Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Heatmap depicting significant gene expression differences (Log2 fold-change, p<0.05) between uninfected Lrrk2 KO and HET BMDMs. ( B ) IPA software analysis showing cellular pathways enriched for differentially expressed genes in uninfected Lrrk2 KO vs. HET BMDMs. ( C ) Heatmap depicting significant gene expression differences (Log2 fold-change) between Lrrk2 KO and HET BMDMs during infection with Mtb. ( D ) As in ( B ) but for pathways enriched for differentially expressed genes in Mtb-infected Lrrk2 KO and HET BMDMs, 4 hr post-infection. ( E ) RT-qPCR showing expression of Ifnb and IFN stimulated genes in uninfected and Mtb-infected Lrrk2 KO and HET macrophages. Data are shown as ISG / Actb . ( F ) RT-qPCR of Tnfa in Lrrk2 KO and HET BMDMs. ( G ) RT-qPCR of Apoe and Ldhb normalized to Actb in uninfected BMDMs. Throughout the manuscript, data are expressed as a mean of three or more biological replicates with error bars depicting SEM. Statistical tests used can be found at the end of the legend. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). In ( E–F ) a two-way ANOVA Tukey post-test was applied, and in ( G ) a two-tailed Student’s T test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Gene Expression, Software, Infection, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Heatmap depicting gene expression differences (Log2 fold-change) between Lrrk2 KO and HET BMDMs after infection with Mtb. ( B ) RT-qPCR of Ifit1 and Isg15 in Lrrk2 WT, HET, and KO BMDMs. ( C ) RT-qPCR of Tnf a expression in Lrrk2 WT, HET, and KO BMDMs. RT-qPCR showing expression of Ifnb and IFN stimulated genes in uninfected and Mtb-infected Lrrk2 KO and HET macrophages. Data are shown as ISG / Actb . ( D ) RT-qPCR of LRRK2 in uninfected SCR and LRRK2 KD U937 macrophage-like cells and gene expression of IFNB and IL1B in uninfected and Mtb-infected cells (MOI = 10, 4 hr). ( E ) RT-qPCR of Lrrk2 in uninfected SCR and Lrrk2 KD RAW 264.7 cells and gene expression of Ifnb and Tnfa in uninfected and Mtb-infected cells (MOI = 10, 4 hr). Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( B–E ) One-way and two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Gene Expression, Infection, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) RT-qPCR of Isg15 expression after 4 and 8 hr of infection with M. leprae (MOI = 50) in Lrrk2 KO BMDMs and HET controls. ( B ) RT-qPCR of Ifnb in unstimulated Lrrk2 KO and HET BMDMs alongside cells transfected with 1 μg/ml ISD (dsDNA) for 4 hr. ( C ) As in ( B ) but with peritoneal macrophages (PEMs) from Lrrk2 KO and HET mice elicited for 4 days with 1 ml 3% Brewer’s thioglycolate broth. ( D ) As in ( B ) but with RAW 264.7 Lrrk2 KO cells and WT controls. ( E ) As in ( B ) but with MEFs from day 14.5 Lrrk2 KO or HET embryos. ( F ) As in ( B ) but with RAW 264.7 Lrrk2 KD and scramble (SCR) controls cells. ( G ) RT-qPCR of Ifnb expression in uninfected Lrrk2 KO or HET BMDMs and in cells treated with 50 ng/ml DMXAA for 2 hr. ( H ) Western blot analysis and quantification of IRF3 phosphorylation (Ser396) and STAT1 phosphorylation (Tyr701) in BMDMs from HET and Lrrk2 KO mice compared to total IRF3 and STAT1 with tubulin as a loading control following transfection with 1 μg/ml ISD (dsDNA). ( I ) As in ( G ) but following transfection with 1 μg/ml poly(I:C), 100 ng/ml LPS, transfection with 10 μM CpG 2395, or stimulation with 1 μM CL097, all for 4 hr. ( J ) RT-qPCR of Isg15 expression after treatment with 200 IU IFN-β for 4 hr. ( K ) RT-qPCR of Irf7 gene expression in Lrrk2 HET and KO BMDMs with or without overnight treatment with IFN-β neutralizing antibody (blocking Ab, 1:250). ( L ) RT-qPCR of Irf7 gene expression in BMDMs from WT, Lrrk2 KO, Ifnar KO, and double knockout ( Lrrk2 / Ifnar DKO) mice. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( A–L ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Quantitative RT-PCR, Expressing, Infection, Transfection, Western Blot, Phospho-proteomics, Control, Gene Expression, Blocking Assay, Double Knockout

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) RT-qPCR of Isg15 expression after 4 and 8 hr of infection with M. leprae (MOI = 50) in WT and Lrrk2 KO RAW 264.7. ( B ) Western blot analysis and quantification of IRF3 phosphorylation (Ser396) and STAT1 phosphorylation (Tyr701) in BMDMs from HET and Lrrk2 KO mice compared to total IRF3 and STAT1 with tubulin as a loading control following transfection with 1 μg/ml ISD (dsDNA). ( C ) RT-qPCR of Ifit1 in Lrrk2 HET and KO BMDMs transfected with 1 μg/ml ISD (dsDNA) or 1 μg/ml poly(I:C) or treated with 100 ng/ml LPS for 4 hr. ( D ) RT-qPCR of Ifnb expression in uninfected Lrrk2 KO or HET PEMs treated with 100 ng/ml LPS for 4 hr. ( E ) RT-qPCR of Ifnb expression in uninfected Lrrk2 KO or HET MEFs treated with 100 ng/ml LPS or transfected with 1 μg/ml poly(I:C) for 4 hr. ( F ) RT-qPCR of Irf7 expression in Lrrk2 HET and KO BMDMs after treatment with 200 IU IFN-β for 4 hr. ( G ) RT-qPCR of Isg15 expression in Lrrk2 HET and KO BMDMs with or without overnight blocking with IFN-β neutralizing antibody (blocking Ab, 1:250). ( H ) RT-qPCR of Irf7 gene expression in BMDMs from WT, Lrrk2 KO, Ifnar KO, and double knockout ( Lrrk2 / Ifnar DKO) mice. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); %%p<0.01, ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( A–H ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Quantitative RT-PCR, Expressing, Infection, Western Blot, Phospho-proteomics, Control, Transfection, Blocking Assay, Gene Expression, Double Knockout

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Isg15 gene expression in Lrrk2 WT, Lrrk2 KO, cGas KO, and double KO ( cGas/Lrrk2 DKO) BMDMs treated with 5 μg/ml DMXAA or transfected with 1 μg poly(I:C) for 4 hr. ( B ) Western blot analysis of STAT1 phosphorylation (Tyr701) in BMDMs from WT, Lrrk2 KO, cGas KO, and cGas/Lrrk2 double knockout (DKO) mice compared to total STAT1 with tubulin as a loading control. ( C ) Immunofluorescent images with anti-TOM20 antibody to visualize the mitochondrial network of Lrrk2 HET and KO MEFs. TOM20 (green), nucleus (DAPI, blue); Scale bar = 10 μm ( D ) qPCR of total 16 s and cytB (mitochondrial DNA) relative to Tert (nuclear DNA). ( E ) As in ( D ) but cytosolic mitochondrial DNA. ( F ) Western blot of ACTIN, TFAM, and VDAC protein levels in total, cytosol, and pellet (organelle and membrane) fractions of Lrrk2 KD and SCR RAW 264.7 cells. ( G ) Irf7 gene expression normalized to Actb in untreated BMDMs from Lrrk2 WT, Lrrk2 KO, Tfam HET, and Lrrk2 KO/ Tfam HET mice. ( H ) qPCR of dLoop (mitochondrial DNA) normalized to Tert (nuclear) to confirm mtDNA depletion in WT and Lrrk2 KO RAW 264.7 cells treated with 10 μM ddC for 4 days. ( I ) RT-qPCR of Ifnb gene expression in WT and Lrrk2 KO RAW 264.7 cells with or without ddC treatment, untreated and at 4 hr post-transfection with 1 μg/ml ISD. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); %p<0.05, ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( A and I ) 3-way ANOVA, Tukey post-test; ( D and E ) two-tailed Student’s T test; ( G and H ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Gene Expression, Transfection, Western Blot, Phospho-proteomics, Double Knockout, Control, Membrane, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Rsad2 or Irf7 gene expression in Lrrk2 WT, Lrrk2 KO, cGas KO, and double KO ( cGas/Lrrk2 DKO) BMDMs treated with 5 μg/ml DMXAA or transfected with 1 μg poly(I:C) for 4 hr. ( B ) qPCR of total 16 s and cytB (mitochondrial DNA) relative to Tert (nuclear DNA) in Lrrk2 HET and KO MEFs. ( C ) As in ( B ) but measuring cytosolic mitochondrial DNA. ( D ) RT-qPCR of Irf7 gene expression in WT and Lrrk2 KO RAW 264.7 cells with or without ddC and transfected with 1 μg ISD for 4 hr. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); ##p<0.001 (comparing stimulated to unstimulated of same genotype). ( A and D ) 3-way ANOVA Tukey post-test; ( B and C ) two-tailed Student’s T test. *p<0.05, **p<0.01, ***p<0.005. ( E ) Western blot analysis of STAT1 phosphorylation (Tyr701) in BMDMs from wild-type and Lrrk2 KO mice either untreated (top) or treated with ddC (bottom) at 0, 2, 4, and 6h post-ISD transfection. Total STAT1 and tubulin are shown as controls. Densitometry quantitation of shown under blots.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Gene Expression, Transfection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Phospho-proteomics, Quantitation Assay

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of mitochondrial network TOM20 (green) and DRP1 (red) in MEFs from Lrrk2 HET and KO embryos. Nuclei are stained with DAPI (blue). Scale bar = 10 μm. ( B ) Western blot analysis and quantification of DRP1 phosphorylation (Ser616) in SCR and Lrrk2 KD RAW 264.7 cells compared to total DRP1 and actin as a loading control. ( C ) Histograms showing counts of phospho-S616-DRP1 in Lrrk2 HET and KO MEFs treated with 100 μM H 2 O 2 or 50 μM Mdivi-1 for 4 hr. ( D ) RT-qPCR of Isg15 and Irf7 gene expression Lrrk2 KO and HET BMDMs treated with 50 μM Mdivi-1 for 12 hr. ( E ) qPCR of cytosolic and total 16 s (mitochondrial DNA) relative to Tert (nuclear DNA) in Lrrk2 HET and KO MEFs treated with 50 μM Mdivi-1 for 12 hr. Statistical analysis: As in ; *p<0.05, **p<0.01, ***p<0.005.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Immunofluorescence, Staining, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR, Gene Expression

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Histograms showing counts of phospho-S616-DRP1 in SCR and Lrrk2 KD RAW 264.7 cells as measured by flow cytometry. ( B ) Western blot analysis and quantification of DRP1 phosphorylation (Ser616) in SCR and Lrrk2 KD RAW 264.7 cells compared to total DRP1 and actin as a loading control. ( C ) As in ( A ) but for BMDMs. ( D ) As in ( A ) but for MEFs. ( E ) Basal gene expression of Isg15 and Irf7 in SCR and Lrrk2 KD RAW 267.4 cells treated with Mdivi-1 50 μM for 12 hr. ( F ) qPCR of cytosolic and total 16 s (mitochondrial DNA) relative to Tert (nuclear DNA) in SCR and Lrrk2 KD RAW 264.7 cells treated with 50 μM Mdivi-1 for 12 hr. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. ( A, C, and D ) Two-tailed Student’s T test; ( E and F ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Flow Cytometry, Western Blot, Phospho-proteomics, Control, Gene Expression, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Mitochondrial membrane potential in Lrrk2 HET and KO BMDMs as measured by JC-1 dye by flow cytometry. Aggregates (610/20) indicate normal mitochondrial membrane potential and monomers (520/50) indicate low membrane potential. ( B ) Histogram of ( A ) displaying cell counts of JC-1 aggregates for Lrrk2 HET and KO BMDMs. ( C ) JC-1 aggregates measured by flow cytometry in BMDMs treated for 3 hr with 2.5 μM rotenone followed by 5 μM ATP for 0, 5, or 30 min. Histogram of cell counts is on the right. ( D ) Flow cytometry of mitochondrial membrane potential measured by TMRE (585/15) in SCR and Lrrk2 KD RAW 264.7 cells treated for 3 hr with 2.5 μM rotenone followed by 5 μM ATP for 15 min or 50 μM FCCP for 15 min. ( E ) JC-1 aggregates measured by flow cytometry in Lrrk2 KO BMDMs treated with 10 or 50 μM Mdivi-1 for 4 hr. ( F ) The same as in ( E ) but with TMRE. ( G ) Basal gene expression of Irf7 and Isg15 in Lrrk2 HET and KO BMDMs treated overnight with 200 μM mitoTEMPO. JC-1 flow cytometry assays are representative of 3 independent experiments. Statistical analysis: **p<0.01, ***p<0.005, ****p<0.001. ( F ) Two-tailed Student’s T test; ( G ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Membrane, Flow Cytometry, Gene Expression, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Flow cytometry of SCR or Lrrk2 KD RAW 264.7 cells measuring mitochondrial membrane potential by JC-1 dye with 2.5 μM rotenone followed by 5 μM ATP for the indicated times. ( B ) As in ( A ) but with Lrrk2 HET and KO MEFs. Histogram of cell counts on the right. ( C ) As in ( A ) with 50 μM FCCP as a positive control in Lrrk2 HET and KO BMDMs (left) and SCR and Lrrk2 KD RAW 264.7 cells (right). Histogram of cell counts below. ( D ) Flow cytometry of mitochondrial membrane potential measured by TMRE (585/15) in BMDMs (left), MEFs (center), and RAW 264.7 cells (right). Histogram plots with quantifications below. ( E ) Flow cytometry of mitochondrial membrane potential measured by TMRE (585/15) in SCR or Lrrk2 KD RAW 264.7 cells treated with 2.5 μM rotenone followed by 5 μM ATP or 50 μM FCCP. JC-1 flow cytometry assays are representative of 3 independent experiments. Statistical analysis: As in ; *p<0.05.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Flow Cytometry, Membrane, Positive Control

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Irf7 gene expression in HET and Lrrk2 KO BMDMs cultured with or without 1 mM sodium pyruvate. ( B ) Ifnb and Irf7 gene expression in SCR and Lrrk2 KD RAW 264.7 cells cultured with or without 1 mM sodium pyruvate. ( C ) BMDMs from Lrrk2 HET and KO mice were treated with 200 μM mitoTEMPO, IFN-β blocking antibody, and the combination of both overnight followed by analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured using the Seahorse Metabolic Analyzer (Agilent). ( D ) Quantification of maximal respiration and spare respiratory capacity from ( C ). Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. (A, B, and D) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Gene Expression, Cell Culture, Blocking Assay

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) RT-qPCR of Ifnb expression in Lrrk2 HET and KO MEFs cultured with 1 mM sodium pyruvate for 24 hr. ( B ) Seahorse metabolic analysis of Lrrk2 HET and KO BMDMs treated with increasing concentrations of sodium pyruvate (0, 1, and 2 mM). ( C ) Quantitation of maximal respiration and spare respiratory capacity shown in ( B ). Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. ( A and C ) two-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Quantitation Assay

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Chromatogram depicting targeted metabolomic analysis of Lrrk2 HET (n = 3) and KO BMDMs (n = 3) with pure molecular weight standard to IMP (top) and hypoxanthine (bottom). Replicate experiments are shown as individual lines (n = 2). Coefficient of variance (CV) for IMP = 8.8% (KO) and 21.7% (HET). CV for hypoxanthine = 9.1% (KO) and 14.0% (HET). ( B ) Diagram of key metabolites produced during purine metabolism oriented to the major steps of the pathway. De novo synthesis (green), salvage (red), breakdown (blue). ( C ) Representative immunofluorescence microscopy image of purinosome formation measured by PFAS puncta (green) in Lrrk2 HET and KO MEFs. Nuclei stained with DAPI (blue). ( D ) Quantification of number of PFAS puncta per cell. 100 cells were counted per coverslip from three coverslips. ( E ) RT-qPCR of Irf7 and Isg15 gene expression in Lrrk2 HET and KO BMDMs treated with increasing concentrations of urate (10, 50, 100, and 250 μM for 24 hr). ( F ) Basal gene expression of Ifnb and Ifit1 in SCR and Lrrk2 KD RAW 264.7 cells treated with 250 μM urate overnight. ( G ) JC-1 aggregate vs. monomer formation measured by flow cytometry in Lrrk2 HET and KO BMDMs treated with 100 μM urate or 200 μM mitoTEMPO overnight. Histograms shown below and merged histograms shown to the right. ( H ) Histograms of Lrrk2 HET and KO BMDMs treated with 100 μM urate or 200 μM mitoTEMPO overnight and then treated with 2.5 μM rotenone for 3 hr followed by 5 μM ATP for 15 min. ( I ) Histograms of DRP1 p-S616 flow cytometry analysis for Lrrk2 KO BMDMs following treatment with 100 μM urate or 200 μM mitoTEMPO. Quantification is shown on the right. JC-1 flow cytometry assays are representative of three independent experiments. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); #p<0.005, ##p<0.001 (comparing treated to untreated of same genotype). ( D ) Two-tailed Student’s T test; ( E and F ) two-way ANOVA Tukey post-test; ( I ) one-way ANOVA Tukey post-test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Molecular Weight, Produced, Immunofluorescence, Microscopy, Staining, Quantitative RT-PCR, Gene Expression, Flow Cytometry, Two Tailed Test

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) LC-MS/MS analysis of BMDMS from Lrrk2 HET and KO mice showing IMP peak. ( B ) As in ( A ) but for hypoxanthine.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet: ( A ) Mtb colony forming units (CFUs) recovered from Lrrk2 HET and KO BMDMs over the course of 5 days (MOI = 1). ( B ) Survival curves for Lrrk2 HET (n = 8) and KO (n = 11) mice over a 250 day Mtb infection. Survival times not statistically different based on log-rank Mantel-Cox test. ( C ) CFUs recovered from lungs and spleens of Mtb-infected Lrrk2 HET and KO mice at Day 7, 21, 63, and 126 post-infection. ( D ) Circulating serum cytokines measured at Day 21 in Lrrk2 HET and KO mice. ( E ) RT-qPCR of inflammatory cytokines from total RNA recovered from lung homogenates from Day 21 Mtb-infected Lrrk2 HET and KO mice. ( F ) As in ( E ) but detecting ISGs. ( G ) Hematoxylin and eosin (H&E) stain of inflammatory nodules in the lungs of Lrrk2 KO and HET mice 21 days after infection with Mtb. Small scale bar, 500 μm; large scale bar 1 mm. ( H ) Semi-quantitative score of pulmonary inflammation with a score of 0, 1, 2, 3 or 4 assigned based on granulomatous nodules in none, up to 25%, 26–50%, 51–75% or 76–100% of fields, respectively. Perivascular and peribronchial inflammation was scored using an analogous scale based on percentage of medium-caliber vessels or bronchioles with adjacent inflammatory nodules. ( I ) H&E stain of neutrophils within an inflammatory nodule in the lung of Lrrk2 HET and KO mice 21 days after infection with Mtb. Left panel bar is 20 μm. Right panel bar is 200 μm. ( J ) Quantification of neutrophils in the lungs of Lrrk2 HET and KO mice infected with Mtb for 21 or 63 days. Total neutrophil scores were determined by the percentage of fields of view at 20X magnification containing neutrophils. Degenerate neutrophil scores were determined by the percentage of PMN positive fields containing degenerate neutrophils. Statistical analysis: *p<0.05, **p<0.01, ***p<0.005, ****p<0.001 (comparing indicated data points); #p<0.005, ##p<0.001 (comparing infected to uninfected of same genotype). ( A ) Two-way ANOVA Tukey post-test; ( B ) Mantel-Cox log-rank; ( C–J ) Mann-Whitney test.

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Infection, Quantitative RT-PCR, Staining, MANN-WHITNEY

Journal: eLife

Article Title: LRRK2 maintains mitochondrial homeostasis and regulates innate immune responses to Mycobacterium tuberculosis

doi: 10.7554/eLife.51071

Figure Lengend Snippet:

Article Snippet: Cell line ( Mus musculus) , RAW 264.7 Lrrk2 Parental; WT , ATCC , ATCC SC-6003 , .

Techniques: Sequencing, Recombinant

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Cystine/Cysteine transporters are upregulated in CRC cells to promote tumor growth . (A) Schematic diagram of exogenous 13 C 2 -cystine or 13 C 3 -cysteine uptake through various transporters. (B) Colorectal cancer cells import both cystine and cysteine. HCT116 or DLD1 cells was treated by either 13 C 2 -cystine (100 μM) or 13 C 3 -cysteine (100 μM). Intracellular cysteine was labeled as 13 C 1 -cysteine (M1) by 13 C 2 -cystine treatment, or as 13 C 3 -cysteine (M3) by 13 C 3 -cysteine treatment. Both unlabeled and 13 C-labeled cysteine were presented (n = 6). (C) The transporter genes of both cystine and cysteine were significantly upregulated in CRC compared with paired non-tumor colon tissues in TCGA database (n = 50). SLC7A11, the light chain of cystine/glutamate antiporter system; SLC3A2, the heavy chain of cystine/glutamate antiporter system; SLC1A4, alanine/serine/cysteine/threonine transporter 1 (ASCT1); SLC1A5, alanine/serine/cysteine/threonine transporter 2 (ASCT2). mRNA levels represent FPKM value downloaded from TCGA database. (D and E) Knockdown of SLC1A4, SLC1A5, SLC7A11 or SLC3A2 reduced intracellular cysteine abundance in CRC cells. SLC1A4, SLC1A5, SLC7A11 or SLC3A2 was knocked down by siRNAs mixture in HCT116 or DLD1 cells. The knockdown efficiency was validated by qRT-PCR (n = 3) (D), and intracellular cysteine abundance was measured by UHPLC-QTRAP/MS (n = 6 for DLD1 SLC1A4 knockdown cells; n = 3 for others) (E). (F–H) Knockdown of SLC7A11 or SLC1A5 reduced the proliferation of CRC cells. SLC1A5 or SLC7A11 was knocked down by shRNA in HCT116 or DLD1 cells. The knockdown efficiency of SLC1A5 (F) and SLC7A11 (G) was validated by qRT-PCR (n = 3) and Western blots. The proliferation rates were measured by CCK-8 (n = 3) (H). (I–L) Knockdown of SLC1A5 on top of SLC7A11 knockdown further reduced intracellular cysteine content and significantly impaired CRC tumor growth. SLC1A5 was knockdown by siRNAs in DLD1 cells or DLD1 shRNA-SLC7A11 knockdown cells, and the cells were assayed for abundance of intracellular cysteine by UHPLC-QTRAP/MS (n = 3) (I); cell proliferation by CCK-8 (n = 3) (J); xenograft tumor (n = 10) growth rate (K) and weight (L). Two-way ANOVA was used for statistical analyses of H, J, K. Student t-test was used for others. Data of K is presented as mean ± SEM, and data of others are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant.

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Labeling, Knockdown, Quantitative RT-PCR, shRNA, Western Blot, CCK-8 Assay

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Hypoxia induces the transcription of cystine/cysteine transporters through ATF4 . (A) SLC1A4, SLC1A5, SLC7A11 and SLC3A2 were upregulated concurrently in CRC tumors. The RNA-seq data of SLC1A4, SLC1A5, SLC7A11 and SLC3A2 in CRC and paired non-tumor tissues from TCGA (n = 50) and GSE223120 (n = 20) were analyzed. The fold change (tumor/non-tumor) >1.1 was considered as upregulation. The percentage of tumors which upregulated 4 genes, 3 genes, 2 genes, or 1 gene were presented. (B and C) Hypoxia induced the expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2. HCT116 and DLD1 cells were incubated in hypoxia chamber (1.0 % O 2 , 5.0 % CO 2 , 94 % N 2 37 °C) or normoxia chamber overnight respectively. qRT-PCR was performed to examine the expression levels of SLC1A4, SLC1A5, SLC7A11 and SLC3A2 (n = 3) (B). Two independent sets of HCT116 cells were transfected with indicated pGL3-promoter constructs and internal control Renilla luciferase construct. 2 days post-transfection, two sets of cells were incubated under hypoxia or normoxia conditions respectively overnight. Luciferase activities were then assayed by dual-luciferase reporter assay system (n = 3) (C). (D) The flowchart to screen transcription factors which are responsible for co-expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2. ChEA3, ChIP-X Enrichment Analysis Version 3; GTRD, Gene Transcription Regulation Database; Hypoxia, the transcription factors which were reported as hypoxia inducible genes; STRING, functional protein association networks. (E and F) Hypoxia-related ATF4 regulates the expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2. ATF4 was knocked down by shRNAs in HCT116 cells. Cells were incubated under hypoxia condition overnight and the expression levels of indicated genes were examined by qRT-PCR (n = 3) (E). pCMV-ATF4 or empty vector was transfected into HCT116 cells. 48 h post-transfection, the expression levels of indicated genes were examined by qRT-PCR (n = 3) (F). (G) ROS induced the expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2. Cells were treated with H 2 O 2 , and the expression levels of indicated genes were examined by qRT-PCR (n = 3). (H) ER stress regulates the expression of SLC1A4, SLC1A5, SLC7A11 and SLC3A2 via ATF4. HCT116 cells were treated with Tg (thapsigargin), and the expression levels of indicated genes were examined by Western blots. (I) Fold enrichment of DNA fragments surrounding putative ATF4 binding sites of indicated gene promoters by ChIP-qPCR with or without H 2 O 2 (250 μM) treatment (n = 3). (J) Knockdown of ATF4 significantly inhibited the expression of cystine and cysteine transporter genes induced by hypoxia (overnight), H 2 O 2 (250 μM, 6 h), or Tg (250 nM, 6 h). (K and L) Hypoxia induced ATF4 expression through PERK. ATF4 protein levels in PERK knockdown cells (K) or ISRIB-treated cells ( p -eIF2α inhibition) (L) were detected. Student t-test was used for statistical analyses. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant.

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: RNA Sequencing, Expressing, Incubation, Quantitative RT-PCR, Transfection, Construct, Control, Luciferase, Reporter Assay, Functional Assay, Plasmid Preparation, Western Blot, Binding Assay, ChIP-qPCR, Knockdown, Inhibition

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: The flux from cysteine to GSH increases in CRC . (A) Volcano plots of differential expressed metabolites of HCT116 cells cultured with or without cystine/cysteine. Metabolites in cysteine-GSH pathway are highlighted as red. (B) KEGG pathway analysis of DEMs from HCT116 cells with or without cystine/cysteine. Y axis represents -log 10 (p value) of pathway analysis. (C) GSH rescued the growth of CRC cells in cystine/cysteine depletion condition. 200 μM of cystine, cysteine, or NAC (N-acetyl cysteine), 400 μM GSH (reduced glutathione) or taurine, nucleotides (1 × EmbyoMax Nucleosides), NEAA (1 × non-essential amino acids), 2 mM glutamate or α-KG (dimethyl α-ketoglutarate), 1 mM sodium pyruvate, 200 μM cystathionine or homocysteine were individually supplemented into cystine/cysteine deficient medium. 48 h later, cell numbers were counted with hemocytometer by Trypan-Blue exclusive assay (n = 3). (D–F) The flux from cysteine to GSH is elevated in CRC cells. Cells were supplied with both 100 μM 13 C 2 -cystine (GSH labeled as M1) and 100 μM 13 C 3 -cysteine (GSH labeled as M3) for indicated time points, fraction of 13 C-labeled GSH was measured (n = 6) (D). Schematic diagram of 13 C 3 -cysteine flux and related enzymes is shown in (E). Cells were cultured in the presence of 100 μM 13 C 3 -cysteine for 2 h, and percentages of labeled metabolites in total pool were plotted (n = 6) (F). (G) The flux from cysteine to GSH is elevated by hypoxia induced ER stress. DLD1 cells were treated with Tg (250 nM), H 2 O 2 (250 μM), or hypoxia overnight, and then incubated with 100 μM 13 C 3 -cysteine for 30 min. Percentages of labeled GSH in total pool were plotted (n = 8). (H) Spontaneous colon tumors were generated with CAKP mice. After infusion with 13 C 3 -cysteine for 4 h, the serum, colorectal tumors and adjacent non-tumor colon tissues were collected, and percentages of labeled metabolites were plotted (n = 8). Student t-test was used for statistical analyses. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Cell Culture, Labeling, Incubation, Generated

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Overexpression of GSS plays important role in CRC growth . (A and B) Glutathione synthetase (GSS) was significantly upregulated in CRC samples compared with normal colon tissues. The mRNA levels of indicated genes of paired CRC and normal tissues in TCGA (n = 50, A) and GSE223120 (n = 20, B) datasets were plotted. (C–H) Knockdown of GSS reduced the growth of colorectal cancer cells. HCT116 or DLD1 cells were transfected with siRNAs against GSS or control siRNA. 48 h post-transfection, the cells were assayed for knockdown efficiency by qRT-PCR (n = 3) (C) or Western blots (D); relative abundance of γ-glutamylcysteine (E) or GSH (F) by LC-MS (n = 6); cell proliferation rate by CCK-8 (n = 3) (G); subcutaneous xenograft tumor growth (n = 10) (H). Two-way ANOVA was used for statistical analyses of G, H. Student t-test was used for others. Data of H is presented as mean ± SEM, and data of others are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant.

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Over Expression, Knockdown, Transfection, Control, Quantitative RT-PCR, Western Blot, Liquid Chromatography with Mass Spectroscopy, CCK-8 Assay

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Scavenging exogenous cystine/cysteine represses CRC growth . (A) Relative survival of colorectal cancer cells and normal colon epithelial cells under cystine/cysteine depletion condition (n = 3). (B and C) Cyst(e)inase efficiently depletes cystine and cysteine. HCT116 cells were treated with 1 mg/ml cyst(e)inase for 24 h. Intracellular (cells) and exogenous (medium) cystine or cysteine were measured by UHPLC-QTRAP/MS (n = 6) (B). C57BL/6 mice (n = 6) were intraperitoneally injected with cyst(e)inase (50 mg/kg). 24 h later, mice serum was collected to measure cystine or cysteine levels by UHPLC-QTRAP/MS (C). (D) The fold changes of cysteine-related metabolites abundance after HCT116 cells were treated with cystine/cysteine depletion condition or cyst(e)inase for 24 h. (E–G) Cyst(e)inase inhibited the CRC tumor growth. Nude mice were subcutaneous injected with either HCT116 cells (E) or small pieces of patient-derived xenograft tumors (F). Once tumors reached 100–150 mm 3 , mice were divided into two groups to intraperitoneally inject with PBS vehicle or cyst(e)inase (50 mg/kg) thrice a week. CAKP mice were administrated with tamoxifen. 40 days post injection, mice were divided into two groups to intraperitoneally inject with PBS vehicle or cyst(e)inase (50 mg/kg) thrice a week. Kaplan-Meier analyses was used to show the survival of mice with or without cyst(e)inase treatment (G). Student t-test was used for statistical analyses. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant.

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Injection, Derivative Assay

Journal: Redox Biology

Article Title: Hypoxia-induced cysteine metabolism reprogramming is crucial for the tumorigenesis of colorectal cancer

doi: 10.1016/j.redox.2024.103286

Figure Lengend Snippet: Depletion of cystine/cysteine induces autophagy of CRC cells through mTOR-ULK pathway . (A) Depletion of cystine/cysteine affects cellular autophagy, apoptosis or ferroptosis. Cells were cultured under cystine/cysteine depletion condition (-cyst(e)ine) or treated with cyst(e)inase (+cyst(e)inase) for 48 h. Cell lysates were collected and cell death markers were detected by Western blot assays. (B) Depletion of cystine/cysteine, but not glutamine or methionine, significantly induced autophagy in CRC cells but not in normal epithelial cells. (C–E) Cystine/cysteine depletion significantly induces autophagy in CRC cells. mCherry-GFP-LC3-expressing HCT116 and DLD1 cells were cultured under cystine/cysteine depletion condition or treated with cyst(e)inase for 24 h. Representative images of fluorescent LC3 puncta were show in (C). Mean number of GFP and mcherry dots per cell (n = 3) (D); and mean number of autophagosomes (yellow dots in merged images) and autolysosomes (red dots in merged images) per cell were counted (n = 3) (E). (F and G) Cystine/cysteine depletion induces autophagy of CRC cells through mTOR-ULK1 pathway. The dose-dependence (F) and time-dependence (G) of autophagy (the expression of LC3A and LC3B), mTOR activity (p-p70S6K) and ULK activity ( p -ULK) upon cystine/cysteine depletion were assayed by Western blots. Student t-test was used for statistical analyses. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; n.s., not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: HCT116 or DLD1 cells were transduced with the lentivirus expressing mCherry-EGFP-LC3B fusion protein (GeneCopoeia, Guangzhou, China) at 15 MOI, and stable mCherry-EGFP-LC3B cells were selected with puromycin (Invitrogen) (0.8 μg/ml for HCT116 and 2.0 μg/ml for DLD1).

Techniques: Cell Culture, Western Blot, Expressing, Activity Assay