Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitor WT161 modulates the VLA-4/FAK pathway by inhibiting PKA activity in acute lymphoblastic leukemia

doi: 10.1038/s41598-025-23887-y

Figure Lengend Snippet: HDAC6 expression in ALL cell lines and the effect of WT161 on HDAC6, acetyl-α-tubulin, and cell proliferation. ( a ) HDAC6 protein expression in four ALL cell lines (BALL-1, NALM-6, Jurkat, and MOLT-4) as detected by Western blot. ( b ) Relative HDAC6 mRNA levels measured by quantitative PCR (qPCR), showing expression trends consistent with ( a ). ( c ) Western blot analysis of HDAC6 and acetyl-α-tubulin protein levels in cell lines treated with or without 5 µM WT161 for 72 h. ( d ) Concentration- and time-dependent inhibition of cell proliferation by WT161 across all four ALL cell lines. IC 50 values were calculated to quantify compound sensitivity.

Article Snippet: WT161 was obtained from MedChemExpress (USA), vincristine from Abmole Bioscience Inc. (USA), fibronectin from Solarbio Life Sciences (China), and the CellTracker Green CMFDA (5-Chloromethylfluorescein Diacetate) from YEASEN (China).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitor WT161 modulates the VLA-4/FAK pathway by inhibiting PKA activity in acute lymphoblastic leukemia

doi: 10.1038/s41598-025-23887-y

Figure Lengend Snippet: WT161 induces apoptosis and cell cycle arrest in ALL cells. ( a ) Apoptosis analysis by flow cytometry with Annexin V/PI staining in four ALL cell lines after treatment with 5 µM WT161 for 72 h. ( b ) Cell cycle distribution assessed by PI staining following WT161 treatment (5 µM, 72 h), showing G₁-phase accumulation and reduced S-phase population. ( c ) Quantitative analysis of the percentage of cells in G₁ and S phases. Data are presented as the mean ± SD ( n = 3). *** P < 0.001 vs. control. ( d ) Caspase-3, -8, and − 9 activity in ALL cells treated with WT161 (5 µM, 72 h). *** P < 0.001.

Article Snippet: WT161 was obtained from MedChemExpress (USA), vincristine from Abmole Bioscience Inc. (USA), fibronectin from Solarbio Life Sciences (China), and the CellTracker Green CMFDA (5-Chloromethylfluorescein Diacetate) from YEASEN (China).

Techniques: Flow Cytometry, Staining, Control, Activity Assay

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitor WT161 modulates the VLA-4/FAK pathway by inhibiting PKA activity in acute lymphoblastic leukemia

doi: 10.1038/s41598-025-23887-y

Figure Lengend Snippet: WT161 impairs adhesion, migration, and VLA-4 expression in ALL cells. ( a ) Adhesion assay results showing reduced adhesion capacity of ALL cells after treatment with 5 µM WT161 for 72 h, as measured by fluorescence intensity. ( b ) Transwell migration assay demonstrating decreased migration ability of ALL cells treated with 5 µM WT161 for 72 h, as indicated by fewer cells migrating to the lower chamber. ( c ) Flow cytometry analysis showing downregulated surface expression of VLA-4 subunit (CD49d and CD29) in ALL cells treated with WT161 (5 µM, 72 h). ( d ) qRT-PCR data showing the decreased mRNA level of VLA-4 subunits (CD49d/CD29) in ALL cells in response to 5 µM WT161 (72 h treatment). ( e ) Protein expression of VLA-4 subunits assessed by Western blot in ALL cells following WT161 treatment (5 µM, 72 h). Data are presented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control.

Article Snippet: WT161 was obtained from MedChemExpress (USA), vincristine from Abmole Bioscience Inc. (USA), fibronectin from Solarbio Life Sciences (China), and the CellTracker Green CMFDA (5-Chloromethylfluorescein Diacetate) from YEASEN (China).

Techniques: Migration, Expressing, Cell Adhesion Assay, Fluorescence, Transwell Migration Assay, Flow Cytometry, Quantitative RT-PCR, Western Blot, Control

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitor WT161 modulates the VLA-4/FAK pathway by inhibiting PKA activity in acute lymphoblastic leukemia

doi: 10.1038/s41598-025-23887-y

Figure Lengend Snippet: WT161 suppresses PKA signaling and impairs ALL cell adhesion and migration. ( a ) Western blot analysis of phospho-PKA substrates and PKA-Cα in ALL cells treated with 5 µM WT161 for 72 h. ( b ) Intracellular cAMP levelsmeasured by ELISA in WT161-treated cells (5 µM, 72 h). ( c , d ) Expression and phosphorylation of α4-integrin at Ser988 in ( c ) B-ALL and ( d ) T-ALL cells treated with WT161 (5 µM), H-89 (PKA inhibitor), or their combination for 72 h. ( e , f ) FAK and phospho-FAK (Tyr397) levels in ( e ) B-ALL and ( f ) T-ALL cells under the same treatment conditions. ( g , h ) EGFR and phospho-EGFR (Tyr1068) expression in ( g ) B-ALL and ( h ) T-ALL cells. ( i ) Quantification of adherent cells by fluorescence intensity. ( j ) Ratio of migrated cells (measured by Transwell assay) showing decreased migration. Data are presented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control.

Article Snippet: WT161 was obtained from MedChemExpress (USA), vincristine from Abmole Bioscience Inc. (USA), fibronectin from Solarbio Life Sciences (China), and the CellTracker Green CMFDA (5-Chloromethylfluorescein Diacetate) from YEASEN (China).

Techniques: Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Phospho-proteomics, Fluorescence, Transwell Assay, Control

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitor WT161 modulates the VLA-4/FAK pathway by inhibiting PKA activity in acute lymphoblastic leukemia

doi: 10.1038/s41598-025-23887-y

Figure Lengend Snippet: WT161 suppresses the VLA-4/FAK signaling pathway in ALL cells. ( a ) Western blot analysis of phosphorylated α4-integrin (Ser988), FAK (Tyr397), and EGFR (Tyr1068) in ALL cells treated with 5 µM WT161 for 72 h. ( b ) Immunofluorescence staining of active β1 integrin (green) and nuclei (blue, DAPI) in ALL cells following WT161 treatment (5 µM, 72 h), showing reduced membrane localization of active β1 integrin.

Article Snippet: WT161 was obtained from MedChemExpress (USA), vincristine from Abmole Bioscience Inc. (USA), fibronectin from Solarbio Life Sciences (China), and the CellTracker Green CMFDA (5-Chloromethylfluorescein Diacetate) from YEASEN (China).

Techniques: Western Blot, Immunofluorescence, Staining, Membrane

Journal: Scientific Reports

Article Title: Selective HDAC6 inhibitor WT161 modulates the VLA-4/FAK pathway by inhibiting PKA activity in acute lymphoblastic leukemia

doi: 10.1038/s41598-025-23887-y

Figure Lengend Snippet: WT161 suppresses tumor growth and enhances vincristine efficacy in ALL xenograft models. ( a – c ) Tumor volume changes in mice treated with WT161 (80 mg/kg, daily), vincristine (1 mg/kg, weekly), or their combination for 15 days. ( d ) Tumor weight at endpoint showing reduced tumor burden, particularly in the combination group. ( e ) H&E staining of tumor sections revealing increased necrosis, nuclear condensation, and decreased cellular density in treated groups. ( f ) TUNEL staining demonstrating enhanced apoptosis in tumor tissues after treatment. ( g ) Quantitative analysis of TUNEL-positive cells confirming the pro-apoptotic effect of WT161 in vivo. ( h ) Immunofluorescence staining showing increased acetylated α-tubulin (red) in tumor tissues following WT161 treatment. Data are presented as mean ± SD ( n = 3). ** P < 0.01, *** P < 0.001 vs. control.

Article Snippet: WT161 was obtained from MedChemExpress (USA), vincristine from Abmole Bioscience Inc. (USA), fibronectin from Solarbio Life Sciences (China), and the CellTracker Green CMFDA (5-Chloromethylfluorescein Diacetate) from YEASEN (China).

Techniques: Staining, TUNEL Assay, In Vivo, Immunofluorescence, Control

Journal: Frontiers in Pharmacology

Article Title: Identification of WT161 as a Potent Agent for the Treatment of Colitis by Targeting the Nucleotide-Binding Domain-Like Receptor Family Pyrin Domain Containing 3 Inflammasome

doi: 10.3389/fphar.2022.780179

Figure Lengend Snippet: WT161 alleviated intestinal inflammation in DSS-induced colitis model. (A–F) The colitis model was induced in C57BL/6J WT mice by administrating 3% DSS in drinking water from day 1 to day 10. WT161 (10 or 20 mg/kg) or dimethyl sulfoxide (DMSO) were given by intraperitoneal injection from day 1 to day 10 (DMSO + H 2 O group, n = 5; other groups, n = 12). Weight change (A) and disease activity index (DAI) (B) were monitored every day. Gross morphology imaging of the colons (C) , measurement of the colon lengths (D) , representative imaging of H&E-stained colons (E) , and histological analysis (F) of colitis were performed at the end of animal experiments. Damaged crypt structure (red arrow), destroyed epithelial layer (black arrow). Scale bars: 50 μm (magnification ×300). The image and data shown in (A–F) are representative of three independent experiments. For (A,B) , the p -values were calculated using two-way ANOVA with the Bonferroni’s multiple comparisons test (compared with the DMSO + DSS group); for (D,F) , the p -values were calculated using one-way ANOVA with the Bonferroni’s test (compared with the DMSO + DSS group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; while p > 0.05 displayed as ns. DSS, dextran sulfate sodium; DMSO, dimethyl sulfoxide.

Article Snippet: WT161 (cat. no. 1206731-57-8) from Selleck Chemicals; DSS (cat. no. 0216011080, 36,000–50,000 molecular weight) from MP Biomedicals; ultrapure lipopolysaccharide (LPS) ( E. coli 0111: B4, cat. no. tlrl-3pelps), ATP (cat. no. tlrl-atpl), and nigericin (cat. no. tlrl-nig), monosodium urate (MSU) (cat. no. tlrl-msu) from InvivoGen; Protein A/G PLUS-Agarose (cat. no. sc-2003) from Santa Cruz; mouse immunoglobin IgG protein (cat. no. ab198772) from Abcam, and cell lysis buffer (CLB) (cat. no. 9803) from Cell Signaling Technology; mouse IL-1β (cat. no. 88–7013), IL-6 (cat. no. 88-7064-88) ELISA kits (Thermo Fisher).

Techniques: Injection, Activity Assay, Imaging, Staining

Journal: Frontiers in Pharmacology

Article Title: Identification of WT161 as a Potent Agent for the Treatment of Colitis by Targeting the Nucleotide-Binding Domain-Like Receptor Family Pyrin Domain Containing 3 Inflammasome

doi: 10.3389/fphar.2022.780179

Figure Lengend Snippet: WT161 decreased the levels of the pro-inflammatory factors in the colonic tissue of DSS-induced colitis mice and activated peritoneal macrophages induced by LPS + DSS. (A,B) The colonic explants isolated from mice in the animal experiments were cultured for 24 h, and the interleukin (IL)-1β (A) and IL-6 (B) levels in the supernatant was detected by ELISA. For comparison, we extracted the same weight of colon explants (DMSO + H 2 O group, n = 3; other groups, n = 7). (C,D) The IL-1β (C) and IL-6 (D) levels in the supernatant culture of LPS + DSS-activated peritoneal macrophages. For (A–D) , the p -values were calculated using one-way ANOVA with the Bonferroni’s test. ** p < 0.01; **** p < 0.0001. DSS, dextran sulfate sodium; DMSO, dimethyl sulfoxide.

Article Snippet: WT161 (cat. no. 1206731-57-8) from Selleck Chemicals; DSS (cat. no. 0216011080, 36,000–50,000 molecular weight) from MP Biomedicals; ultrapure lipopolysaccharide (LPS) ( E. coli 0111: B4, cat. no. tlrl-3pelps), ATP (cat. no. tlrl-atpl), and nigericin (cat. no. tlrl-nig), monosodium urate (MSU) (cat. no. tlrl-msu) from InvivoGen; Protein A/G PLUS-Agarose (cat. no. sc-2003) from Santa Cruz; mouse immunoglobin IgG protein (cat. no. ab198772) from Abcam, and cell lysis buffer (CLB) (cat. no. 9803) from Cell Signaling Technology; mouse IL-1β (cat. no. 88–7013), IL-6 (cat. no. 88-7064-88) ELISA kits (Thermo Fisher).

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Pharmacology

Article Title: Identification of WT161 as a Potent Agent for the Treatment of Colitis by Targeting the Nucleotide-Binding Domain-Like Receptor Family Pyrin Domain Containing 3 Inflammasome

doi: 10.3389/fphar.2022.780179

Figure Lengend Snippet: WT161 decreased NLRP3 inflammasome activation in the peritoneal macrophages. (A) Mouse peritoneal macrophages were pre-treated with WT161, and then primed with lipopolysaccharide (LPS), followed by stimulation with nigericin. The interleukin (IL)-1β in the supernatant was detected by ELISA. (B,C) The level of cleaved caspase-1 (p10) and mature IL-1β (p17) upon nigericin stimulation in the supernatant (SN) by WT161 treatment was analyzed by western blot (B) and quantitative analysis (C) . (D) Immunoblot analysis of ASC oligomerization in lysates of LPS-primed peritoneal macrophages treated with WT161 and then stimulated with nigericin. (E) Quantitative analysis of ASC oligomerization. (F) Immunofluorescence microscopy analysis of ASC Speck (arrow) in LPS-primed peritoneal macrophages treated with or without WT161, and then stimulated with ATP or nigericin. Scale bars: 10 μm. (G) Percentage of cells with ASC specks. The image and data shown in (A–F) are representative of three independent experiments. For (A,C,E,G) , the p -values were calculated using two-way ANOVA with the Bonferroni’s multiple comparisons test. * p < 0.05; **** p < 0.0001; while p > 0.05 displayed as ns. DMSO, dimethyl sulfoxide; LN, LPS + nigericin; SN, supernatant; MOCK, blank control.

Article Snippet: WT161 (cat. no. 1206731-57-8) from Selleck Chemicals; DSS (cat. no. 0216011080, 36,000–50,000 molecular weight) from MP Biomedicals; ultrapure lipopolysaccharide (LPS) ( E. coli 0111: B4, cat. no. tlrl-3pelps), ATP (cat. no. tlrl-atpl), and nigericin (cat. no. tlrl-nig), monosodium urate (MSU) (cat. no. tlrl-msu) from InvivoGen; Protein A/G PLUS-Agarose (cat. no. sc-2003) from Santa Cruz; mouse immunoglobin IgG protein (cat. no. ab198772) from Abcam, and cell lysis buffer (CLB) (cat. no. 9803) from Cell Signaling Technology; mouse IL-1β (cat. no. 88–7013), IL-6 (cat. no. 88-7064-88) ELISA kits (Thermo Fisher).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Microscopy, Control

Journal: Frontiers in Pharmacology

Article Title: Identification of WT161 as a Potent Agent for the Treatment of Colitis by Targeting the Nucleotide-Binding Domain-Like Receptor Family Pyrin Domain Containing 3 Inflammasome

doi: 10.3389/fphar.2022.780179

Figure Lengend Snippet: WT161 inhibited the expression of NLRP3 and negatively modulated NF-κB signaling. (A–C) Relative mRNA expression of NLRP3 (A) , ASC (B) , and caspase-1 (C) in the primary peritoneal macrophages treated with WT161 (5 μM) for 30 min and primed with lipopolysaccharide (LPS) for the indicated hours. (D) Immunoblot analysis of p-p65, p65, NLRP3, ASC, and caspase-1 in the mouse peritoneal macrophages with same treatment in (A–C) . (E) Relative expression to β-actin of NLRP3, ASC, caspase-1 in the primary peritoneal macrophages. (F) Ratio of p-p65/p65 in the primary peritoneal macrophages. (G) Immunoblot analysis of NLRP3, ASC, and caspase-1 in the colonic tissue isolated from DSS-induced colitis mice. (H) Relative expression to β-actin of NLRP3, ASC, and caspase-1 in the colonic tissue isolated from DSS-induced colitis mice. The image and data shown in (A –H) are representative of three independent experiments. For (A– C, E, F, H) , the p -values were calculated using two-way ANOVA with the Bonferroni’s multiple comparisons test. ** p < 0.01; *** p < 0.001; **** p < 0.0001; while p > 0.05 displayed as ns. DMSO, dimethyl sulfoxide; WCL, whole cell lysate.

Article Snippet: WT161 (cat. no. 1206731-57-8) from Selleck Chemicals; DSS (cat. no. 0216011080, 36,000–50,000 molecular weight) from MP Biomedicals; ultrapure lipopolysaccharide (LPS) ( E. coli 0111: B4, cat. no. tlrl-3pelps), ATP (cat. no. tlrl-atpl), and nigericin (cat. no. tlrl-nig), monosodium urate (MSU) (cat. no. tlrl-msu) from InvivoGen; Protein A/G PLUS-Agarose (cat. no. sc-2003) from Santa Cruz; mouse immunoglobin IgG protein (cat. no. ab198772) from Abcam, and cell lysis buffer (CLB) (cat. no. 9803) from Cell Signaling Technology; mouse IL-1β (cat. no. 88–7013), IL-6 (cat. no. 88-7064-88) ELISA kits (Thermo Fisher).

Techniques: Expressing, Western Blot, Isolation