Journal: Nature Communications

Article Title: Ubiquinol-mediated suppression of mitochondria-associated ferroptosis is a targetable function of lactate dehydrogenase B in cancer

doi: 10.1038/s41467-025-57906-3

Figure Lengend Snippet: Immunoblot analysis of LDHB, GPX4, DHODH, LDHA, SLC7A11, and FSP1 in HT1080, MSTO-211H, A549, PANC-1 siRNAs cells, n = 3–6 independent repeats, and the statistical analysis was shown in Supplementary Fig. ( a ). Colocalization analysis by immunofluorescence in HT1080 siRNAs cells after 72 h of transfection ( b , scale bar, 20 µm). Images of microscopic analysis ( c , 20x objective) and assessment of mitochondrial lipid peroxidation by flow cytometry in HT1080 sgNTC and sgGPX4 cells after 72 h of transfection with siRNAs, n = 3 independent replicates ( d , e ). Clonogenic assays of parental HT1080 (HT1080-P) and HT1080 cells treated with 4 µM RSL3 after ten cycles (HT1080-R) ( f ). Immunoblot analysis of LDHB, GPX4, DHODH, and LDHA in HT1080 parental and RSL3 resistant cells. The experiment was repeated three times, yielding consistent results ( g ). Cell viability of HT1080-P and HT1080-R cells after transfection with siRNAs treated with DMSO or RSL3 alone or in combination with 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h, n = 3 independent replicates ( h ). Immunoblot analysis of LDHB, GPX4, LDHA in HT1080 control and LDHB overexpression cells ( i ). Cell viability assays of HT1080 cells and HT1080 LDHB ORF cells treated with RSL3 with or without 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h. n = 3 independent replicates ( j ). Clonogenic assay of HT1080 siRNAs cells treated with DMSO or RSL3 alone or in combination with 2 mM glutathione reduced ethyl ester (GSH-mee) for 48 h after pretreatment with vehicle, 2 mM GSH-mee for 24 h ( k ). Tumor volume and weight of A549 sgNTC shCTRL ( n = 12), sgNTC shLDHB ( n = 11), sgGPX4 shCTRL ( n = 12), sgGPX4 shLDHB ( n = 12), sgGPX4 shLDHB treated with liproxstatin-1 ( n = 12) xenograft tumors from different mice ( l , m ), tumor volume and weight of HT1080 sgNTC shCTRL ( n = 10), sgNTC shLDHB ( n = 10), sgGPX4 shCTRL ( n = 10), sgGPX4 shLDHB ( n = 12), sgGPX4 shLDHB treated with liproxstatin-1 ( n = 12) xenograft tumors from different mice ( o , p ), 4-HNE expression of A549 and HT1080 sgNTC shCTRL, sgNTC shLDHB, sgGPX4 shCTRL, and sgGPX4 shLDHB xenograft tumors treated with or without liproxstatin-1, n = 10 or n = 12 different regions from different tumors from different mice respectively ( n , q ). Data were presented as mean ± SEM ( l , o ) or mean ± SD ( e , h , j , m , n , p , q ). Two-way ANOVA ( h , j , l , o ), Unpaired, two-tailed t -test ( e , m , n , p , q ). ns no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Subsequently, the membranes were incubated with the primary antibodies against LDHB (1:10,000, R&D Systems MAB9205-100), LDHA (1:1000, Cell Signaling Technology 2012S), GPX4 (1:1000, Abcam ab125066), DHODH (1: 1000, Cell Signaling Technology 26381S), FSP1 (1:1000, Proteintech 20886-1-AP), SLC7A11 (1:1000, Cell Signaling Technology 12691S), beta-actin (1:5000, Cell Signaling Technology 3700S) overnight on a rotating wheel (3 rpm) at 4 °C.

Techniques: Western Blot, Immunofluorescence, Transfection, Flow Cytometry, Control, Over Expression, Clonogenic Assay, Expressing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Targeting pancreatic cancer glutamine dependency confers vulnerability to GPX4-dependent ferroptosis

doi: 10.1016/j.xcrm.2025.101928

Figure Lengend Snippet: Upregulation of H3K4me3 is essential for cellular response and adaptation to prolonged glutamine restriction (A) Levels of indicated histone modifications were determined by using western blotting in Low Gln and Cont groups. (B) Levels of SAM (S-adenosylhomocysteine) and acetyl-CoA were determined in Low Gln and Cont groups. (C) Heatmap of CUT&Tag-seq signals of H3K4me3, H3K27Ac, H3K27me3, and H2AK119ub from 3-kb upstream and downstream of transcription start site (TSS) and H3K36me3 from 3-kb upstream of TSS to 3 kb downstream of transcription end site (TES) in Low Gln and Cont groups. (D) Heatmap illustrated the correlation between transcriptional levels and indicated histone modification levels (Low Gln versus Cont). (E) Boxplots showed alternations of indicated histone modification depositions for open DEGs ( n = 1,037 in Figure 1 C) and all genes. (F) Integrated analysis of open DEGs and genes with increased H3K4me3 at promoter ( n = 4,017, CUT&Tag-seq, FC_RPKM ≥ 1.5). Right, the KEGG enrichment analysis of the 163 H3K4me3-related open DEGs. (G) Heatmap illustrated mRNA levels and H3K4me3 depositions at the promoters of indicated genes in Low Gln and Cont groups. Transcripts per kilobase of exon model per million mapped reads (TPMs) were shown. (H) Representative Integrative Genomics Viewer (IGV) screenshot of ATAC-seq signals, H3K4me3 signals (CUT&Tag-seq), and Fosl1/Junb signals (ChIP-seq, GSE134233 ) of indicated genes. (I) Top 5 known motifs at H3K4me3-gain zones of 163 overlapping genes. (J and K) KPC1199 cells were treated with DON (1 μM), DON and MM-102 (25 μM), or DMSO as control for 72 h. (J) The mRNA levels of Hmox1 , Steap3 , Atg7 , Slc7a11 , and Gpx4 were determined by RT-qPCR. (K) Western blot analysis of Hmox1, Slc7a11, Gpx4, Atg7, β-Actin, H3K4me3, and Histone H3 in the indicated groups. Data represent mean ± SEM (B), mean with range (E), or mean ± SD (J) with n = 3 biological replicates. p values were determined using unpaired Student’s t test (B and E) or one-way ANOVA with Tukey’s multiple comparison test (J).

Article Snippet: Rabbit anti-xCT/SLC7A11(D2M7A) , Cell Signaling Technology , Cat# 12691; RRID: AB_2687474.

Techniques: Western Blot, Modification, ChIP-sequencing, Control, Quantitative RT-PCR, Comparison

Journal: Cell Reports Medicine

Article Title: Targeting pancreatic cancer glutamine dependency confers vulnerability to GPX4-dependent ferroptosis

doi: 10.1016/j.xcrm.2025.101928

Figure Lengend Snippet: CRISPR-Cas9 screening reveals that PAXIP1 mediates increased H3K4 trimethylation under glutamine restriction (A) Schematic diagram of the experimental design for the epigenome-wide CRISPR-Cas9 screen. (B) Bubble-rank plot illustrated genes whose knockout significantly enhanced (positive, red) or reduced (negative, blue) the relative cell viability under low-glutamine treatment (Low Gln) compared to the control (Cont). (C) Western blot analysis of Paxip1, β-Actin, H3K4me3, and Histone H3 in sg Lacz , sg Paxip1 #1, and sg Paxip1 #3 cells treated with or without DON (1 μM) for 72 hours. (D) Heatmap of CUT&Tag-seq signals of H3K4me3 from 3-kb upstream and downstream of TSS in sg Lacz and sg Paxip1 #1 cells with or without DON treatment (1 μM). (E) Correlation between CUT&Tag signals of PAXIP1 and H3K4me3. (F) Left, heatmap of DON-induced PAXIP1 peaks at promoter in wild-type KPC1199 (sg Lacz ) cells under DON treatment (1 μM). Right, heatmap of H3K4me3 peaks within DON-induced PAXIP1-binding domains in sg Lacz and sg Paxip1 #1 cells with or without DON treatment (1 μM). (G) Representative IGV screenshot of H3K4me3 CUT&Tag signals at the indicated genes from sg Lacz and sg Paxip1 #1 cells with or without DON treatment (1 μM). (H) Relative quantitative PCR analysis of H3K4me3 occupancies in the zones indicated in (G). The input was used as the control. (I and J) sg Lacz , sg Paxip1 #1, and sg Paxip1 #3 cells were treated with or without DON (1 μM) for 72 h. (I) The mRNA levels of Hmox1 , Steap3 , Atg7 , Slc7a11 , and Gpx4 were determined by RT-qPCR. (J) Western blot analysis of Hmox1, Slc7a11, Gpx4, and β-Actin in the indicated groups. Data represent mean ± SD (H and I) with n = 3 biological replicates. p values were determined using two-sided Pearson’s correlation test (E) and one-way ANOVA with Tukey’s multiple comparison test (H and I). ∗, 0.01 < p ≤ 0.05; ∗∗, 0.001 < p ≤ 0.01; ∗∗∗, p ≤ 0.001.

Article Snippet: Rabbit anti-xCT/SLC7A11(D2M7A) , Cell Signaling Technology , Cat# 12691; RRID: AB_2687474.

Techniques: CRISPR, Knock-Out, Control, Western Blot, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison

Journal: Cell Reports Medicine

Article Title: Targeting pancreatic cancer glutamine dependency confers vulnerability to GPX4-dependent ferroptosis

doi: 10.1016/j.xcrm.2025.101928

Figure Lengend Snippet: DON treatment induces lipid peroxidation in human PDAC (A) Cell viability of PANC-1, MIA PaCa-2, BxPC-3, and AsPC-1 cells under DON treatment for 72 hours. (B and C) Relative lipid peroxidation levels (B) and cell viability (C) were assessed in AsPC-1 and MIA PaCa-2 cells under treatment of indicated concentrations of DON. (D) The mRNA levels of Hmox1 , Steap3 , Atg7 , Slc7a11 , and Gpx4 were determined by RT-qPCR in AsPC-1 and MIA PaCa-2 cells under treatment of DON (AsPC-1 10 μM; MIA PaCa-2 4 μM) and/or MM-102 (25 μM). (E) Western blot analysis of HMOX1, SLC7A11, GPX4, β-Actin, H3K4me3, and Histone H3 in AsPC-1 and MIA PaCa-2 cells. (F and G) Relative levels of labile iron pool (F) and lipid peroxidation (G) were assessed in AsPC-1 and MIA PaCa-2 cells under treatment of DON (AsPC-1 10 μM; MIA PaCa-2 4 μM) and/or DFO (100 μM) for 72 hours. (H and I) Relative levels of labile iron pool (H) and lipid peroxidation (I) were assessed in AsPC-1 and MIA PaCa-2 cells under treatment of DON (AsPC-1 10 μM; MIA PaCa-2 4 μM) and/or ZnPP (10 μM) for 72 h. Data were represented as mean ± SD (B–D and F–I) with n = 3 biological replicates. p values were determined using one-way ANOVA with Tukey’s multiple comparison test (B–D) or two-way ANOVA with Tukey’s multiple comparison test (F–I). ∗, 0.01 < p ≤ 0.05; ∗∗, 0.001 < p ≤ 0.01; ∗∗∗, p ≤ 0.001.

Article Snippet: Rabbit anti-xCT/SLC7A11(D2M7A) , Cell Signaling Technology , Cat# 12691; RRID: AB_2687474.

Techniques: Quantitative RT-PCR, Western Blot, Comparison

Journal: Cell Reports Medicine

Article Title: Targeting pancreatic cancer glutamine dependency confers vulnerability to GPX4-dependent ferroptosis

doi: 10.1016/j.xcrm.2025.101928

Figure Lengend Snippet:

Article Snippet: Rabbit anti-xCT/SLC7A11(D2M7A) , Cell Signaling Technology , Cat# 12691; RRID: AB_2687474.

Techniques: Virus, CRISPR, Recombinant, Protease Inhibitor, Lysis, Ligation, Reverse Transcription, SYBR Green Assay, Glo Assay, Sample Prep, DNA Extraction, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Flow Cytometry, Software