Journal: Communications Biology

Article Title: Retinoic acid receptor alpha inhibits ferroptosis by promoting thioredoxin and protein phosphatase 1F in lung adenocarcinoma

doi: 10.1038/s42003-024-06452-7

Figure Lengend Snippet: a CCK8 assays to detect the cell viability of A549 and PC9 cells treated with RSL3 (2 μM), RSL3 (2 μM) plus DFO (100 μM) or fer-1 (20 μM) for 24 h after incubating with DMSO, Ch55 (5 μM), AM580 (5 μM) ATRA (20 μM) or AGN193109 (5 μM) for 24 h. ( n = 4 biologically independent experiments, Student t -test). b – e MDA level ( b ) lipid peroxidation ( c ), mitochondrial membrane potential (MMP) by TMRE ( d ), and labile iron pool (LIP) by CA-AM ( e ) were accessed in A549 and PC9 cells treated with DMSO or RSL3 (1 μM) for 24 h after incubating with DMSO, Ch55 (5 μM), AM580 (5 μM) ATRA (20 μM) or AGN193109 (5 μM) for 24 h. ( n = 3 biologically independent experiments, Student t -test). f Light microscopy images showed the degrees of “ballooning phenotype” in A549 and PC9 cells treated with DMSO or RSL3 (1 μM) for 24 h after incubating with DMSO, Ch55 (5 μM), AM580 (5 μM) ATRA (20 μM) or AGN193109 (5 μM) for 24 h. Scale bar:50 μm. The zoomed-in figures, scale bar: 250 μm. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The RSL3, Imidazole Ketone Erastin (IKE), Erastin, Ferric ammonium citrate (FAC), RARA inhibitor AGN193109, RARA activator Ch55 and AM580, PAK inhibitor FRAX486, cisplatin (CDDP), and pemetrexed were acquired from Topscience (Shanghai, China) and were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Membrane, Light Microscopy

Journal: Communications Biology

Article Title: Retinoic acid receptor alpha inhibits ferroptosis by promoting thioredoxin and protein phosphatase 1F in lung adenocarcinoma

doi: 10.1038/s42003-024-06452-7

Figure Lengend Snippet: a qRT-PCR and WB assays confirmed the mRNA and protein expression level of RARA in A549 and PC9 cells after transfection with NC (control shRNA), RARA-SH1, or RARA-SH2 lentivirus. ( n = 3 biologically independent experiments, Student t -test) ( b ) CCK8 assays to detect the cell viability of NC, RARA-SH1, or RARA-SH2 groups in A549 and PC9 cells treated with a gradient dose of RSL3 or IKE for 24 h. ( n = 4 biologically independent experiments, two-way ANOVA) ( c ) CCK8 assays to detect the cell viability of NC, RARA-SH1, or RARA-SH2 groups in A549 and PC9 cells treated with DMSO or RSL3 (2 μM) for 24 h after incubating with DMSO, Ch55 (5 μM), AM580 (5 μM) ATRA (20 μM) or AGN193109 (5 μM) for 24 h. ( n = 4 biologically independent experiments, Student t -test). d – g MDA level ( d ), lipid peroxidation ( e ), MMP by TMRE ( g ) and LIP by CA-AM ( g ) were accessed in NC, RARA-SH1 groups of A549 and PC9 cells treated with DMSO or RSL3 (1 μM) for 24 h after incubating with DMSO, Ch55 (5 μM), AM580 (5 μM) ATRA (20 μM) or AGN193109 (5 μM) for 24 h. ( n = 3 biologically independent experiments, Student t -test) ns, not significant * p < 0.05; ** p < 0.01; *** p < 0.001;**** p < 0.0001.

Article Snippet: The RSL3, Imidazole Ketone Erastin (IKE), Erastin, Ferric ammonium citrate (FAC), RARA inhibitor AGN193109, RARA activator Ch55 and AM580, PAK inhibitor FRAX486, cisplatin (CDDP), and pemetrexed were acquired from Topscience (Shanghai, China) and were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, shRNA

Journal: Communications Biology

Article Title: Retinoic acid receptor alpha inhibits ferroptosis by promoting thioredoxin and protein phosphatase 1F in lung adenocarcinoma

doi: 10.1038/s42003-024-06452-7

Figure Lengend Snippet: a The volcano plot showed the differentially expressed genes in the NC group vs. the RARA-KD group and showed that TXN and PPM1F were down-regulated in the RARA-knockdown group. b Venn plot displayed the predicted targets of RARA by the intersection of DEGs of RNA-Seq, CHIP-Seq, and ferroptosis-related gene set. c qRT-PCR verified the expression of key downstream genes: TXN, PPM1F, HMOX1, and GCLC in the NC group vs. the RARA-KD group in two cell lines. ( n = 3 biologically independent experiments, Student t -test). d qRT-PCR revealed the mRNA expression of TXN and PPM1F in A549 and PC9 cells after treatment with ATRA (20 μM) or AGN193109 (5 μM) for 24 h. ( n = 3 biologically independent experiments, Student t -test). e WB showed the protein level of TXN and PPM1F in the NC group vs. the RARA-knockdown group in A549 and PC9 cells as well as the two cells after treatment with ATRA (20 μM) or AGN193109 (5 μM) for 24 h. f qRT-PCR showed the expression level of GPX4, FSP1, and SLC7A11 in A549 and PC9 cells. ( n = 3 biologically independent experiments, Student t -test). g WB showed the protein level of GPX4, FSP1, and SLC7A11 in A549 and PC9 cells treated with ATRA (20 μM) or AGN193109 (5 μM) for 24 h. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The RSL3, Imidazole Ketone Erastin (IKE), Erastin, Ferric ammonium citrate (FAC), RARA inhibitor AGN193109, RARA activator Ch55 and AM580, PAK inhibitor FRAX486, cisplatin (CDDP), and pemetrexed were acquired from Topscience (Shanghai, China) and were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Knockdown, RNA Sequencing, ChIP-sequencing, Quantitative RT-PCR, Expressing

Journal: Communications Biology

Article Title: Retinoic acid receptor alpha inhibits ferroptosis by promoting thioredoxin and protein phosphatase 1F in lung adenocarcinoma

doi: 10.1038/s42003-024-06452-7

Figure Lengend Snippet: a Dose-toxicity curves showed relative viability of NC and RARA-SH1 A549 and PC9 cells transfected with or without TXN-OE + PPM1F-OE upon a gradient dose CDDP or pemetrexed treatment for 24 h. ( n = 3 biologically independent experiments, two-way ANOVA). b CCK8 assays showed the cell viability in NC, RARA-SH, inhibitor AGN193109 (5 μM, 24 h) groups of A549 and PC9 cells treated with RSL3 (2 μM) or IKE (5 μM), with or without CDDP (2 μM), pemetrexed (1 μM) for 24 h. ( n = 3 biologically independent experiments, Student t -test). c Flow chart of the process of subcutaneous tumor formation in male nude mice and the treatment measures. d Representative tumors formed in nude mice by NC, RARA-SH1, NC + TXN-OE + PPM1F-OE, RARA-SH1 + TXN-OE + PPM1F-OE. Tumors were resected after 4 weeks. e Tumor volumes were measured weekly. ( n = 6 biologically independent animals, two-way ANOVA). f The tumor weight of each group was weighed after 4 weeks. ( n = 6 biologically independent animals, two-way ANOVA). g Representative images of immunohistochemistry staining of mice tumor. Scale bar:100 μm. h Representative images of immunohistochemistry staining of 100 LUAD patient samples. Scale bar:100 μm. i Mechanism diagram for RARA inhibiting ferroptosis through TXN and PPM1F. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The RSL3, Imidazole Ketone Erastin (IKE), Erastin, Ferric ammonium citrate (FAC), RARA inhibitor AGN193109, RARA activator Ch55 and AM580, PAK inhibitor FRAX486, cisplatin (CDDP), and pemetrexed were acquired from Topscience (Shanghai, China) and were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunohistochemistry, Staining