Journal: Cell death & disease

Article Title: Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC.

doi: 10.1038/s41419-023-06238-5

Figure Lengend Snippet: Fig. 1 Inhibition of LSD1 induces ferroptosis. A Cell viability was detected by SRB assay in NSCLC cell lines treated with ORY at increasing concentrations for 96 h. B Lipid peroxides were measured by flow cytometry in NSCLC cells treated with 10 μM ORY for 96 h. C–F After A549 and H1975 cells were transfected with LSD1 siRNA for 48 h, LSD1 expression (C) was analyzed by Western blotting, Lipid peroxides (D), cell death (E), and total ROS accumulation (F) were measured by flow cytometry. G After NSCLC cell lines were treated with 10 μM ORY for 48 h and 5 μM Fer-1, 5 μM ZVAD, 2 μM Nec1, and 10 nM BA1 were added again for 48 h as indicated, cell viability was measured by SRB assay. H–J After A549 and H1975 cells were treated with 5 μM, 10 μM or 20 μM ORY for 96 h, 20 μM ERA for 48 h or 2 μM RSL for 48 h as indicated, lipid peroxides (H), cell death (I), and total ROS accumulation (J) were analyzed by flow cytometry. K, L After A549-KOLSD1 cell lines were transfected with pIRES2-EGFP-LSD1 plasmid and were treated with 10 μM ORY for 96 h, cell viability (K) was detected by SRB, and production of lipid peroxides (L) was analyzed by flow cytometry. n ≥3; Mean ± SEM; nsP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The primary antibodies used here include: GAPDH (Immunoway, YT5052), β-actin (Sungene biotech, KM9001T), ACSL4 (Invitrogen, PA527137), TFRC (Abcam, ab214039), GPX4 (Abcam, ab125066), xCT (Abcam, ab37185), ATF4 (Cell Signaling Technology, 11815), LSD1 (Cell Signaling Technology, 2184), H3K9me2 (Cell Signaling Technology, 4658), YAP (Cell Signaling Technology, 14074), p-YAP (Cell Signaling Technology, 13008), FPN (Abcam, ab1239583), DMT1 (Abcam, ab55735), Ferritin (Abcam, 287968), p-STAT1 (Cell Signaling Technology, 9167), STAT1 (Cell Signaling Technology, 9172), PARP (Cell Signaling Technology, 9542), and LC3B (Cell Signaling Technology, 3868).

Techniques: Inhibition, Sulforhodamine B Assay, Cytometry, Transfection, Expressing, Western Blot, Plasmid Preparation

Journal: Cell death & disease

Article Title: Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC.

doi: 10.1038/s41419-023-06238-5

Figure Lengend Snippet: Fig. 2 Inhibiting LSD1 regulates intracellular iron overload through iron-regulating proteins. A The content of Fe2+ was measured in NSCLC cells treated with 10 μM ORY for 96 h. B Fe2+ levels were determined in A549 and H1975 cell lines treated with gradient concentrations of ORY for 96 h. C After NSCLC cell lines were treatment with 10 μM ORY for 48 h and 10 μM DFO was added again for 48 h as indicated, cell viability was measured by SRB. D The expression of TFRC, DMT1, ferritin and FPN was examined by western blotting in NSCLC cells treatment with 10 μM ORY for 96 h. E TFRC expression was determined by western blotting in A549 and H1975 cell lines treated with 10 μM GSK, 10 μM GSK-2879552 or 10 μM ORY for 96 h as indicated. F TFRC expression was determined by western blotting in A549 and H1975 cell lines treated with increasing concentrations of ORY for 96 h. G–I After A549 and H1975 cell lines were transfected with TFRC siRNA and were treated with 10 μM ORY for 96 h, TFRC expression (G) was determined by western blotting, cell viability (H) was detected by SRB, and production of lipid peroxides (I) was analyzed by flow cytometry. J Quantitative ChIP studies were conducted to characterize the enrichment of H3K4me2 and H3K9me2 at promoters of TFRC gene in A549 cells treated with 10 μM ORY for 96 h. n ≥3; Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The primary antibodies used here include: GAPDH (Immunoway, YT5052), β-actin (Sungene biotech, KM9001T), ACSL4 (Invitrogen, PA527137), TFRC (Abcam, ab214039), GPX4 (Abcam, ab125066), xCT (Abcam, ab37185), ATF4 (Cell Signaling Technology, 11815), LSD1 (Cell Signaling Technology, 2184), H3K9me2 (Cell Signaling Technology, 4658), YAP (Cell Signaling Technology, 14074), p-YAP (Cell Signaling Technology, 13008), FPN (Abcam, ab1239583), DMT1 (Abcam, ab55735), Ferritin (Abcam, 287968), p-STAT1 (Cell Signaling Technology, 9167), STAT1 (Cell Signaling Technology, 9172), PARP (Cell Signaling Technology, 9542), and LC3B (Cell Signaling Technology, 3868).

Techniques: Expressing, Western Blot, Transfection, Cytometry

Journal: Cell death & disease

Article Title: Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC.

doi: 10.1038/s41419-023-06238-5

Figure Lengend Snippet: Fig. 3 Upregulating ACSL4 is involved in LSD1 inhibitor-induced ferroptosis. A The expression of ACSL4 was examined in NSCLC cells treatment with 10 μM ORY for 96 h. B ACSL4 expression was determined by western blotting in A549 and H1975 cell lines treated with 10 μM GSK, 10 μM GSK-2879552, or 10 μM ORY for 96 h as indicated. C ACSL4 expression was determined by western blotting in A549 and H1975 cell lines treated with increasing concentrations of ORY for 96 h. D ACSL4 mRNA was determined by qRT-PCR in A549 and H1975 cell lines treated with increasing concentrations of ORY for 96 h. E The expression of LSD1 and ACSL4 was analyzed by western blotting in A549 and H1975 cell lines transfected with ACSL4 siRNA for 48 h. F–H After A549 and H1975 cell lines transfected with ACLS4 siRNA and were treated with 10 μM ORY for 96 h, ACSL4 expression (F) was determined by western blotting, cell viability (G) was detected by SRB, and the production of lipid peroxides (H) was analyzed by flow cytometry. I Quantitative ChIP studies were conducted to characterize the enrichment of H3K4me2 and H3K9me2 at promoters of ACSL4 gene in A549 cells treated with 10 μM ORY for 96 h. n ≥3; Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The primary antibodies used here include: GAPDH (Immunoway, YT5052), β-actin (Sungene biotech, KM9001T), ACSL4 (Invitrogen, PA527137), TFRC (Abcam, ab214039), GPX4 (Abcam, ab125066), xCT (Abcam, ab37185), ATF4 (Cell Signaling Technology, 11815), LSD1 (Cell Signaling Technology, 2184), H3K9me2 (Cell Signaling Technology, 4658), YAP (Cell Signaling Technology, 14074), p-YAP (Cell Signaling Technology, 13008), FPN (Abcam, ab1239583), DMT1 (Abcam, ab55735), Ferritin (Abcam, 287968), p-STAT1 (Cell Signaling Technology, 9167), STAT1 (Cell Signaling Technology, 9172), PARP (Cell Signaling Technology, 9542), and LC3B (Cell Signaling Technology, 3868).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Cytometry

Journal: Cell death & disease

Article Title: Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC.

doi: 10.1038/s41419-023-06238-5

Figure Lengend Snippet: Fig. 4 Decreased xCT is critical for LSD1 inhibitor to induce ferroptosis. A The levels of GSH were detected in NSCLC cell lines treated with 10 μM GSK for 96 h. B, C After A549 and H1975 cell lines were treatment with 10 μM ORY for 48 h and 5 mM NAC or 1 mM DTT was added again for 48 h as indicated, the levels of GSH (B) and cell viability (C) were measured. D xCT expression was determined by western blotting in NSCLC cell lines treated with 10 μM ORY for 96 h. E The expression of xCT and LSD1 was analyzed by western blotting in A549 and H1975 cell lines transfected with LSD1 siRNA for 48 h. F xCT expression was analyzed by western blotting in A549 and NCI-H1975 cell lines transfected with LSD1 siRNA for 48 h. G, H After A549 and H1975 cell lines were transfected with xCT siRNA for 48 h and 5 mM NAC or 1 mM DTT was added again for 48 h as indicated, the levels of GSH (G) and cell viability (H) were measured. I xCT expression was analyzed by western blotting in xCT-overexpressing cells. J After xCT-overexpressing cells were treated with 10 μM ORY for 48 h and 5 mM NAC or 1 mM DTT was added again for 48 h as indicated, the levels of GSH were measured. K Cell viability was determined by SRB in xCT-overexpressing cells treated with ORY at increasing concentrations for 96 h. n ≥3; Mean ± SEM; nsP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The primary antibodies used here include: GAPDH (Immunoway, YT5052), β-actin (Sungene biotech, KM9001T), ACSL4 (Invitrogen, PA527137), TFRC (Abcam, ab214039), GPX4 (Abcam, ab125066), xCT (Abcam, ab37185), ATF4 (Cell Signaling Technology, 11815), LSD1 (Cell Signaling Technology, 2184), H3K9me2 (Cell Signaling Technology, 4658), YAP (Cell Signaling Technology, 14074), p-YAP (Cell Signaling Technology, 13008), FPN (Abcam, ab1239583), DMT1 (Abcam, ab55735), Ferritin (Abcam, 287968), p-STAT1 (Cell Signaling Technology, 9167), STAT1 (Cell Signaling Technology, 9172), PARP (Cell Signaling Technology, 9542), and LC3B (Cell Signaling Technology, 3868).

Techniques: Expressing, Western Blot, Transfection

Journal: Cell death & disease

Article Title: Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC.

doi: 10.1038/s41419-023-06238-5

Figure Lengend Snippet: Fig. 5 LSD1 inhibition reduces xCT expression via ATF4. A ATF4 mRNA levels were determined by qRT-PCR in NSCLC cell lines treated with 10 μM ORY for 96 h. B ATF4 expression was determined by western blotting in NSCLC cell lines treated with 10 μM ORY for 96 h. C The expression of xCT and ATF4 were measured by western blotting in ATF4-overexpressing A549 and H1975 cells treated with 10 μM ORY for 96 h. D ATF4 expression were measured by Western blotting in ATF4-overexpressing A549 and H1975 cells. E Cell viability was determined by SRB assay in ATF4-overexpressing A549 and H1975 cells treated with ORY at increasing concentrations for 96 h. F The expression of xCT and ATF4 were measured by western blotting in ATF4-overexpressing A549 and H1975 cells transfected with xCT siRNA. G, H After ATF4-overexpressing A549 and H1975 cells were transfected with xCT siRNA and were treated with ORY, cell viability (G) and the levels of GSH (H) were detected. I ChIP primers were designed spanning from −1000 to +400 bp around the transcription start sites of ATF4. J Quantitative ChIP studies were conducted to characterize the enrichment of H3K4me2 and H3K9me2 at promoters of ATF4 gene in A549 cells treated with 10 μM ORY for 96 h. n ≥3; Mean ± SEM; nsP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The primary antibodies used here include: GAPDH (Immunoway, YT5052), β-actin (Sungene biotech, KM9001T), ACSL4 (Invitrogen, PA527137), TFRC (Abcam, ab214039), GPX4 (Abcam, ab125066), xCT (Abcam, ab37185), ATF4 (Cell Signaling Technology, 11815), LSD1 (Cell Signaling Technology, 2184), H3K9me2 (Cell Signaling Technology, 4658), YAP (Cell Signaling Technology, 14074), p-YAP (Cell Signaling Technology, 13008), FPN (Abcam, ab1239583), DMT1 (Abcam, ab55735), Ferritin (Abcam, 287968), p-STAT1 (Cell Signaling Technology, 9167), STAT1 (Cell Signaling Technology, 9172), PARP (Cell Signaling Technology, 9542), and LC3B (Cell Signaling Technology, 3868).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Western Blot, Sulforhodamine B Assay, Transfection

Journal: Cell death & disease

Article Title: Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC.

doi: 10.1038/s41419-023-06238-5

Figure Lengend Snippet: Fig. 6 LSD1 inhibition sensitizes cancer cells to ferroptosis. A Cell viability was determined by SRB in A549 and H1975 cells treated with 10 μM ORY for 96 h and 2 μM RSL for 48 h alone or in combination as indicated. B The synergistic effect of ORY combination with RSL was analyzed in A549 and H1975 cell lines. Combination index (CI) values were calculated at the drug concentration of ORY (2.5, 5, 10, 20 μM) plus RSL (1, 2, 4, 8 μM) using the Chou-Talalay method. C–G After A549 and H1975 cells were treated with 10 μM ORY for 96 h and 2 μM RSL for 48 h alone or in combination as indicated, production of lipid peroxides (C), cell death (D), and total ROS accumulation (E) were analyzed by flow cytometry, the levels of GSH (F) were detected by Total Glutathione Assay Kit, and the expression of TFRC, ACSL4 xCT, and ATF4 (G) was analyzed by western blotting. n ≥3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The primary antibodies used here include: GAPDH (Immunoway, YT5052), β-actin (Sungene biotech, KM9001T), ACSL4 (Invitrogen, PA527137), TFRC (Abcam, ab214039), GPX4 (Abcam, ab125066), xCT (Abcam, ab37185), ATF4 (Cell Signaling Technology, 11815), LSD1 (Cell Signaling Technology, 2184), H3K9me2 (Cell Signaling Technology, 4658), YAP (Cell Signaling Technology, 14074), p-YAP (Cell Signaling Technology, 13008), FPN (Abcam, ab1239583), DMT1 (Abcam, ab55735), Ferritin (Abcam, 287968), p-STAT1 (Cell Signaling Technology, 9167), STAT1 (Cell Signaling Technology, 9172), PARP (Cell Signaling Technology, 9542), and LC3B (Cell Signaling Technology, 3868).

Techniques: Inhibition, Concentration Assay, Cytometry, Glutathione Assay, Expressing, Western Blot

Journal: Cell death & disease

Article Title: Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC.

doi: 10.1038/s41419-023-06238-5

Figure Lengend Snippet: Fig. 7 LSD1 inhibitors can enhance the tumor suppressive effect of ferroptosis inducers in vivo. A Images of xenograft tumors. B Relative tumor volume during the administration period. C Tumor weight. D Body weight of mice. E The content of GSH in tumor tissue. F The content of MDA in tumor tissue. G The content of Fe2+ in tumor tissue. H The mRNA levels of PTGS2 in tumor tissue. I Western blotting analysis of TFRC, ACSL4, and xCT in tumor tissue. n ≥3; mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The primary antibodies used here include: GAPDH (Immunoway, YT5052), β-actin (Sungene biotech, KM9001T), ACSL4 (Invitrogen, PA527137), TFRC (Abcam, ab214039), GPX4 (Abcam, ab125066), xCT (Abcam, ab37185), ATF4 (Cell Signaling Technology, 11815), LSD1 (Cell Signaling Technology, 2184), H3K9me2 (Cell Signaling Technology, 4658), YAP (Cell Signaling Technology, 14074), p-YAP (Cell Signaling Technology, 13008), FPN (Abcam, ab1239583), DMT1 (Abcam, ab55735), Ferritin (Abcam, 287968), p-STAT1 (Cell Signaling Technology, 9167), STAT1 (Cell Signaling Technology, 9172), PARP (Cell Signaling Technology, 9542), and LC3B (Cell Signaling Technology, 3868).

Techniques: In Vivo, Western Blot

Journal: Cell death & disease

Article Title: Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC.

doi: 10.1038/s41419-023-06238-5

Figure Lengend Snippet: Fig. 8 A working model. Proposed working model of inhibiting LSD1 on ferroptosis in cancer cells. (Left) LSD1 enhances the demethylation of H3K9me2 at the ATF4 promoter and activates ATF4 expression, which leads to the upregulation of xCT, thereby inhibiting ferroptosis in cancer cells. (Right) Inhibition of LSD1 results in the gain of H3K9me2 at ATF4 promoters, which silences ATF4 gene expression, leading to the reduction of xCT and ferroptosis. In addition, the suppression of LSD1 leads to an increase in H3K4me2 levels at the promoter sequences of TFRC and ACSL4, which promotes the expression of TFRC and ACSL4, which will promote the synthesis of unsaturated fatty acid and the accumulation of intracellular iron, respectively, thus promoting ferroptosis.

Article Snippet: The primary antibodies used here include: GAPDH (Immunoway, YT5052), β-actin (Sungene biotech, KM9001T), ACSL4 (Invitrogen, PA527137), TFRC (Abcam, ab214039), GPX4 (Abcam, ab125066), xCT (Abcam, ab37185), ATF4 (Cell Signaling Technology, 11815), LSD1 (Cell Signaling Technology, 2184), H3K9me2 (Cell Signaling Technology, 4658), YAP (Cell Signaling Technology, 14074), p-YAP (Cell Signaling Technology, 13008), FPN (Abcam, ab1239583), DMT1 (Abcam, ab55735), Ferritin (Abcam, 287968), p-STAT1 (Cell Signaling Technology, 9167), STAT1 (Cell Signaling Technology, 9172), PARP (Cell Signaling Technology, 9542), and LC3B (Cell Signaling Technology, 3868).

Techniques: Expressing, Inhibition, Gene Expression