Journal: bioRxiv

Article Title: Infection with Mycobacterium tuberculosis orchestrates the PRMT5-dependent methylation of NCOA4 to govern host ferroptosis

doi: 10.1101/2025.10.23.684158

Figure Lengend Snippet: ( A ) Estimation of intracellular labile ferrous iron in murine macrophages pretreated with PRMT5 inhibitor (for 1 hour) at 24 hours post infection. ( B ) Confocal images of murine macrophages pretreated with PRMT5 inhibitor (for 1 hour) stained for 4HNE (green) and nuclear staining (blue) at 24 hours post infection. Scale bars: 5 µm (left), and its quantification (right). ( C ) Assessment of necrotic cell death measured by LDH released in the supernatants from the macrophage pretreated with PRMT5 inhibitor (for 1 hour) at 4 days post infection (left). Examination of the release of live mycobacteria from necrotic cells by CFU quantification in culture supernatants of macrophages pretreated with PRMT5 inhibitor (for 1 hour) at 4 days post infection (right). ( D-J ) BALB/c mice were infected with Mtb H37Rv and treated with PRMT5 inhibitor EPZ015666 (4mg/kg) q.o.d. as indicated in ( D ) (Number of mice per group = 5) ( E ) Analysis of TB pathology (granulomatous lesions) in the lung sections with H and E staining (left) and its quantification by a pathologist in a blinded manner (right). ( F-H ) Enumeration of mycobacterial burden in the lungs ( F ), spleen ( G ), liver ( H ) of infected and PRMT5 inhibitor treated mice by plating homogenates on 7H11 plates. ( I ) Measurement of lipid peroxidation (malondialdehyde) in lung homogenates from uninfected, Mtb infected and PRMT5 inhibitor treated mice. All immunofluorescence data are representative of three independent experiments. Specific regions of the H and E-stained sections were analysed by the pathologist for evaluation of the granuloma fraction. Accordingly, the portions have been demarcated in the images; Magnification, 4X (left panel); 10X (right panel); Scale bar for H and E images, 100μm; G, granuloma; AS, alveolar space; MFI, mean fluorescence intensity; DAPI, 4’,6-diamidino-2-phenylindole; CTCF, corrected total cell fluorescence; LDH, lactose dehydrogenase; CFU, colony forming units; q.o.d., quaque altera die , every other day. EPZ015666 (20 μM), PRMT5 inhibitor. *, p<0.05; **, p<0.01; ****, p < 0.0001 (One-way ANOVA in A, B, I; Student’s t-test in C, E, F, G, H; GraphPad Prism 10.0).

Article Snippet: Ltd. siRNAs were obtained from Eurogentec against Ncoa4 , Prmt5 .

Techniques: Infection, Staining, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Infection with Mycobacterium tuberculosis orchestrates the PRMT5-dependent methylation of NCOA4 to govern host ferroptosis

doi: 10.1101/2025.10.23.684158

Figure Lengend Snippet: ( A ) Confocal images of murine macrophages treated with FeSO 4 for 24 hours and then treated with DFO (iron chelator) for 1 hour, stained for ferritin heavy chain (red) and nuclear staining (blue) at 24 hours post infection. Scale bars: 5 µm (left), and its quantification (right). ( B ) Assessment of ferritin heavy chain levels in RAW 264.7 cells transfected with NCOA4 OE vectors at 24 hours post infection by immunoblotting. ( C ) Assessment of ferritin heavy chain levels in murine macrophages pretreated with PRMT5 inhibitor (for 1 hour) at 24 hours post infection (left); and in murine macrophages transfected with NT or Prmt5 siRNAs at 24 hours post infection (right) by immunoblotting. ( D ) Confocal images of murine macrophages pretreated with PRMT5 inhibitor (for 1 hour) stained for ferritin heavy chain (green), lysosomes LAMP1 (red) and nuclear staining (blue) at 24 hours post infection. Scale bars: 2 µm (left), and its quantification (right). ( E ) BALB/c mice were infected with Mtb H37Rv and treated with PRMT5 inhibitor EPZ015666 (4mg/kg) q.o.d. Assessment of ferritin heavy chain levels in the lung homogenates of uninfected and infected mice by immunoblotting. All immunoblotting and immunofluorescence data are representative of three independent experiments. ACTB was used as a loading control. NT, non-targeting; MFI, mean fluorescence intensity; DAPI, 4’,6-diamidino-2-phenylindole; OE, over expression; CTCF, corrected total cell fluorescence; CFU, colony forming units; q.o.d., quaque altera die , every other day. *, p<0.05; **, p<0.01; ****, p < 0.0001 (Student’s t-test in A, D; GraphPad Prism 10.0). EPZ015666 (20 μM), PRMT5 inhibitor, FeSO 4 (100 μM); DFO (100 μM). Below each panel, the quantification of the blots normalized to the loading control has been indicated.

Article Snippet: Ltd. siRNAs were obtained from Eurogentec against Ncoa4 , Prmt5 .

Techniques: Staining, Infection, Transfection, Western Blot, Immunofluorescence, Control, Fluorescence, Over Expression

Journal: bioRxiv

Article Title: Infection with Mycobacterium tuberculosis orchestrates the PRMT5-dependent methylation of NCOA4 to govern host ferroptosis

doi: 10.1101/2025.10.23.684158

Figure Lengend Snippet: ( A ) Assessment of the interaction between NCOA4 and ferritin heavy chain by co-immunoprecipitation of whole cell lysates from murine macrophages pretreated with PRMT5 inhibitor (for 1 hour) at 24 hours post infection ( B ) Confocal images of murine macrophages pretreated with PRMT5 inhibitor (for 1 hour) stained for ferritin heavy chain (green), NCOA4 (red) lysosomes LAMP1 (blue) and nuclear staining (grey) at 24 hours post infection. Scale bars: 2 µm (left), and its quantification (right). ( C ) Assessment of extent of arginine methylation on NCOA4 by immunoprecipitation of whole cell lysates from murine macrophages pretreated with PRMT5 inhibitor (for 1 hour) at 24 hours post infection. ( D ) The putative arginine residue that is methylated in NCOA4 is conserved across different species. ( E ) Assessment of extent of arginine methylation on NCOA4 and its interaction with ferritin heavy chain by co-immunoprecipitation of whole cell lysates from RAW 264.7 cells transfected with NCOA4 OE vectors at 24 hours post infection. All immunoblotting is representative of three independent experiments. ACTB was used as a loading control. DAPI, 4’,6-diamidino-2-phenylindole; IP, immunoprecipitation; OE, overexpression. **, p<0.01; (Student’s t-test in B; GraphPad Prism 10.0). EPZ015666 (20 μM), PRMT5 inhibitor.

Article Snippet: Ltd. siRNAs were obtained from Eurogentec against Ncoa4 , Prmt5 .

Techniques: Immunoprecipitation, Infection, Staining, Methylation, Residue, Transfection, Western Blot, Control, Over Expression

Journal: bioRxiv

Article Title: Infection with Mycobacterium tuberculosis orchestrates the PRMT5-dependent methylation of NCOA4 to govern host ferroptosis

doi: 10.1101/2025.10.23.684158

Figure Lengend Snippet: ( A, B ) RAW 264.7 cells were transfected with NCOA4 OE vectors. Confocal images stained for NCOA4 (magenta) and nuclear staining (blue) at 24 hours post infection ( A ) and the quantification of the nuclear cytoplasmic localisation ( B ). ( C ) Assessment of the interaction between NCOA4 and vitamin D3 receptor by co-immunoprecipitation of whole cell lysates from murine macrophages pretreated with Calcitriol and PRMT5 inhibitor (for 1 hour) at 24 hours post infection. (D) Quantification of the nuclear cytoplasmic localisation of NCOA4 in murine macrophages pretreated with Calcitriol and PRMT5 inhibitor (for 1 hour) at 24 hours post infection by immunostaining. All immunofluorescence and immunoblotting data are representative of three independent experiments. DAPI, 4’,6-diamidino-2-phenylindole; CTCF, corrected total cell fluorescence; EPZ015666 (20 μM), PRMT5 inhibitor; Calcitriol (10nM), 1,25(OH)₂D₃. ***, p<0.001; ****, p<0.0001 (One-way ANOVA in B, D; GraphPad Prism 10.0).

Article Snippet: Ltd. siRNAs were obtained from Eurogentec against Ncoa4 , Prmt5 .

Techniques: Transfection, Staining, Infection, Immunoprecipitation, Immunostaining, Immunofluorescence, Western Blot, Fluorescence

Journal: bioRxiv

Article Title: Infection with Mycobacterium tuberculosis orchestrates the PRMT5-dependent methylation of NCOA4 to govern host ferroptosis

doi: 10.1101/2025.10.23.684158

Figure Lengend Snippet: ( A, B ) Confocal images stained for lysosomes LAMP1 (red) and nuclear staining (blue) infected with Mtb H37Rv-GFP (green) in murine macrophages pretreated with PRMT5 inhibitor (for 1 hour) at 24 hours post infection ( A ) and its respective quantification ( B ). ( C-F ) BALB/c mice were infected with Mtb H37Rv and treated with Calcitriol (1μg/kg) q.d and PRMT5 inhibitor EPZ015666 (4mg/kg) q.o.d. as indicated in ( C ) (Number of mice per group = 5). ( D, E ) Enumeration of mycobacterial burden in the lungs ( D ) and spleen ( E ) of infected and inhibitor treated mice by plating organ homogenates on 7H11 plates. ( F ) Analysis of TB pathology (granulomatous lesions) in the lung sections with H and E staining (left) and its quantification by a pathologist in a blinded manner (right). Specific regions of the H and E-stained sections were analysed by the pathologist for evaluation of the granuloma fraction. Accordingly, the portions have been demarcated in the images; Magnification, 4X (left panel); 10X (right panel); Scale bar for H and E images, 100μm; G, granuloma; AS, alveolar space; DAPI, 4’,6-diamidino-2-phenylindole; CFU, colony forming units; q.o.d., quaque altera die , every other day; q.d., quaque die , once a day. EPZ015666 (20 μM), PRMT5 inhibitor; Calcitriol (10nM), 1,25(OH)₂D₃. *, p<0.05; ***, p<0.001 (One-way ANOVA in E; Student’s t-test in C; GraphPad Prism 10.0).

Article Snippet: Ltd. siRNAs were obtained from Eurogentec against Ncoa4 , Prmt5 .

Techniques: Staining, Infection