Journal: International Journal of Molecular Sciences

Article Title: Relationship of Iron Metabolism and Short-Term Cuprizone Treatment of C57BL/6 Mice

doi: 10.3390/ijms20092257

Figure Lengend Snippet: mRNA and protein analyses of NFS1, frataxin (FRX), and ferrochelatase (Fc) genes in CZ treated groups. Real Time PCR was performed with SYBR green protocol using gene specific primers. The relative expression of the controls was regarded as 1. Western blot analyses were done from protein extracts of the liver tissue samples. The same amount of protein (15 µg) from each sample was loaded onto 10% SDS-PAGE and transferred to nitrocellulose membranes and probed with NFS1 or Fc polyclonal rabbit antibodies. β-actin was used as loading control. ( A ) Relative mRNA expression values of NFS1, FRX, and Fc in CC. ( B ) Relative mRNA levels of NFS1, FRX, and Fc in the liver. ( C ) Protein expression levels of NFS1 and Fc in the liver. The Western blot experiments were repeated at least three times in each group. The bars represent mean values and error bars represent standard errors of the mean (SEM) for three independent measurements. For statistical analyses, Student’s t -test and two-sample t -tests were used. The asterisk marks p < 0.05 compared to non-treated animals, while the cross indicates a difference p < 0.05 between the treated groups, n = 6 in each group. Optical density analyses can be seen in .

Article Snippet: The same amount of protein from each sample was loaded onto 12% or 14% SDS-PAGE and transferred to nitrocellulose membranes (BioTrace NT, Pall Life Sciences, Port Washington, NY) and probed with rabbit polyclonal antibodies produced against mouse ferrochelatase (Fc 1:1000; Novus Biologicals), mitochondrial ferritin (FTMT 1:1000; Thermo-Fischer Scientific Inc.), ferritin heavy chain (FTH 1:1000; Cell Signaling Technology Europe, Leiden, The Netherlands), NFS1 (1:1000; Novus Biologicals), transferrin receptor-1 (TfR1 1:1000; Thermo-Fischer Scientific Inc.) and alpha-1 antitrypsin (A1AT 1:1000; Abcam, Cambridge, UK). β-actin (1:2000; Sigma-Aldrich Kft.) was used as loading control.

Techniques: Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing, Western Blot, SDS Page, Control

Journal: Cancers

Article Title: The Circadian Rhythm of Intracellular Protoporphyrin IX Accumulation Through Heme Synthesis Pathway in Bladder Urothelial Cancer Cells Exposed to 5-Aminolevulinic Acid

doi: 10.3390/cancers16234112

Figure Lengend Snippet: Association between intracellular PPIX accumulation and eight enzymes in the heme synthesis pathway. Eight main enzymes are involved in the heme synthesis pathway from 5-ALA to heme or PPIX export from cells ( A ). Tumors obtained from nude mice were used to evaluate the intracellular concentration of PPIX and the expression of genes related to heme synthesis. When mRNA levels exhibited a circadian rhythm, a sinusoidal curve was observed. ( B – I ) show the shifts in the intracellular PPIX concentration and mRNA levels for each target gene: ALAD ( B ), HMBS ( C ), UROS ( D ), UROD ( E ), CPOX ( F ), PPOX ( G ), FECH ( H ), and ABCG2 ( I ). The goodness-of-fit values that could not be calculated were represented as “NA”, indicating “not applicable”.

Article Snippet: TaqMan probes with the following IDs were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and used for the quantification of target gene expression: Hs01007553_m1 (for PER2 ), Hs00154147_m1 (for BMAL1 ), Hs00765604_m1 (for ALAD ), Hs00609296_g1 (for HMBS ), Hs03405152_m1 (for UROS ), Hs01099757_g1 (for UROD ), Hs01071019_m1 (for CPOX ), Hs00970229_g1 (for PPOX ), Hs01555261_m1 (for FECH ), and Hs01053790_m1 (for ABCG2 ).

Techniques: Concentration Assay, Expressing

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

Journal: Cell

Article Title: Hypoxia rescues frataxin loss by restoring iron sulfur cluster biogenesis

doi: 10.1016/j.cell.2019.03.045

Figure Lengend Snippet: (A) Aconitase activity assay from control or FXN KO K562 cells grown in 21% O2, 1% O2 or 21% O2 with 75μM FG-4592. Bar plots show mean ± SD. ****=p < 0.0001. One-way ANOVA with Bonferroni’s post-test. (B) Immunoblot of control or FXN KO cells grown in 21% O2, 1% O2 or 21% O2 with 75μM FG-4592, blotted for OXPHOS subunits or Tubulin. (C) Immunoblot of control or FXN KO cells grown in 21% O2, 1% O2 or 21% O2 with 75μM FG-4592, blotted for Lipoic acid, which is conjugated to PDH-E2, KGDH-E2 and GCSH. Additional blots against OGDH (i.e. KGDH-E1) and Actin. (D) Immunoblot of control or FXN KO cells grown in 21% O2, 1% O2 or 21% O2 with 75μM FG-4592, blotted against enzymes in the heme biosynthesis pathway- FECH and ALAD- or TOMM20. (E) Immunoblot of control or FXN KO cells grown in 21% O2, 1% O2 or 21% O2 with 75μM FG-4592, blotted against POLD1, MMS19 or Actin. See also Figure S3.

Article Snippet: Ferrochetelase (FECH) , GTX113435 , Genetex.

Techniques: Activity Assay, Control, Western Blot

Journal: Cell

Article Title: Hypoxia rescues frataxin loss by restoring iron sulfur cluster biogenesis

doi: 10.1016/j.cell.2019.03.045

Figure Lengend Snippet:

Article Snippet: Ferrochetelase (FECH) , GTX113435 , Genetex.

Techniques: