantitative RT-PCR, Western Blot, Isolation, Infection, Staining, Expressing, Comparison

Journal: Cell Death & Disease

Article Title: Impaired SUMOylation of FoxA1 promotes nonalcoholic fatty liver disease through down-regulation of Sirt6

doi: 10.1038/s41419-024-07054-1

Figure Lengend Snippet: Primary hepatocytes extracted from FoxA1-LKO mice were transduced with Ad-LacZ or Ad-Sirt6, followed by PAL treatment. A Primary hepatocytes were received BODIPY staining to observe lipid droplets (scale bar = 100 µm). After feeding with HFD for 2 weeks, Ad-LacZ were injected into FoxA1 flox/flox mice, and Ad-LacZ or Ad-Sirt6 were injected into FoxA1-LKO mice. 14 days after injection, the liver tissues were collected. B , C Sirt6, FoxO1, Ac-FoxO1, Pparα, Acox1 and Lpl levels were detected by RT-qPCR and Western blotting, respectively. D , E Liver cholesterol and triglycerides levels were determined. F Liver steatosis was evaluated by HE staining (scale bar = 50 µm). G Lipid accumulation in livers was analyzed by Oil-Red O staining (scale bar = 50 µm). N = 6, * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA followed by Tukey’s multiple comparison test was performed.

Article Snippet: The blots were blocked by skim milk and incubated using the primary antibodies against Sirt6 (A18468, 1:500, ABclonal), Cpt1a (ab234111, 1:1000, Abcam), Acox1 (ab184032, 1:1000, Abcam), Lpl (ab91606, 1:1000, Abcam), Pparα (bs-3614R, 1:500, Bioss), Cyp4a14 (ab3573, 1:1000, Abcam), FoxA1 (A15278, 1:500, ABclonal), Sae1 (ab185949, 1:1000, Abcam), Sae2 (ab185955, 1:1000, Abcam), Ubc9 (ab33044, 1:1000, Abcam), Pias1/2 (ab77231, 1:1000, Abcam), Pias3 (ab105178, 1:1000, Abcam), Pias4 (ab137500, 1:1000, Abcam), FoxO1 (ab70382, 1:2000, Abcam), Ac-FoxO1 (PA5-104560, 1:1000, Thermo Fisher), Ac-K (ab190479, 1:1000, Abcam), Cpt2 (ab181114, 1:1000, Abcam), β-actin (bs-0061R, 1:5000, Bioss) at 4 °C overnight.

Techniques: Transduction, Staining, Injection, Quantitative RT-PCR, Western Blot, Comparison

Journal: Cell Death & Disease

Article Title: FBP1 orchestrates keratinocyte proliferation/differentiation and suppresses psoriasis through metabolic control of histone acetylation

doi: 10.1038/s41419-024-06706-6

Figure Lengend Snippet: a 1 × 10 6 Ctrl or shFBP1-2 HaCaT cells were harvested and acetyl-CoA levels in HaCaT cells were detected by acetyl-CoA Assay. Values were expressed as fold differences compared to Ctrl, n = 3 independent experiments. b Ctrl or shFBP1-2 HaCaT cells were exposed to 0.06 mM (−) or 1.2 mM (+) calcium for 6 days before collection. The indicated proteins were detected by western blots. c Immunostaining for H3K9-Ac (green) in Ctrl or shFBP1-2 HaCaT cells. The nuclei were stained by DAPI. Scale bars: 100 μm. d Ctrl or shFBP1-2 HaCaT cells were treated with 0 mM (−), 1 mM (+) or 2 mM (++) 2-DG for 2 days, and then exposed to 0.06 mM (−) or 1.2 mM (+) calcium for 6 days before collection. The indicated proteins were detected by western blots. e Ctrl or shFBP1-2 HaCaT cells were treated with 0 mM (−), 2.5 mM (+) or 5 mM (++) 2-HC for 2 days, and then exposed to 0.06 mM (−) or 1.2 mM (+) calcium for 6 days before collection. The indicated proteins were detected by western blots. f Ctrl or shFBP1-2 HaCaT cells were plated (1000 cells/well) and cultured with or without 2.5 mM 2-HC for 2 weeks. The colonies were stained with 0.1% crystal violet and counted, n = 3 independent experiments. g H3K9-ac abundance on indicated genes in Ctrl or shFBP1-2 HaCaT cells was detected by H3K9-ac immunoprecipitation and qPCR. Values were expressed as fold differences compared to Ctrl, n = 4 independent experiments. h Relative mRNA levels of CDC6 and TP63 in Ctrl and shFBP1 HaCaT cells, n = 4 independent experiments. Data are shown as mean ± s.d. Statistical analyses in ( a ), ( f ), ( g ) and ( h ) were performed with Student’s t -tests. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant.

Article Snippet: Cells were incubated with primary antibodies against H3K9-ac (Zen-bio, 340016) overnight at 4 °C.

Techniques: Western Blot, Immunostaining, Staining, Cell Culture, Immunoprecipitation

Journal: Heliyon

Article Title: Electroacupuncture regulates histone acetylation of Bcl-2 and Caspase-3 genes to improve ischemic stroke injury

doi: 10.1016/j.heliyon.2024.e27045

Figure Lengend Snippet: EA treatment enhances histone acetylation levels in the ischemic hemisphere 1 day after MCAO. The tissue was collected to isolate proteins for ELISA and Western blot. HAT and HDAC activity were measured by enzyme-linked immunosorbent assay (ELISA). ( A ) HAT activity (n = 5). ( B ) HDAC activity (n = 4). ( C–I ) Histone acetylation (H3, H3K9ace, and H3K27ace) levels were detected by Western blot and quantification analysis. Protein loading was normalized to Histone3 or GAPDH, which served as a control. For original immunoblot images, please see Supplementary material 2. The quantitation of blots was performed by densitometric analysis (n = 4). The information is presented as mean standard deviation (SD). The error bars represent standard deviation, and the statistical symbol indicates a statistical difference between the indicated columns, based on one-way ANOVA. * P < 0.05 vs. the sham group, # P < 0.05 vs. the MCAO group, ^ P < 0.05 vs. the EA group.

Article Snippet: The membranes were then incubated with primary antibodies against Ace-H3 (Millipore, cat #9717, 1:1000), H3K9ace (Cell Signaling Technology, cat #6949, 1:1000), H3K27ace (Cell Signaling Technology, cat #4353, 1:1000), Histone3 (Abcam, ab1791, 1:1000), Bcl-2 (Abcam, ab59348, 1:1000), Bax (Cell Signaling Technology, cat #2772, 1:1000), caspase-3 (Cell Signaling, cat #9662, 1:1000), cleaved caspase-3 (Cell Signaling, cat #9664S, 1:1000) and GAPDH (SAB, cat #21612S; 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Activity Assay, Control, Quantitation Assay, Standard Deviation