Journal: Angiogenesis

Article Title: Exploiting porphyrin metabolism to inhibit angiogenesis

doi: 10.1007/s10456-026-10034-y

Figure Lengend Snippet: Heme synthesis is up regulated during retinal angiogenesis a Schematic representation of heme synthesis and catabolism pathways. Up-regulated genes are displayed in red. b–h Quantitative real-time reverse transcription PCR (qRT-PCR) analysis of heme-related genes (i.e. Alas1 , Alad , Hmbs , Uros , Cpox , Fech , Hmox-1 ) in CD31+ (EC, red line) and CD31- (retina parenchyma, grey line) cells isolated from developing retina at P3, P6 and P15. Data are representative of at least 3 independent experiments and are expressed as mean ± SEM. * p < 0.05; ** p < 0.01; **** p < 0.0001. For statistical analyses ordinary two-way ANOVA test with Sidak’s multiple comparisons was used. i – l Single-cell RNA sequencing data from mouse retina on P6. A colour-coded UMAP of cell type identity is shown ( i ) alongside UMAPs illustrating the expression levels of the indicated genes in each cell ( j and k ). The corresponding violin plots illustrate the expression levels of the indicated genes for each cell cluster ( l ). Each data point represents the value for one cell. FC, fold change

Article Snippet: FECH , Hs00164616_m1.

Techniques: Reverse Transcription, Quantitative RT-PCR, Isolation, Single Cell, RNA Sequencing, Expressing

Journal: Toxicological Sciences

Article Title: The Isoniazid Metabolites Hydrazine and Pyridoxal Isonicotinoyl Hydrazone Modulate Heme Biosynthesis

doi: 10.1093/toxsci/kfy294

Figure Lengend Snippet: Isoniazid decreases human FECH protein levels, but not mRNA levels. Western blot analysis of FECH in primary human hepatocytes (PHHs) treated with 10 µM rifampicin (RIF), 10 µM SPA70, 200 µM isoniazid (INH), or a combination of these drugs (A) and a dose-response assay of isoniazid for 72 h (B). C, Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) analysis of FECH expression in (A). Western blot analysis (D) and qRT-PCR analysis (E) of FECH expression in HepG2/C3A cells treated with isoniazid for 72 h. For PHHS, the results are presented as the mean fold change in expression±SEM. For HepG2/C3A cells, the results are presented as the mean fold change in expression for 3 biological repeats, each with 3 technical repeats. Two-way ANOVA followed by Tukey’s post hoc analysis was used to compare group means for qRT-PCR analysis. One-way ANOVA followed by Dunnett’s post hoc analysis was used to compare group means for Western blot analysis. **p < .01; *p < .05.

Article Snippet: The following TaqMan probes were used: CYP3A4 (Hs00604506_m1), ALAS1 (Hs00963534_m1), Alas1 (Mm01235914_m1), Fech (Mm00500394_m1), FECH (Hs01555261_m1), Cyp3a11 (Mm00731567), hPXR (Hs01114267_m1), 18S (Hs03928990_g1), and Gapdh (Mm99999915_g1).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

Journal: Toxicological Sciences

Article Title: The Isoniazid Metabolites Hydrazine and Pyridoxal Isonicotinoyl Hydrazone Modulate Heme Biosynthesis

doi: 10.1093/toxsci/kfy294

Figure Lengend Snippet: Iron levels affect FECH protein levels. Western blot analysis of FECH in HepG2/C3A cells treated for 24 h with deferoxamine mesylate in 10% fetal bovine serum (FBS)-containing medium (A), with FeCl3 in 1% FBS (B), and with FeCl3 in 10% FBS in the presence or absence of 4.7 µM deferoxamine (C). The results are presented as the mean fold change in expression±SEM for 3 biological repeats, each with 3 technical repeats. One-way ANOVA followed by Dunnett’s post hoc analysis was used to compare group means for Western blot analysis. ****p < .0001; **p < .01; *p < .05.

Article Snippet: The following TaqMan probes were used: CYP3A4 (Hs00604506_m1), ALAS1 (Hs00963534_m1), Alas1 (Mm01235914_m1), Fech (Mm00500394_m1), FECH (Hs01555261_m1), Cyp3a11 (Mm00731567), hPXR (Hs01114267_m1), 18S (Hs03928990_g1), and Gapdh (Mm99999915_g1).

Techniques: Western Blot, Expressing

Journal: Toxicological Sciences

Article Title: The Isoniazid Metabolites Hydrazine and Pyridoxal Isonicotinoyl Hydrazone Modulate Heme Biosynthesis

doi: 10.1093/toxsci/kfy294

Figure Lengend Snippet: Detection of pyridoxal isonicotinoyl hydrazone (PIH) after isoniazid (INH) application. (A) Pyridoxal isonicotinoyl hydrazone is formed from isoniazid and pyridoxal. The LC/MS/MS analysis of pyridoxal isonicotinoyl hydrazone in the livers of mPxr−/−-C57BL/6 mouse with (hPXR) and without (Pxr-/-) hPXR-transgene, treated for 6 weeks with control chow, control water, chow containing 100 mg/kg rifampicin, and/or water containing 400 mg/l isoniazid (B); in primary human hepatocyte (PHH) cell culture medium treated for 72 h with DMSO, 10 µM rifampicin, 200 µM isoniazid, 10 µM SPA70, or a combination of these drugs (C); and in HepG2/C3A cell culture medium in a time-dependent (D) and a dose-dependent (E) manner after treatment with isoniazid or other compounds as indicated. The LC/MS/MS analysis of pyridoxal isonicotinoyl hydrazone in HepG2/C3A cell culture medium treated for 24 h with various concentrations of pyridoxal 5′-phosphate (F), pyridoxal HCl (G), or pyridoxine HCl (H). The LC/MS/MS analysis of pyridoxal isonicotinoyl hydrazone in phosphate-buffered saline treated with the indicated vitamin B6 analog (1 mM) at the indicated times and temperatures in the presence (I) or absence (J) of 1 mM isoniazid. Western blot analysis of FECH in HepG2/C3A cells treated for 24 h with pyridoxal 5′-phosphate (K) or pyridoxal HCl (L) in the presence or absence of 200 µM isoniazid. The results for PIH levels are presented as the mean fold change for 3 biological replicates±SEM for the in vitro data and as the mean (n = 4–5 per group)±SEM for the in vivo data. The results for Western blot analysis of FECH are presented as the mean fold change in expression±SEM for 3 biological repeats, each with 3 technical repeats. One-way ANOVA followed by Dunnett’s post hoc analysis was used to compare group means for Western blot analysis and LC/MS/MS analysis. ****p < .0001; ***p < .001; **p < .01; *p < .05.

Article Snippet: The following TaqMan probes were used: CYP3A4 (Hs00604506_m1), ALAS1 (Hs00963534_m1), Alas1 (Mm01235914_m1), Fech (Mm00500394_m1), FECH (Hs01555261_m1), Cyp3a11 (Mm00731567), hPXR (Hs01114267_m1), 18S (Hs03928990_g1), and Gapdh (Mm99999915_g1).

Techniques: Liquid Chromatography with Mass Spectroscopy, Cell Culture, Western Blot, In Vitro, In Vivo, Expressing

Journal: Toxicological Sciences

Article Title: The Isoniazid Metabolites Hydrazine and Pyridoxal Isonicotinoyl Hydrazone Modulate Heme Biosynthesis

doi: 10.1093/toxsci/kfy294

Figure Lengend Snippet: The effect of isoniazid metabolites on FECH and aminolevulinic acid synthase (ALAS1) levels. Western blot analysis of FECH (A) and ALAS1 (B) in HepG2/C3A cells treated for 16 h with the indicated compound at a concentration of 100 µM. Western blot analysis (C) and qRT-PCR analysis (D) of FECH in HepG2/C3A cells treated for 24 h with the indicated dose of pyridoxal isonicotinoyl hydrazone. Western blot analysis (E) and qRT-PCR analysis (F) of ALAS1 in HepG2/C3A cells treated for 24 h with the indicated dose of hydrazine. Western blot analysis (G) and qRT-PCR analysis (H) of ALAS1 in primary human hepatocytes (PHHs) treated for 24 h with the indicated dose of hydrazine. For qRT-PCR analysis and Western blot analysis of FECH, the results are presented as the mean fold change in expression±SEM for 3 biological repeats, each with 3 technical repeats. For Western blot analysis of ALAS1, the results are presented as the mean fold change in expression±SEM for 3 biological repeats. Two-way ANOVA followed by Tukey’s post hoc analysis was used to compare group means for qRT-PCR analysis. One-way ANOVA followed by Dunnett’s post hoc analysis was used to compare group means for Western blot analysis. ****p < .0001; ***p < .001; **p < .01; *p < .05. DEF, Deferoxamine mesylate; INA, isonicotinic acid; A-INH, acetyl-isoniazid; Ac-HZ, acetyl-hydrazine; DiacHZ, diacetylhydrazine; HZ, hydazine; PIH, pyridoxal isonicotinoyl hydrazone.

Article Snippet: The following TaqMan probes were used: CYP3A4 (Hs00604506_m1), ALAS1 (Hs00963534_m1), Alas1 (Mm01235914_m1), Fech (Mm00500394_m1), FECH (Hs01555261_m1), Cyp3a11 (Mm00731567), hPXR (Hs01114267_m1), 18S (Hs03928990_g1), and Gapdh (Mm99999915_g1).

Techniques: Western Blot, Concentration Assay, Quantitative RT-PCR, Expressing

Journal: Toxicological Sciences

Article Title: The Isoniazid Metabolites Hydrazine and Pyridoxal Isonicotinoyl Hydrazone Modulate Heme Biosynthesis

doi: 10.1093/toxsci/kfy294

Figure Lengend Snippet: Pyridoxal isonicotinoyl hydrazone decreases FECH levels by decreasing iron availability in EMEM containing 10% fetal bovine serum. Western blot analysis of HepG2/C3A cells (A) and primary human hepatocytes (PHHs) (B) treated with increasing doses of FeCl3 in the presence or absence of pyridoxal isonicotinoyl hydrazone (PIH) (in HepG2/C3A cells) and in the presence or absence of PIH and isoniazid (in PHHs). Spectrophotometric analysis of CAS shuttle assays of deferoxamine (C), PIH (D), biochemical reaction products of pyridoxal HCl and isoniazid (E), and HepG2/C3A cell culture medium (F) treated with pyridoxal in the presence or absence of isoniazid (100 µM). For Western blot analysis of FECH in HepG2/C3A cells, the results are presented as the mean fold change in expression±SEM for 3 biological repeats, each with 3 technical repeats. For Western blot analysis of FECH in PHHs from donors, the results are presented as the mean fold change in expression±SEM. The results of spectrophotometric analysis of the background-subtracted absorbance (630 nm) of the CAS shuttle solution are presented as the mean±SEM of 3 repeats for deferoxamine and PIH dilutions and biochemical reaction products and as the mean±SEM for 3 biological repeats, each with 3 technical repeats, for the HepG2/C3A cell culture medium. One-way ANOVA followed by Dunnett’s post hoc analysis was used to compare group means for Western blot analysis and spectrophotometric analysis. ****p < .0001; ***p < .001; **p < .01; *p < .05.

Article Snippet: The following TaqMan probes were used: CYP3A4 (Hs00604506_m1), ALAS1 (Hs00963534_m1), Alas1 (Mm01235914_m1), Fech (Mm00500394_m1), FECH (Hs01555261_m1), Cyp3a11 (Mm00731567), hPXR (Hs01114267_m1), 18S (Hs03928990_g1), and Gapdh (Mm99999915_g1).

Techniques: Western Blot, Cell Culture, Expressing

Journal: Toxicological Sciences

Article Title: The Isoniazid Metabolites Hydrazine and Pyridoxal Isonicotinoyl Hydrazone Modulate Heme Biosynthesis

doi: 10.1093/toxsci/kfy294

Figure Lengend Snippet: The proposed mechanism of regulation of heme biosynthesis. Vitamin B6 analogs form pyridoxal isonicotinoyl hydrazone (PIH) in the presence of isoniazid. Pyridoxal isonicotinoyl hydrazone chelates iron and prevents the stabilization of FECH. Isoniazid breaks down to hydrazine, and hydrazine upregulates aminolevulinic acid synthase at the transcription level. Both effects may be expected to increase the levels of liver protoporphyrin IX and lead to cholestatic liver injury. ALA, aminolevulinic acid.

Article Snippet: The following TaqMan probes were used: CYP3A4 (Hs00604506_m1), ALAS1 (Hs00963534_m1), Alas1 (Mm01235914_m1), Fech (Mm00500394_m1), FECH (Hs01555261_m1), Cyp3a11 (Mm00731567), hPXR (Hs01114267_m1), 18S (Hs03928990_g1), and Gapdh (Mm99999915_g1).

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: KDM4C works in concert with GATA1 to regulate heme metabolism in head and neck squamous cell carcinoma

doi: 10.1007/s00018-025-05693-x

Figure Lengend Snippet: Heme-metabolism genes are regulated by KDM4C and GATA1. A Top six KDM4C-correlated pathways from GSEA analysis of KDM4C knockdown (KD) RNA-seq result. B KDM4C-KD SAS cells enrich for gene signatures characteristic of heme-metabolism loss, as identified by mRNA-seq followed by GSEA. C Enrichr analysis of key transcription factors in heme-metabolism genes derived from ( A ). D Global binding profile of KDM4C at transcription start sites (TSSs) within ±3 kb regions, visualized as a heatmap and peak density plot derived from KDM4C CUT&Tag-seq analysis. The heatmap highlights the binding intensity of KDM4C across TSSs, with color gradients representing varying binding levels. Data visualization was generated using the Galaxy plot heatmap tool, allowing for a clear illustration of KDM4C enrichment at promoter regions, indicative of its regulatory role in gene expression. E Identification of heme metabolism genes regulated by KDM4C. The Venn diagram depicts the overlap between KDM4C-downregulated genes and KDM4C-bound promoter genes. The intersecting set represents KDM4C-regulated genes involved in heme metabolism. An Enrichr analysis ( https://maayanlab.cloud/Enrichr/ ) was performed on these genes to identify consensus transcription factors, with GATA1 emerging as the top-ranking transcription factor, highlighted in the enrichment bar chart to indicate its pivotal role in the KDM4C regulatory network. F KDM4C (this study), GATA1 (SRX4172742_HCT116), and H3K9me3 (ENCFF794 WNF) at proximal promoter regions of FECH and E2F2 . G , H Analysis of occupancy changes at the promoter region of FECH ( G ) and E2F2 ( H ) via ChIP-qPCR. ChIP was conducted with specific antibodies (anti-GATA1 and anti-H3K9me3) in LKO and KDM4C-KD SAS cells. I, J The relative mRNA levels of heme-metabolism genes ( FECH and E2F2 ) in the LKO and KDM4C-KD SAS cells. Data in ( G – J ) are represented in individual points and mean. P-values are determined by one-way ANOVA with Tukey’s multiple comparisons test ( G – I ), or two-way ANOVA with Tukey’s multiple comparisons test ( J ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant

Article Snippet: E Relative mRNA levels of heme metabolism genes ( FECH and E2F2 ) in SAS-LN cells following 24-hour treatment with control (DMSO, 0.1%), myricetin (12.5 μM), or 22S0 (6.25 μM).

Techniques: Knockdown, RNA Sequencing, Derivative Assay, Binding Assay, Generated, Gene Expression, ChIP-qPCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: KDM4C works in concert with GATA1 to regulate heme metabolism in head and neck squamous cell carcinoma

doi: 10.1007/s00018-025-05693-x

Figure Lengend Snippet: Effects of KDM4 inhibitors on heme metabolism and tumor growth in HNSCC. A , B SAS-LN cells were treated with varying concentrations of myricetin ( A ) or 22S0 ( B ) for 3 days. Cell survival rates were measured using the MTT assay and are shown as dose-response curves. C , D Analysis of H3K9me3 levels in inhibitor-treated SAS-LN cells for 3 days. The H3K9me3 signals were detected by Western blot analysis. E Relative mRNA levels of heme metabolism genes ( FECH and E2F2 ) in SAS-LN cells following 24-hour treatment with control (DMSO, 0.1%), myricetin (12.5 µM), or 22S0 (6.25 µM). F Representative images of SAS-LN cells xenografted in zebrafish and treated with drugs. Treatments include control (DMSO, 0.1%), myricetin (11.07 µM), 22S0 (6.17 µM), and docetaxel (0.38 nM). Scale bar: 200 µm. G Quantification of cell migration of SAS-LN cells under various drug treatments from ( F ). Each data point represents the percentage of embryos exhibiting tumor cell migration at 3 dpi in one of three independent biological experiments. The total number of embryos analyzed per group is as follows: control (n = 33), myricetin (n = 41), 22S0 (n = 32), and docetaxel (n = 31). H ‒ J SAS cells (1×10 6 cells) were subcutaneously injected into the BALB/cAnN.Cg-Foxn1nu/CrlNarl mice. When the tumors had grown to approximately 100 mm 3 , the mice received treatment. They were given two injections per week, with either 75 mg/kg of 22S0 or a control vehicle solution (DMSO/PEG300/PBS), administered via intra-tumor injection. Tumor volumes ( H ) and body weights ( I ) were measured at each treatment session, and tumor weights were measured at sacrificed endpoint ( J ). Data in ( E , G , J ) are represented as individual points and mean. Data in ( A , B ) are represented in mean ± SD, and data in ( H , I ) are mean ± SEM. P-values are determined by two-way ANOVA with Tukey’s multiple comparisons test ( E , I ), one-way ANOVA with Tukey’s multiple comparisons test ( G ), and two-tailed Student’s t–test ( J ). * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant

Article Snippet: E Relative mRNA levels of heme metabolism genes ( FECH and E2F2 ) in SAS-LN cells following 24-hour treatment with control (DMSO, 0.1%), myricetin (12.5 μM), or 22S0 (6.25 μM).

Techniques: MTT Assay, Western Blot, Control, Migration, Injection, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: KDM4C works in concert with GATA1 to regulate heme metabolism in head and neck squamous cell carcinoma

doi: 10.1007/s00018-025-05693-x

Figure Lengend Snippet: Schematic illustration of KDM4C-GATA1 axis in heme metabolism and cancer progression. In the active state (top panel), KDM4C interacts with GATA1, demethylating H3K9me3 marks at heme metabolism gene promoters such as FECH , opening chromatin, and promoting gene transcription. This upregulation supports cancer cell growth and migration. Upon KDM4C inhibition (bottom panel), H3K9me3 demethylation is blocked, leading to a repressive chromatin state, downregulating heme metabolism genes, and reducing cancer growth and migration

Article Snippet: E Relative mRNA levels of heme metabolism genes ( FECH and E2F2 ) in SAS-LN cells following 24-hour treatment with control (DMSO, 0.1%), myricetin (12.5 μM), or 22S0 (6.25 μM).

Techniques: Migration, Inhibition