Fig 1: FAM60A is a candidate regulator of ferroptosis, and its expression is negatively correlated with PDAC prognosis. (A) This heatmap showed the expression differences of different gene sets between subgroups of PDAC patients identified by the ssGSEA algorithm. The 2 major subgroups were referred to as the “High-ferroptosis” and “Low-ferroptosis” groups. The top differential genes between the 2 groups and their prognosis in the TCGA database. (B) FAM60A expression in the “High-ferroptosis” and “Low-ferroptosis” groups. (C) FAM60A expression in tumor and normal pancreatic tissues in the TCGA and GTEx database. (D) FAM60A expression in tumor and normal pancreatic tissues in GSE16515, GSE28735, and GSE15471. (E) Fam60a expression in the KPC mice pancreatic tissues sequencing database (GSE33322). (F) IHC image of FAM60A expression in the KPC mice model during each pathological period. (G) qRT-PCR experiment measured FAM60A mRNA expression levels in the hTERT-HPNE and different PDAC cell lines; n = 3. (H) Western blotting experiment measured FAM60A protein expression levels in the hTERT-HPNE and different PDAC cell lines. (I) FAM60A staining was divided into 4 grades according to the depth of tissue chip IHC staining, with 2 grades with lower staining set to the low-expression group and 2 stronger grades to the high-expression group. (J) Survival analysis of FAM60A expression and patient survival time in TMA. (K) IHC image of FAM60A staining from patient-derived sections in PDAC and para-cancerous tissues. ***P < 0.001.
Fig 2: FAM60A constrains ferroptosis in PDAC cells through the PPAR and GPX4 pathway. (A) PPARA, PPARG, and PPARD fold changes in RNA-Seq data. (B) Western blotting experiment examined the effect of FAM60A knockdown on PPARA and PPARG protein expression levels. (C) The ChIP-Seq database predicted regulatory genes downstream of PPAR. (D) Western blotting experiment examined the effect of FAM60A knockdown on ACSL1 protein expression levels. (E) An IHC staining of paraffin sections from nude mouse orthotopic xenograft tumors was performed in order to determine whether FAM60A knockdown affected PPARA, PPARG, ACSL1, ACSL4, and GPX4 levels. (F) Schematic diagram of FAM60A binding site with promoter regions of PPARA, PPARG, ACSL1, ACSL4, and GPX4 as a transcription factor in CUT&Tag sequencing data.
Fig 3: A schematic diagram illustrating the potential mechanism by which FAM60A regulates ferroptosis in PDAC cells. PE, phosphatidyl ethanolamine.
Fig 4: FAM60A is a repressor of ferroptosis. (A) The viability of cells treated with different concentrations of erastin in FAM60A-knockdown AsPC-1 cells was assessed; n = 3. (B) Effects of ferroptosis inhibitor Ferrostatin-1 (2 μM), apoptosis inhibitor Z-VAD-FMK (5 μM), and necrosis inhibitor Necrostatin-1 (2 μM) on the proliferation capacity of FAM60A knockdown AsPC-1 cells; n = 3. (C) Heatmap of ferroptosis-related genes changes in RNA-Seq data. (D) Transmission electron microscopy to observe the effect of FAM60A knockdown on mitochondrial morphology in PDAC cells. (E) Effect of FAM60A knockdown on ROS content in PDAC cells; n = 3. (F) Effect of FAM60A knockdown on GSH/GSSG content in PDAC cells; n = 3. (G) Western blotting experiment examined the effect of FAM60A knockdown on GPX4 protein expression levels. (H) Western blotting experiment examined the effect of FAM60A knockdown on ACSL4 protein expression levels. ***P < 0.001, **P < 0.01, *P < 0.05.
Fig 5: Pharmacological experiments verify that FAM60A regulates ferroptosis via the PPAR pathway. (A) A comparison of serial tissue sections of PDAC patient samples stained with IHC revealed differences in expression of ACSL1, ACSL4, and GPX4. (B) Wild-type PDAC cells were treated with 2 inhibitors, and Western blotting experiments detected changes in the expression of downstream ACSL1 and ACSL4. (C) Western blotting experiments detected expression changes of ACSL1 and ACSL4 caused by FAM60A knockdown and PPAR inhibitor treatment. (D) FAM60A-knockdown cells were treated with 2 inhibitors, and detect changes in GSH/GSSG levels in each group of cells; n = 3. (E) FAM60A-knockdown cells were treated with 2 inhibitors, and detect changes in ROS levels in each group of cells; n = 3. ***P < 0.001, **P < 0.01, *P < 0.05.
Supplier Page from Novus Biologicals, a Bio-Techne Brand for FAM60A Antibody - Azide and BSA Free
Available conjugates: Available conjugates: UnconjugatedSizes Available: 0.05 mg