Journal: Journal of Translational Medicine

Article Title: JMJD6 K375 acetylation restrains lung cancer progression by enhancing METTL14/m6A/SLC3A2 axis mediated cell ferroptosis

doi: 10.1186/s12967-025-06241-8

Figure Lengend Snippet: JMJD6 inhibits SLC3A2 expression by METTL14 mediated m6A manner. ( A ) The TCGA database was used to analyze the ferroptosis-related genes significantly related to JMJD6 in lung cancer cells. ( B ) TCGA database was used to analyze the expression level of SLC3A2 in lung cancer tissues and normal tissues. ( C ) TCGA data set was used to analyze the Kaplan-Meier survival of lung cancer patients with high SLC3A2 expression. ( D ) Heat map analysis of m6A modication-related enzyme genes was performed. ( E ) Protein-protein interaction network (PPI) was used to analyze the interaction of JMJD6 with METTL14 and SLC3A2. ( F , G ) The expression levels of METTL14 and SLC3A2 in H1299 cells after JMJD6 knockdown were verified by Western blot and RT-qPCR at the protein ( F ) and mRNA ( G ) levels, respectively. ( H , I ) The expression levels of METTL14 and SLC3A2 after JMJD6 overexpression were verified by Western blot and RT-qPCR at the protein level ( H ) and mRNA level ( I ), respectively. ( J ) CUT&Tag detection, representative trace plots showing the region of JMJD6 binding METTL14 and peak plots regulating changes in its H4R3m2a modification levels. The arrow marks the promoter region. ( K ) ChIP-qPCR was used to verify the binding of JMJD6 and H4R3me2a to the promoter region of METTL14 (500–1000). ( L ) Western blot was used to verify the expression levels of JMJD6 and SLC3A2 after knockdown of METTL14. ( M ) Western blot was used to verify the expression levels of JMJD6 and METTL14 after SLC3A2 knockdown. ( N , O ) METTL14 was knocked down in H1299 cells, and the effect of METTL14 on SLC3A2 expression was verified by detecting the m6A level ( N ) and mRNA expression ( O ) of SLC3A2, respectively. ( P ) The effects of JMJD6 and METTL14 on SLC3A2 expression were verified by detecting the mRNA stability of si-JMJD6 group, METTL14 overexpression group and si-JMJD6/si-METTL14 group. ( Q ) The effects of JMJD6 and METTL14 on SLC3A2 expression were verified by detecting the stability of SLC3A2 mRNA in JMJD6 overexpression group, si-METTL14 group and Flag-JMJD6/Flag-METTL14 group. ( R ) SLC3A2 3’UTR containing either wild-type or mutant (A-to-C mutation) m6A sites was cloned into luciferase reporter vector. ( S ) Relative luciferase activity of the wild-type and mutant form of SLC3A2 3’UTR reporter vectors in A549 cells transfected with si-control or si-METTL14, respectively. ( T , U , V ) The correlation between JMJD6, METTL14 and SLC3A2 was analyzed by TCGA database. Data are presented as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used in this study were listed as follows: JMJD6 antibody (Santa Cruz Biotechnology, Cat# sc-28348, 1:1000), METTL14 antibody (CST, Cat# 48699, 1:1000), SLC3A2 antibody (Abways, Cat# CY8416, 1:1000), GAPDH antibody (Abways, Cat# AB2000, 1:6000), Flag-tag antibody (Abways, Cat# AB0030, 1:1000), HA-tag antibody (proteintech, Cat# 51064-2-AP, 1:3000), Acetylated-Lysine Antibody (CST, Cat# 9441, 1:1000), JMJD6-acK375 antibody (Jiaxuan Biotech Cat#JXR7611, 1:1000), Rabbit Anti-Goat IgG (H + L) HRP (Abways, Cat# AB0101, 1:8000).

Techniques: Expressing, Knockdown, Western Blot, Quantitative RT-PCR, Over Expression, Binding Assay, Modification, ChIP-qPCR, Mutagenesis, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Transfection, Control

Journal: Journal of Translational Medicine

Article Title: JMJD6 K375 acetylation restrains lung cancer progression by enhancing METTL14/m6A/SLC3A2 axis mediated cell ferroptosis

doi: 10.1186/s12967-025-06241-8

Figure Lengend Snippet: JMJD6 acetylation inhibits NSCLC progression and xenograft growth by promoting ferroptosis. ( A ) H1299 cells stably expressing JMJD6-WT, JMJD6-K375R and JMJD6-K375Q were established, and the expression of target proteins was confirmed by Western blot analysis with JMJD6 antibody. ( B ) CCK-8 was used to detect the effect of JMJD6-K375 acetylation on the proliferation of H1299 cells. ( C ) EdU was used to detect the effect of JMJD6-K375 acetylation on the proliferation of H1299 cells. Representative images are shown on the left and statistical data analysis on the right. The scale bars represent 100 μm. ( D ) Colony formation assay was used to detect the effect of JMJD6-K375 acetylation on the proliferation of H1299 cells. Representative pictures are shown on the left and statistical data analysis on the right. ( E ) Transwell assay was used to detect the effect of JMJD6-K375 acetylation on the migration and invasion of H1299 cells. Representative pictures are shown on the left and statistical data analysis on the right. ( F , G , H ) Mice were injected with H1299 cells expressing JMJD6-WT, JMJD6-K375R and JMJD6-K375Q. ( H ) The growth curve of transplanted tumor in nude mice was drawn. ( F , G ) On the 30th day, the images of transplanted tumors were dissected and taken, and the average tumor weight was measured. ( I ) The effect of JMJD6 acetylation on ROS level in NSCLC cells. ( J ) JMJD6 acetylated NSCLC cells were treated with erastin and ferrostain-1, and cell viability was detected. ( K ) JC-1 fluorescent probe was used to detect the change of mitochondrial membrane potential in H1299 cells after acetylation of JMJD6. Representative pictures are shown on the left and statistical data analysis on the right. The scale bars represent 50 μm. ( L ) Expression of JMJD6, GPX4, SLC3A2, xCT and Ki67 in serial sections of subcutaneous xenograft tumors. Representative images are shown at the top and statistical data analysis is shown at the bottom. The scale bars represent 100 μm. Data are expressed as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used in this study were listed as follows: JMJD6 antibody (Santa Cruz Biotechnology, Cat# sc-28348, 1:1000), METTL14 antibody (CST, Cat# 48699, 1:1000), SLC3A2 antibody (Abways, Cat# CY8416, 1:1000), GAPDH antibody (Abways, Cat# AB2000, 1:6000), Flag-tag antibody (Abways, Cat# AB0030, 1:1000), HA-tag antibody (proteintech, Cat# 51064-2-AP, 1:3000), Acetylated-Lysine Antibody (CST, Cat# 9441, 1:1000), JMJD6-acK375 antibody (Jiaxuan Biotech Cat#JXR7611, 1:1000), Rabbit Anti-Goat IgG (H + L) HRP (Abways, Cat# AB0101, 1:8000).

Techniques: Stable Transfection, Expressing, Western Blot, CCK-8 Assay, Colony Assay, Transwell Assay, Migration, Injection, Membrane

Journal: Journal of Translational Medicine

Article Title: JMJD6 K375 acetylation restrains lung cancer progression by enhancing METTL14/m6A/SLC3A2 axis mediated cell ferroptosis

doi: 10.1186/s12967-025-06241-8

Figure Lengend Snippet: Acetylation weakens the activity of JMJD6 in regulating METTL14 expression and affecting its mediated m6A modification to regulate SLC3A2. ( A , B ) Western blot ( A ) and RT-qPCR ( B ) were used to detect the effect of JMJD6 acetylation on METTL14 expression at the protein and mRNA levels, respectively. ( C ) CHIP assay was used to verify the regulation of JMJD6 acetylation on the enrichment level of H4R3me2a in the promoter region of METTL14. ( D , E ) Western blot ( D ) and RT-qPCR ( E ) were used to detect the effect of JMJD6 acetylation on SLC3A2 expression at the protein and mRNA levels, respectively. ( F ) MeRIP was used to detect the m6A level of SLC3A2 in JMJD6-WT, JMJD6-K375R and JMJD6-K375Q groups, and to verify the effect of JMJD6 acetylation on the m6A modification level of SLC3A2 mRNA. ( G ) MeRIP was used to detect the effect of METTL14 knockdown and JMJD6 mimics K375 deacetylation on the m6A modification of SLC3A2 mRNA. ( H ) CCK-8 was used to detect the effect of SLC3A2 knockdown and JMJD6 acetylation deletion on cell proliferation. ( I , J ) Cell viability ( I ) and ROS levels ( J ) were detected in H1299 cells to assess whether SLC3A2 knockdown would alter the sensitivity to erastin. Data are expressed as mean ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: The antibodies used in this study were listed as follows: JMJD6 antibody (Santa Cruz Biotechnology, Cat# sc-28348, 1:1000), METTL14 antibody (CST, Cat# 48699, 1:1000), SLC3A2 antibody (Abways, Cat# CY8416, 1:1000), GAPDH antibody (Abways, Cat# AB2000, 1:6000), Flag-tag antibody (Abways, Cat# AB0030, 1:1000), HA-tag antibody (proteintech, Cat# 51064-2-AP, 1:3000), Acetylated-Lysine Antibody (CST, Cat# 9441, 1:1000), JMJD6-acK375 antibody (Jiaxuan Biotech Cat#JXR7611, 1:1000), Rabbit Anti-Goat IgG (H + L) HRP (Abways, Cat# AB0101, 1:8000).

Techniques: Activity Assay, Expressing, Modification, Western Blot, Quantitative RT-PCR, Knockdown, CCK-8 Assay

Journal: Journal of Translational Medicine

Article Title: JMJD6 K375 acetylation restrains lung cancer progression by enhancing METTL14/m6A/SLC3A2 axis mediated cell ferroptosis

doi: 10.1186/s12967-025-06241-8

Figure Lengend Snippet: Elevated JMJD6 acetylation predicts a favorable survival in NSCLC patients. ( A ) The acetylation level of JMJD6 was detected by immunohistochemistry in 68 cases of lung cancer and precancerous lesions. Representative images are shown on the right and statistical data analysis on the left. The scale bars represent 100 μm. ( B ) Sixty-eight lung cancer patients were clinically selected and divided into two groups, low (0–7) and high (8–12), according to the expression level of JMJD6-acK375. Then the expression of JMJD6-acK375 in lung cancer was tested whether it was related to gender, age, differentiation degree, tumour size and TNM stage, respectively. ( C ) The expression of JMJD6-acK375 was examined in 68 lung cancer tissues with TNM stage I-II and stage III-IV. ( D ) JMJD6-acK375 expression was detected in 68 cases of tumor tissues with tumor size greater than or equal to 5 cm and less than 5 cm. ( E ) Kaplan-Meier analysis of JMJD6 hyperacetylation level, log-rank test, P = 0.022. ( F ) The working model elucidates the molecular mechanism through which JMJD6 influences the expression of its downstream target METTL14 both pre- and post-acetylation, consequently impacting the expression of the ferroptosis-related protein SLC3A2. In this process, we demonstrated that acetylated JMJD6 enhances METTL14 expression by elevating H4R3me2a levels, whereas METTL14 overexpression suppresses SLC3A2 expression and impedes the progression of lung cancer cells by facilitating ferroptosis

Article Snippet: The antibodies used in this study were listed as follows: JMJD6 antibody (Santa Cruz Biotechnology, Cat# sc-28348, 1:1000), METTL14 antibody (CST, Cat# 48699, 1:1000), SLC3A2 antibody (Abways, Cat# CY8416, 1:1000), GAPDH antibody (Abways, Cat# AB2000, 1:6000), Flag-tag antibody (Abways, Cat# AB0030, 1:1000), HA-tag antibody (proteintech, Cat# 51064-2-AP, 1:3000), Acetylated-Lysine Antibody (CST, Cat# 9441, 1:1000), JMJD6-acK375 antibody (Jiaxuan Biotech Cat#JXR7611, 1:1000), Rabbit Anti-Goat IgG (H + L) HRP (Abways, Cat# AB0101, 1:8000).

Techniques: Immunohistochemistry, Expressing, Over Expression

Journal: Cell death discovery

Article Title: IFNγ regulates ferroptosis in KFs by inhibiting the expression of SPOCD1 through DNMT3A.

doi: 10.1038/s41420-024-02257-z

Figure Lengend Snippet: Fig. 5 IFNγ could regulate the biological actives of KFs through SPOCD1. A The results of wound healing. B Quantitative analysis of wound healing model. C–J Gene expression of SPOCD1, IFNγ, col1, col3, GPX4, GSH, SLC7A11 and SLC3A2 in KFs treated with si-IFNγ, si-SPOCD1 or si- IFNγ + SPOCD1 for 48 h. Bar=100 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The primary antibodies included rabbit antibodies against β-actin (proteintech, 1:1,000), SPOCD1 (proteintech, 1:5,000), IFNγ (proteintech, 1:1,000), GPX4 (proteintech, 1:10,000), GSH (proteintech, 1:1,000), SLC7A11 (proteintech, 1:1,000), SLC3A2 (proteintech, 1:1,000), DNMT1 (proteintech, 1:1,000), DNMT3A (proteintech, 1:1,000), and DNMT3B (proteintech, 1:2,000).

Techniques: Gene Expression