Journal: Doklady. Biochemistry and Biophysics

Article Title: HDAC3 Regulates Ferroptosis via Nrf2–GPX4 Signaling in Colorectal Cancer Cells

doi: 10.1134/S1607672925600496

Figure Lengend Snippet: Class I HDAC inhibition enhances ferroptosis sensitivity in colorectal cancer cells. (a–e) HCT116 cells were treated with the ferroptosis inducer RSL3 (1 µM) in combination with various HDAC inhibitors for 24 h. Intracellular ferrous iron (Fe 2+ ) levels were quantified using the Iron Assay Kit (MAK025, Sigma-Aldrich). Each treatment group consisted of three technical replicates from a representative experiment; data are mean ± SD. Statistical significance was determined by one‑way ANOVA with Tukey’s post hoc test (* p < 0.05 vs CON; NS = not significant). Bars are color‑coded: **blue** = control (CON), **orange** = RSL3, **red** = TSA, **green** = SAHA, **purple** = MS‑275, **brown** = RGFP966, **yellow** = Tubastatin A. (a) TSA (500 nM, pan‑HDAC inhibitor) significantly increased Fe 2+ accumulation. (b) SAHA (1 µM, pan‑HDAC inhibitor) significantly increased Fe 2+ accumulation. (c) MS‑275 (2 µM, Class I HDAC inhibitor) markedly increased Fe 2+ levels. (d) RGFP966 (10 µM, HDAC3‑specific inhibitor) strongly enhanced Fe 2+ levels, suggesting HDAC3 involvement. (e) Tubastatin A (10 µM, HDAC6‑selective inhibitor) showed no significant effect. (f) Reactive oxygen species (ROS) levels were visualized using CellROX™ Green; representative fluorescence images are shown. Scale bar = 50 µm.

Article Snippet: – RGFP966 (Selleck Chemicals, Cat# S7229) was used as an HDAC3-selective inhibitor at a final concentration of 10 μM.

Techniques: Inhibition, Iron Assay, Control, Fluorescence

Journal: Doklady. Biochemistry and Biophysics

Article Title: HDAC3 Regulates Ferroptosis via Nrf2–GPX4 Signaling in Colorectal Cancer Cells

doi: 10.1134/S1607672925600496

Figure Lengend Snippet: HDAC3 regulates GPX4 expression in colorectal cancer cells. (a, b) Quantitative PCR analysis of ferroptosis-related gene expression in HCT116 cells treated with RGFP966 (10 µM, 24 h) (a) or subjected to lentiviral shRNA-mediated HDAC3 knockdown (b). Among the tested genes (COX2, ACSL4, PTGS2, NOX1, FTH1, SLC7A11, and GPX4), only GPX4 mRNA was significantly reduced upon HDAC3 inhibition or silencing (* p < 0.05), while others remained unchanged (NS, not significant). Data represent mean ± SD from three independent experiments. (c, d) Western blot analysis showing decreased GPX4 protein levels following RGFP966 treatment (c) or HDAC3 knockdown using two independent shRNAs (d). ACTIN served as a loading control. Blots are representative of three independent experiments. (e, f) Overexpression of HDAC3 using FLAG-tagged plasmid in 293T (e) and HCT116 (f) cells induced a dose-dependent increase in GPX4 protein expression.

Article Snippet: – RGFP966 (Selleck Chemicals, Cat# S7229) was used as an HDAC3-selective inhibitor at a final concentration of 10 μM.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, shRNA, Knockdown, Inhibition, Western Blot, Control, Over Expression, Plasmid Preparation

Journal: Doklady. Biochemistry and Biophysics

Article Title: HDAC3 Regulates Ferroptosis via Nrf2–GPX4 Signaling in Colorectal Cancer Cells

doi: 10.1134/S1607672925600496

Figure Lengend Snippet: HDAC3 regulates GPX4 expression via NRF2 in colorectal cancer cells. (a) qPCR analysis confirming siRNA-mediated knockdown efficiency of transcription factors NRF2, SP1, ELK1, and CREB in HCT116 cells. Cells were transfected with individual siRNAs for 48 h. Data are presented as mean ± SD ( n = 3). * p < 0.05 vs siNC. (b) GPX4 mRNA levels measured by qPCR following knockdown of each transcription factor and treatment with the HDAC3 inhibitor RGFP966 (10 μM, 24 h). Among the tested factors, only NRF2 knockdown significantly attenuated the RGFP966-induced changes in GPX4 expression, suggesting that NRF2 is required for HDAC3-mediated regulation of GPX4. Data are shown as mean ± SD ( n = 3). * p < 0.05. (c, d) NRF2 mRNA (c) and protein (d) levels after HDAC3 knockdown (shHDAC3) or RGFP966 treatment (10 μM, 24 h) in HCT116 cells. Both interventions led to significant downregulation of NRF2. β-actin was used as the loading control. Data represent three independent experiments. * p < 0.05.

Article Snippet: – RGFP966 (Selleck Chemicals, Cat# S7229) was used as an HDAC3-selective inhibitor at a final concentration of 10 μM.

Techniques: Expressing, Knockdown, Transfection, Control

Journal: Leukemia

Article Title: GNAS knockout potentiates HDAC3 inhibition through viral mimicry-related interferon responses in lymphoma.

doi: 10.1038/s41375-024-02325-4

Figure Lengend Snippet: Fig. 4 GNAS KO synergizes with HDAC3 inhibition to enhance CD8+ T cell-induced cytotoxicity. A Schematic representation of the T cell co-culture cytotoxicity assay. B Quantification of percentage cell death in DLBCL cells. Error bars represent ± SEM (n = 2 independent experiments). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (log2 transformed values or values were compared; one-way ANOVA with Tukey’s HSD test for multiple comparisons). C Flow cytometry-based determination of cell death. Single cells and non-debris were included for analysis of 5- (and 6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) and propidium iodide (PI) signals. CFSE positive and negative populations represent DLBCL cells and CD8+ T cells, respectively. PI positive and negative groups represent dead and live cells, respectively. The total number of single DLBCL cells acquired per sample was 10,000. The percentages shown are percentage cell death within the total DLBCL cell population per sample. PBMCs, peripheral blood mononuclear cells.

Article Snippet: The selective HDAC3 inhibitors RGFP966 and BRD3308 (Selleckchem), the pan-HDAC inhibitor romidepsin (Selleckchem), the cAMP analog 8-Br-cAMP (Tocris), the TBK1 degrader TBK1 PROTAC 3i (Tocris), the JAK1/2 inhibitor ruxolitinib (Selleckchem), and the DNA methyltransferase inhibitor 5-Aza2’-deoxycytidine (5-AZA-CdR; MedChemExpress) were reconstituted and stored according to the manufacturer’s instructions.

Techniques: Inhibition, Co-Culture Assay, Cytotoxicity Assay, Transformation Assay, Flow Cytometry

Journal: Leukemia

Article Title: GNAS knockout potentiates HDAC3 inhibition through viral mimicry-related interferon responses in lymphoma.

doi: 10.1038/s41375-024-02325-4

Figure Lengend Snippet: Fig. 5 GNAS KO plus RGFP966 is associated with dsRNA formation and TE expression for viral mimicry induction. A Representative images of SU-DHL-4 cells stained by Hoechst 33342 (for cell nuclei; blue) and J2 antibody (for dsRNAs; green) from immunofluorescence analysis. Control LacZ cells omitting primary J2 antibody and treated with 300 nM 5-AZA-CdR were the negative and positive control, respectively. Scale bar, 25 µm. B Quantification of results in A. **p < 0.01; ****p < 0.0001 (Kruskal–Wallis test with Wilcoxon rank sum test for pairwise comparisons). C Volcano plots showing normalized expression dynamics of differentially expressed TEs (FDR < 0.05 and |Log2 fold change | > 0) identified from TEtranscripts analysis of cell-line bulk RNA-seq data. Upregulated and downregulated TEs are highlighted in red and blue, respectively. The horizontal and vertical dotted lines indicate FDR = 0.05 and Log2 fold change = 0, respectively. D Normalized TE expression under each comparison by a ratio of upregulated versus downregulated TEs shown in C. The dotted line represents normalized TE expression = 1 (equal numbers of upregulated and downregulated TEs).

Article Snippet: The selective HDAC3 inhibitors RGFP966 and BRD3308 (Selleckchem), the pan-HDAC inhibitor romidepsin (Selleckchem), the cAMP analog 8-Br-cAMP (Tocris), the TBK1 degrader TBK1 PROTAC 3i (Tocris), the JAK1/2 inhibitor ruxolitinib (Selleckchem), and the DNA methyltransferase inhibitor 5-Aza2’-deoxycytidine (5-AZA-CdR; MedChemExpress) were reconstituted and stored according to the manufacturer’s instructions.

Techniques: Expressing, Staining, Control, Positive Control, RNA Sequencing, Comparison

Journal: PLoS ONE

Article Title: Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension

doi: 10.1371/journal.pone.0213186

Figure Lengend Snippet: IC 50 [μM] values for TMP269, RGFP966, and TSA.

Article Snippet: To evaluate the HDAC enzyme inhibitory activity of MS-275 and RGFP966, we determined enzyme activities of HDAC1, HDAC2, HDAC3, and HDAC8 using enzyme assay kits (BPS Bioscience, San Diego, CA, USA) according to the manufacturer’s protocols.

Techniques:

Journal: PLoS ONE

Article Title: Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension

doi: 10.1371/journal.pone.0213186

Figure Lengend Snippet: (A) After angiotensin II infusion (1.3 mg·kg -1 ·day -1 ) to mice for 1 week, we injected MS-275 or RGFP966 (both 3 mg·kg -1 ·day -1 ) daily to mice for additional 7 days. Systolic blood pressures were measured in awake mice. (B and C) Transcript levels of aortic AT1 and ACE1 were quantified using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). GAPDH was used to normalize the values. Data are presented as the means ± SE (n = 8 per group). ***p < 0.001 versus sham group; # p < 0.05 and ### p < 0.001 versus angiotensin II group; NS, not significant.

Article Snippet: To evaluate the HDAC enzyme inhibitory activity of MS-275 and RGFP966, we determined enzyme activities of HDAC1, HDAC2, HDAC3, and HDAC8 using enzyme assay kits (BPS Bioscience, San Diego, CA, USA) according to the manufacturer’s protocols.

Techniques: Injection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension

doi: 10.1371/journal.pone.0213186

Figure Lengend Snippet: (A) Representative images of H&E stained aortas from sham, angiotensin II group (Ang II), MS-275-treated angiotensin II group (Ang II + MS-275), and RGFP966-treated angiotensin II group (Ang II + RGFP966). Scale bar = 100 μm. (B) Arterial wall thickness was quantified. Data are means ± SE (n = 7/group). ***p < 0.001 versus sham group; ## p < 0.01 and ### p < 0.001 versus angiotensin II group. (C‒D) Masson’s trichrome and orcein staining of representative aorta sections. Scale bar = 100 μm. Collagen deposition in aorta is shown as blue staining. (E‒H) The transcript levels of cyclin D1, cyclin E1, E2F3, and GATA6 were quantified using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). * p < 0.05 versus sham group; # p < 0.05 versus angiotensin II group; NS, not significant.

Article Snippet: To evaluate the HDAC enzyme inhibitory activity of MS-275 and RGFP966, we determined enzyme activities of HDAC1, HDAC2, HDAC3, and HDAC8 using enzyme assay kits (BPS Bioscience, San Diego, CA, USA) according to the manufacturer’s protocols.

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension

doi: 10.1371/journal.pone.0213186

Figure Lengend Snippet: The transcript levels for arginase 1 (A), arginase 2 (B), GTPCH1 (C), PRMT1 (D), DDAH1 (E), and DDAH2 (F) were determined using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) in aortas of sham and angiotensin II treated with vehicle, MS-275, or RGFP966. Data are presented as the means ± SE (n = 8 per group). *p < 0.05 and ***p < 0.001 versus sham group; # p < 0.05 versus angiotensin II group; NS, not significant.

Article Snippet: To evaluate the HDAC enzyme inhibitory activity of MS-275 and RGFP966, we determined enzyme activities of HDAC1, HDAC2, HDAC3, and HDAC8 using enzyme assay kits (BPS Bioscience, San Diego, CA, USA) according to the manufacturer’s protocols.

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension

doi: 10.1371/journal.pone.0213186

Figure Lengend Snippet: The transcript levels of Nox1 (A), Nox2 (B), Nox4 (C), p22phox (D), p47phox (E), Cox-2 (F), and SOD3 (G) were determined using qRT-PCR in aortas of sham and Ang II-induced mice treated with vehicle, MS-275, or RGFP966. Results are ± SE (n = 8 per group). * p < 0.05 and ** p < 0.01 versus sham group; NS, not significant.

Article Snippet: To evaluate the HDAC enzyme inhibitory activity of MS-275 and RGFP966, we determined enzyme activities of HDAC1, HDAC2, HDAC3, and HDAC8 using enzyme assay kits (BPS Bioscience, San Diego, CA, USA) according to the manufacturer’s protocols.

Techniques: Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Class I histone deacetylase inhibitor MS-275 attenuates vasoconstriction and inflammation in angiotensin II-induced hypertension

doi: 10.1371/journal.pone.0213186

Figure Lengend Snippet: The transcript levels of iNOS (A), TNF-α (B), IL-1β (C), MCP-1 (D), VCAM-1 (E), and ICAM-1 (F) were determined using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) in aortas of sham and Ang II-induced mice treated with vehicle, MS-275, or RGFP966. Results are means ± SE (n = 8 per group). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus sham group; # p < 0.05 and ## p < 0.01 versus angiotensin II group; NS, not significant. (G) Representative aortic images for macrophage infiltration are shown. Scale bar = 50 μm.

Article Snippet: To evaluate the HDAC enzyme inhibitory activity of MS-275 and RGFP966, we determined enzyme activities of HDAC1, HDAC2, HDAC3, and HDAC8 using enzyme assay kits (BPS Bioscience, San Diego, CA, USA) according to the manufacturer’s protocols.

Techniques: Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Blood Advances

Article Title: Chemical inhibition of PAPD5/7 rescues telomerase function and hematopoiesis in dyskeratosis congenita

doi: 10.1182/bloodadvances.2020001848

Figure Lengend Snippet: RG7834 treatment rescues TERC levels and localization in DKC1- and PARN-depleted cells. (A) Representative northern blots for TERC levels in HeLa cells under indicated conditions. Numbers below panels: average ± standard deviation for 3 biological replicates. (B) Quantification of TERC by quantitative reverse transcription polymerase chain reaction in HeLa cells under the indicated conditions (n = 3; biological replicates). Values are expressed in relation to scrambled control. (C) Representative images for 4′,6-diamidino-2-phenylindole (DAPI) (nucleus), coilin (cajal body), TERC, and merge of individual channels obtained from HeLa cells transfected with indicated siRNAs and treated with RG7834. White arrows indicate TERC localization within the cell. Scale bar, 5 μm. Numbers in image panels: Quantification of fraction of cells with TERC colocalized to cajal bodies from at least 30 independent cells and 3 replicates. Cells with at least one TERC focus colocalized with coilin were counted. (D) Telomerase activity by telomere repeat amplification in HeLa cells transfected with indicated siRNAs and treated with 5 μM of RG7834 or DMSO. Range of protein concentrations represent fourfold serial dilutions. *P < .05; **P < .01. LC, loading control.

Article Snippet: 21 RG7834 was acquired from MedKoo Biosciences (Morrisville, NC) and diluted in dimethyl sulfoxide (DMSO) (MilliporeSigma, Burlington, MA).

Techniques: Northern Blot, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction, Control, Transfection, Activity Assay, Amplification

Journal: Blood Advances

Article Title: Chemical inhibition of PAPD5/7 rescues telomerase function and hematopoiesis in dyskeratosis congenita

doi: 10.1182/bloodadvances.2020001848

Figure Lengend Snippet: RG7834 treatment rescues telomerase activity, increases telomere length, and improves hematopoietic specification in DKC1_A353V mutant hESCs. (A) Quantification of oligo(A) reads at the 3′ end (UGC) of TERC in indicated conditions (average ± standard deviation from 2 independent replicates). TERC reads with more than 2 As at the 3′ end are considered oligoadenylated. (B) Quantification of TERC and TERT by quantitative reverse transcription polymerase chain reaction in DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834 for 30 days (n = 3; biological replicates). (C) Telomerase activity by telomere repeat amplification in DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834. Range of protein concentrations represent fourfold serial dilutions. (D) Telomere length analysis by telomere restriction fragment analysis of DKC1_A353V hESCs treated with DMSO or different concentrations of RG7834 for 90 days. (E) Representative flow cytometric analysis of CD34 and CD43 expression on day 8 of definitive hematopoietic differentiation, following CHIR99021 and SB-431542 treatment in DKC1_A353V cells treated with DMSO or different concentrations of RG7834. (F) Quantification of CD34+CD43– population obtained from day 8 differentiation cultures treated with CHIR99021 and SB-431542, as in panel E. (G) CFC potential of definitive hematopoietic progenitors in WT and DKC1_A353V cells treated with DMSO or 1μM of RG7834 (n = 3; biological replicates). Statistical significance was determined by using one- or two-way analysis of variance following a Bonferroni multiple comparison posttest. *P < .05; **P < .01; ***P < .001. BFU-E, burst forming unit-erythroid; CFC, colony forming cell; MTL, mean telomere length; n.s., not significant.

Article Snippet: 21 RG7834 was acquired from MedKoo Biosciences (Morrisville, NC) and diluted in dimethyl sulfoxide (DMSO) (MilliporeSigma, Burlington, MA).

Techniques: Activity Assay, Mutagenesis, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction, Amplification, Expressing, Comparison

Journal: International Journal of Biological Sciences

Article Title: PPARG Activation of Fatty Acid Metabolism Drives Resistance to Anti-HER2 Therapies in HER2-Positive Breast Cancer

doi: 10.7150/ijbs.99275

Figure Lengend Snippet: ​GW9662 boosts drug sensitivity in HER2-positive breast cancer. (A) Flow cytometry detection of lipid in BT474, SKBR3, HCC1954, JIMT1 cells treated with GW9662; (B) Proliferation curves of different breast cancer cells treated with 10 μM GW9662 (yellow for Luminal type, purple for HER2-positive, and blue for Triple-negative); (C) Gradient concentrations of pyrotinib treatment WT and PR BT474 cells treated with GW9662 were plotted for IC50 curves and IC50 value bar graphs; (D) Expression levels of PI3K/Akt/mTOR signaling pathway proteins in SKBR3 and HCC1954 PPARG-overexpressing cells with or without 10 μM GW9662; (E) Proliferation curves of SKBR3 and HCC1954 PPARG-overexpressing cells with or without 10 μM GW9662; (F) Morphological changes of PDOs with or without GW9662 or with or without trastuzumab/pyrotinib; (G) Schematic representation, and proliferation curves of in situ tumors in Control, P+T, and Combine groups.

Article Snippet: GW9662 and 13(S)-HODE were purchased from GlpBio.

Techniques: Flow Cytometry, Expressing, In Situ, Control