Journal: Viruses

Article Title: Sorting Nexin 10 Mediates Endosomal Acidification and Autophagy to Promote Influenza A Virus Infection

doi: 10.3390/v18040460

Figure Lengend Snippet: SNX10 knockdown inhibited influenza virus infection. ( A , B ) qRT-PCR analysis to verify the knockdown efficiency of siRNA in A549 ( A ) or MLE-12 cells ( B ) at 48 h post siRNA transfection; ( C ) Western blot analysis of NP expression in A549 cells transfected with siSNX10 or siNC and infected with PR8 (MOI = 0.1) for 12 and 24 h. ( D ) Plaque assay to measure viral titers in supernatants from A549 cells transfected with siSNX10 or siNC at 12 h and 24 h post-infection with PR8 (MOI = 0.1). ( E ) Western blot analysis of NP expression in MLE-12 cells transfected with siSNX10 or siNC and infected with PR8 (MOI = 0.1) for 12 h. ( F ) Plaque assay to measure viral titers in supernatants from MLE-12 cells transfected with siSNX10 or siNC at 12 h post-infection with PR8 (MOI = 0.1). Data are presented as the mean ± SD of three independent experiments and analyzed by two-tailed Student’s t -test (* p < 0.05, *** p < 0.001, **** p < 0.0001).

Article Snippet: Madin-Darby canine kidney (MDCK) cells, human lung epithelial (A549) cells, and mouse lung epithelial cells (MLE-12) were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Virus, Infection, Quantitative RT-PCR, Transfection, Western Blot, Expressing, Plaque Assay, Two Tailed Test

Journal: Viruses

Article Title: Sorting Nexin 10 Mediates Endosomal Acidification and Autophagy to Promote Influenza A Virus Infection

doi: 10.3390/v18040460

Figure Lengend Snippet: SNX10 overexpression promoted IAV infection. ( A , B ) Western blot analysis of NP expression in SNX10-overexpressing A549 cells ( A ) and MLE-12 cells ( B ) infected with PR8 (MOI = 0.1) for 12 and 24 h. ( C , D ) Plaque assay to measure viral titers in supernatants from SNX10-overexpressing A549 cells ( C ) and MLE-12 cells ( D ) at 12 and 24 h post-infection with PR8 at an MOI of 0.1. ( E ) Confocal immunofluorescence imaging of NP expression in SNX10-overexpressing A549 cells at 3 h post-infection with PR8 (MOI = 10). Cells were stained with NP antibody to label NP protein (green) and DAPI to label the nucleus (blue), Scale bar = 50 μm. Data are presented as the mean ± SD of three independent experiments and analyzed by two-tailed Student’s t -test (* p < 0.05, *** p < 0.001, **** p < 0.0001).

Article Snippet: Madin-Darby canine kidney (MDCK) cells, human lung epithelial (A549) cells, and mouse lung epithelial cells (MLE-12) were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Over Expression, Infection, Western Blot, Expressing, Plaque Assay, Immunofluorescence, Imaging, Staining, Two Tailed Test

Journal: Viruses

Article Title: Sorting Nexin 10 Mediates Endosomal Acidification and Autophagy to Promote Influenza A Virus Infection

doi: 10.3390/v18040460

Figure Lengend Snippet: Influenza virus infection inhibited the degradation of SNX10 via the ubiquitin–proteasome pathway. ( A ) Western blot analysis of SNX10 expression in A549 cells (left) and MLE-12 cells (right) infected with PR8 (MOI = 1) for the indicated time. ( B ) Western blot analysis of SNX10 expression in A549 cells transfected with poly I:C (1 μg/mL) for the indicated time. ( C ) qRT-PCR analysis of SNX10 mRNA expression in A549 cells ( left ) and MLE12 cells ( right ) infected with PR8 (MOI = 1) for the indicated time. ( D ) Immunoprecipitation analysis of ubiquitinated SNX10 in A549 cells infected with PR8 (MOI = 1) for 20 h and treated with MG-132 (20 μM) for the final 4 h. ( E ) A549 cells were treated with CHX (100 μg/mL) for different times combined with or without MG132 (20 μM).

Article Snippet: Madin-Darby canine kidney (MDCK) cells, human lung epithelial (A549) cells, and mouse lung epithelial cells (MLE-12) were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Virus, Infection, Ubiquitin Proteomics, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Immunoprecipitation

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: High glucose stress activates the mitochondrial repressor MTERF3. A) Quantification of the MTERF3 (Mitochondrial Transcription Termination Factor 3) RNA transcript by taqman quantitative PCR in HDF grown in 5.55 mM or 12 mM glucose. Normalization of the data was performed using GusB (β-glucuronidase) (N = 3). B) Determination by Simple WES of MTERF3 protein expression in HDF cultivated 48H in DMEM with 5.55 mM or 12 mM glucose. Total protein was used for normalization. (N = 3). C) Human Reconstructed Skin (HRS) generation process. HDF were seeded on bovine collagen matrix. After 21 days, Human Equivalent Dermis (HED) was produced. Human Epidermal Keratinocytes were seeded on HED. After 38 days, HRS was produced. D) Immunofluorescence analysis of MTERF3 expression in HRS epidermis grown in 5.55 mM or 12 mM glucose. Signal intensity was quantified by measuring MTERF3 fluorescence (purple color) normalized to the number of cells, as determined by the number of nuclei (DAPI staining; blue color). 10 images were acquired per HRS (N = 3). E) Similar analysis was performed in HRS dermis. F) Determination by Simple WES of the protein expression level of various mitochondrial respiratory chain subunits in human dermal fibroblasts (HDF): complex I (NADH:Ubiquinone Oxidoreductase Subunit B8), complex II (Succinate dehydrogenase [ubiquinone] iron-sulfur subunit), complex III (Ubiquinol-Cyt C Reductase Core Protein 2), complex IV (Cyt C Oxidase Subunit 4) and complex V (ATP synthase alpha subunit) in HDF cultivated 48 h in DMEM with 5.55 mM or 12 mM of glucose. Total protein loading was used for normalization (N = 3). Similar analysis was performed in G) Human Equivalent Dermis (HED, N = 3) and H) Human Reconstructed Skin (HRS, N = 3). All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Unpaired t -test was for all panels.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Produced, Immunofluorescence, Fluorescence, Staining

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: MTERF3 ectopic overexpression represses OXPHOS. A) Proteomic study of HDF overexpressing human MTERF3 (MTERF3OE) as compared to wild-type control expressing the empty plasmid p -lenti (N = 4). B) Schematic representation of the mitochondrial proteins reduced in expression (with Adjp<0.05) as a result of the MTERF3 overexpression (MTERF3OE) in HDF. C) Ingenuity Pathway Analysis (Z-score) of the proteins significantly altered in expression, in response to MTERF3 overexpression. D) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration, non-phosphorylating respiration (oligomycin) and uncoupled respiration (CCCP) were determined in human skin fibroblasts with MTERF3 overexpression, as compared to control cells grown in 5.55 mM or 12 mM of glucose (N = 6). E) Mitochondrial morphology study of HDF overexpressing MTERF3 and control cells grown in DMEM with 5.55 mM. Mitochondrial staining was performed using 50 nM of MitoTracker Red (N = 15). Three parameters of the mitochondrial network were analyzed using Image J: particles count, tubules length and interconnections. F) Quantification of TFAM (Transcription Factor A, Mitochondrial) RNA transcript by Taqman quantitative PCR in HDF overexpressing MTERF3, as compared to plenti-control cells. Normalization of the RT-QPCR data was performed to GusB (β-glucuronidase), N = 3. G) Growth curves of HDF lenti-control and MTERF3 overexpression grown during 7 days in DMEM with 5.55 mM or 12 mM of glucose (N = 12). H) Determination by Simple WES of the protein expression level of mitochondrial respiratory chain subunits: complex I (NADH:Ubiquinone Oxidoreductase Subunit B8), complex II (Succinate dehydrogenase [ubiquinone] iron-sulfur subunit), complex III (Ubiquinol-Cyt C Reductase Core Protein 2), complex IV (Cyt C Oxidase Subunit 4) and complex V (ATP synthase alpha subunit) in HDF control and MTERF3 overexpression cultivated 48 h in DMEM with 5.55 mM or 12 mM of glucose. Protein normalization was performed using total proteins (N = 3). All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Unpaired t -test was used for panels A,E,F and H.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Over Expression, Control, Expressing, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: NR2F2/Coup-TFII glucose-dependent transcription factor inhibition upregulates MTERF3. A) Promoter region of the human MTERF3 gene and regulatory sites for transcription factors binding. Identification of NR2F2 binding site using Swiss Regulon (Expasy). B) Quantification of NR2F2 (Nuclear Receptor Subfamily 2 Group F Member 2) RNA transcript by taqman quantitative PCR in HDF cultivated in DMEM with 5.55 mM, 12 mM or 25 mM of glucose. Normalization of the data was performed to GusB (β-glucuronidase) (N = 3). C) MTERF3 promoter reporter activity assay using a Gaussia-luciferase (Gluc) lentiviral approach. D) Determination by bioluminescence of the MTERF3 promoter activity in HDF with lentiviral transduction of the reporter. Fibroblasts were grown 24 h in DMEM with 5.55 mM,12 mM or 25 mM of glucose, or 5.55 mM of galactose (N = 4). Data were normalized to the number of cells. E) Dose-dependent relationships between MTERF3 promoter activation and glucose concentration in the cell culture medium. F) Quantification of NR2F2 mRNA transcript by taqman quantitative PCR in HDF transfected with esiControl and esiNR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. G) MTERF3 promoter activity determination in HDF transfected with esiNR2F2 (N = 4). H) Quantification of MTERF3 mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2R2. Normalization of data to GusB (β-glucuronidase), N = 3. I) Determination by Simple WES of MTERF3 protein expression level in HDF transfected with esiNR2F2. Protein normalization was performed to total protein loading (N = 3). J) Schematic representation of lentiviral CRISPR guide RNA for the generation of NR2F2 knock-out. K) Quantification of NR2F2 mRNA transcripts by taqman quantitative PCR in stable HDF using sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. L) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration, non-phosphorylating respiration (oligo) and uncoupled respiration (CCCP) were determined in HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. M) Quantification of RNA transcripts by taqman quantitative PCR of COQ9 (coenzyme Q9), TFAM (Transcription Factor A, Mitochondrial) and MT-RNR1 (Mitochondrially Encoded 12S RRNA), in stable HDF sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of data to GusB (β-glucuronidase), N = 3. N) Mitochondrial morphology of HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Imaging was performed using 50 nM of MitoTracker Red (N = 40). O) Schematic representation of the impact of high(12 mM)-glucose medium on NR2F2, MYCN, MTERF3 and OXPHOS. P) Quantification of MTERF3 mRNA transcript by taqman quantitative PCR in HDF transfected with esiMYCN. Normalization of the data to GusB (β-glucuronidase), N = 3. Q) MTERF3 promoter activity assay in HDF transfected with esiMYCN (N = 4). R) Determination by Simple WES of the MTERF3 protein expression level in HDF transfected with esiMYCN (N = 4). All data were expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel B, D, K, L, M and N. Unpaired t -test was used for panels F, G, H, I, P, Q and R.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Inhibition, Binding Assay, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Transduction, Activation Assay, Concentration Assay, Cell Culture, Transfection, Expressing, CRISPR, Knock-Out, Imaging

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: cFOS and NR2F2 transcription factors mediate glucose-dependent repression of GDF15 in human dermis. A) Determination by Simple WES of the protein expression level of proGDF15 in wild-type HDF expressing shcontrol or shGDF15 (N = 3). B) Expression of GDF15 in the skin from human protein expression atlas from EMBL-EBI ( https://www.ebi.ac.uk ) which includes RNA-seq analyses from tissue samples of 122 human individuals, representing 32 different tissues. The results are expressed as TPM (Transcripts Per Kilobase Million). C) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF, A549 and HEPG2 (N = 3). D) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF, 786-O and SN005 cells (N = 3). E) ELISA-Based Quantification of GDF15 secretion in HDFs. HDFs were cultured under conditions of normal (5.5 mM) and high (12 mM) glucose concentrations during 48h. GDF15 levels in the culture supernatants were quantified using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer's instructions. F) Quantification of GDF15 mRNA transcripts by taqman quantitative PCR in HDF grown in 5.55 mM, 12 mM or 25 mM glucose (N = 3). G) GDF15 promoter activity in HDF grown 24 h in DMEM with 5.55 mM,12 mM or 25 mM of glucose or 5.55 mM of galactose (N = 4). H) Dose-dependent relationship between GDF15 promoter activation and glucose concentration in the medium. I) GDF15 gene promoter sequence with identification of the binding site for the FOS transcription factor (Swiss Regulon Expasy). J) Quantification of FOS mRNA transcript by taqman quantitative PCR in HDF cultivated in 5.55 mM or 12 mM glucose (N = 3). K) Determination by Simple WES of FOS protein expression level in HDF cultivated in 5.55 mM or 12 mM glucose (N = 3). L-M) Determination by Simple WES of GDF15 protein expression level in HDF transfected with esiFOS (N = 3). N) Quantification of GDF15 mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2F2 (N = 3). O) Quantification of GDF15 mRNA transcript by taqman quantitative PCR in HDF expressing sgControl, sgRNA 1 targeting NR2F2 and sgRNA2 targeting NR2F2. Normalization of the data to GusB (β-glucuronidase), N = 3. P–S) Quantification of ATF3, ATF4, CHOP and P53 mRNA transcripts by taqman quantitative PCR in HDF expressing siCTRL and esiNR2F2 in 5.5 mM glucose or 12 mM glucose growth medium. Normalization of the data to GusB (β-glucuronidase), N = 3. T) Schematic representation of the NR2F2-MTERF3-GDF15 axis and its control on OXPHOS function in response to glucose stress. All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel C, E and N. Unpaired t -test was used for panels A, B, H, I, J, K, L and M.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Activation Assay, Concentration Assay, Sequencing, Binding Assay, Transfection, Control

Journal: Redox Biology

Article Title: Repression of oxidative phosphorylation by NR2F2, MTERF3 and GDF15 in human skin under high-glucose stress

doi: 10.1016/j.redox.2025.103613

Figure Lengend Snippet: GDF15 inhibition by hyperglycemia or shRNA alters mitochondrial biogenesis. A) Metabolomic profile of HDF grown 48H in DMEM with 5.55 mM or 12 mM + 100 nM GDF15 of glucose and HDF expressing a shGDF15 cultivated in 5.55 mM glucose (N = 3). B) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration and uncoupled respiration (CCCP) were determined in HDF grown in 5.55 mM glucose and HDF expressing shGDF15 grown in 5.55 mM glucose or 5.55 mM glucose supplemented with 100 nM gdf15. C) Oxygen consumption rate (OCR) was measured using the Seahorse XFe96. Routine respiration and uncoupled respiration (CCCP) were determined in HDF grown in 5.55 mM glucose and supplemented with low doses of gdf15: 20pM, 80pM, 1 nM, 10 nM and 100 nM. D) Mitochondrial respiratory chain proteins (gene loci) specifically activated at the level of chromatin accessibility by GDF15 100 nM. E-H) Quantification of mRNA transcripts by taqman quantitative PCR for NR2F2, GDF15, MTERF3 and TFAM in HDF cultivated with 5.55 mM glucose or 5.55 mM glucose supplemented with 100 nM gdf15. Normalization of the data was performed to GusB (β-glucuronidase), N = 3. I-L) Quantification of mRNA transcripts by taqman quantitative PCR for GDF15, MAPK1, MAPK3, PGC1α (Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha) in HDF cultivated with 5.55 mM glucose supplemented with low doses of gdf15. Normalization of the data was performed to GusB (β-glucuronidase), N = 3 M) Quantification of TFAM mRNA transcripts by taqman quantitative PCR in HDF cultivated with 5.55 mM, 12 mM or 25 mM glucose or in HDF expressing shGDF15 grown in 5.55 mM glucose. Normalization of data was performed to GusB (β-glucuronidase), N = 3. N) Quantification of PGC1α mRNA transcript by taqman quantitative PCR in HDF cultivated with 5.55 mM, 12 mM or 25 mM glucose or in HDF expressing shGDF15 grown in 5.55 mM glucose. Normalization of the data to GusB (β-glucuronidase), N = 3. O) Quantification of PGC1α mRNA transcript by taqman quantitative PCR in HDF transfected with esiNR2F2. Normalization of data to GusB (β-glucuronidase), N = 3. P,Q) Quantification of the total Coenzyme Q10 (oxidized and reduced forms) in HDF cultivated with 5.55 mM, 12 mM or 12 mM glucose medium supplemented with 100 nM gdf15. Analysis was also performed in HDF expressing shGDF15 in 5.55 mM glucose. R) Quantification of TFAM in HDF cultivated with 5.55 mM glucose medium or medium supplemented with 20pM, 100 pM and 100 nM rGDF15. S) Summary of the regulatory network linking TFAM, PGC1α, COQ9 and COQ10. All data are expressed as the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Ordinary one-way ANOVA with Dunett's test correction was used for panel A, B, F, G, I-M, P–R. Unpaired t -test was used for panels E, H and O.

Article Snippet: Expression plasmids in lentiviral vectors were purchased for MTERF3 Human Tagged ORF Clone (#RC201030L4, Origene) and GDF15 Human Tagged ORF Clone (#RC201295L2, Origene).

Techniques: Inhibition, shRNA, Expressing, Real-time Polymerase Chain Reaction, Transfection

Journal: Nature cell biology

Article Title: ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signaling in Niemann-Pick type C

doi: 10.1038/s41556-019-0391-5

Figure Lengend Snippet: a , In situ labeling assay for detecting lysosomal membrane cholesterol. b , Cholesterol is deposited at the lysosomal membrane in human NPC1 patient-derived fibroblasts. Control (NPC1 WT ) and NPC1 (NPC1 mut ) fibroblasts were fixed, breached with LN 2 pulse, subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for LAMP2. Scale bar, 10 μm. c , NPC1 deletion via CRISPR/Cas9 genome editing in cells results in cholesterol accumulation at the lysosomal membrane. NPC1-deleted HEK-293T cells expressing FLAG-GFP-Tmem192, either naïve or reconstituted with FLAG-tagged NPC1, were processed for cholesterol labeling. Scale bar, 10 μm. Insets show three examples per genotype. d , Proteomic analysis of affinity-purified lysosomes. Lysosomes were immunopurified by anti-FLAG M2 beads (or anti-HA magnetic beads) from HEK-293T cells expressing LAMP1-mRFP-2xFLAG or Tmem192-mRFP-3xHA and analyzed by mass spectrometry. Unique peptide counts for identified ORPs shown here ( n = 2 independent experiments, 5 biologically independent samples in total). See . e , Pull-down of lysosomes revealing the lysosomal association of ORPs. Lysosomes were purified by anti-HA beads and immunoblotted for the indicated proteins. f , Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in NPC1-null cells depleted of ORPs (box plots showing the min, 1 st quartile, median, 3 rd quartile, and max, 10 fields of view per genotype; n represents cell number: shLuc ( n = 13), shOR8 ( n = 12), shORP11 ( n = 12), shOSBP ( n = 11), shORP5 ( n = 10), ANOVA with Dunnett’s multiple comparison test. ****Adjusted P = 0.0001 vs. NPC1 mut -shLuc). See . g and h , Concomitant depletion of OSBP in NPC1-null cells reduces lysosomal membrane cholesterol levels. Cells were depleted of OSBP via doxycycline-induced shRNA and processed for cholesterol labeling. Scale bar, 10 μm. Insets show three examples per genotype. Quantitation in ( g ) (box plots as in ( f ), 10 fields of view per group; n represents cell number: – Dox ( n = 64), + Dox ( n = 72), two-tailed, unpaired t-test. **** P = 1.16578 × 10 −13 vs. – Dox group). Experiments in b, c performed three times and in e , g two times.

Article Snippet: In cases where indicated, a doxycycline-inducible shRNA system via Tet-pLKO-puro (Addgene #21915) was used to silence OSBP using doxycycline.

Techniques: In Situ, Labeling, Membrane, Derivative Assay, Control, Staining, CRISPR, Expressing, Affinity Purification, Magnetic Beads, Mass Spectrometry, Purification, Quantitation Assay, Comparison, shRNA, Two Tailed Test

Journal: Nature cell biology

Article Title: ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signaling in Niemann-Pick type C

doi: 10.1038/s41556-019-0391-5

Figure Lengend Snippet: a , Depletion of OSBP results in accumulation of PI4P at the lysosomes. HEK-293A cells stably expressing the PI4P-specific probe, GFP-P4M, along with LAMP1-mRFP were depleted of OSBP via doxycycline-induced shRNA. Cells were plated on glass-bottom dishes, allowed to attach overnight and imaged live on a spinning disk confocal microscope. Representative microscopic images are shown. Scale bar, 10 μm. b , Confirmation of lysosomal localization of the lysosome-targeted Sac1 catalytic domain truncations in HEK-293A cells. ( left ) Schematic diagram showing Sac1 catalytic domain is targeted to the lysosome to hydrolyze its target PI4P. ( right ) Representative microscopic images are shown. Scale bar, 10 μm. c , Ectopic expression of a lysosome-targeted Sac1 catalytic domain eliminates excess lysosomal PI4P in OSBP-depleted cells. HEK-293T cells co-expressing mCherry-P4M and either catalytically active (WT) or inactive (CS) forms of lyso-Sac1 were depleted of OSBP and subjected to live-cell imaging. Representative confocal microscopic images are shown. Scale bar, 10 μm. d , Lysosomal PI4P buildup caused by OSBP depletion is not responsible for mTORC1 inhibition. Control cells and cells expressing catalytically active and inactive lyso-Sac1 were depleted of OSBP, followed by cholesterol starvation/restimulation, lysis and immunoblotting for the indicated proteins and phospho-proteins. See unprocessed blots in . Experiments in a – d repeated independently two times.

Article Snippet: In cases where indicated, a doxycycline-inducible shRNA system via Tet-pLKO-puro (Addgene #21915) was used to silence OSBP using doxycycline.

Techniques: Stable Transfection, Expressing, shRNA, Microscopy, Live Cell Imaging, Inhibition, Control, Lysis, Western Blot

Journal: Nature cell biology

Article Title: ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signaling in Niemann-Pick type C

doi: 10.1038/s41556-019-0391-5

Figure Lengend Snippet: a , VAPA/B depletion abolishes the lysosomal recruitment of mTORC1 by cholesterol. HEK-293T cells were depleted for VAPA and VAPB via shRNA and evaluated for mTOR lysosomal localization. Scale bar, 10 μm. b , Quantitation of co-localization between mTOR and LAMP2-positive lysosomes in control cells and cells depleted of VAPA and B (mean ± s.d., 10 fields of view per genotype/condition; n represents cell number: shLuc – chol ( n = 124), shLuc + chol ( n = 74), shVAPA/VAPB – chol ( n = 178), shVAPA/VAPB + chol ( n = 163), ANOVA with Dunnett’s multiple comparison test. **** P = 0.0001 vs. shLuc + chol). c , VAPA and VAPB are necessary for cholesterol-dependent mTORC1 activation. HEK-293T cells were depleted of VAPA and VAPB, either alone or in combination, and evaluated for sterol-induced mTORC1 signaling via immunoblotting. d and e , STARD3 and ORP1L are not required for cholesterol-dependent mTORC1 signaling. Cells depleted of STARD3 or OSBP via shRNA ( d ) and ORP1L knockout cells ( e ) were assayed for sterol-induced mTORC1 activation via immunoblotting. f , Depletion of STARD3 or ORP1L has no effects on both peripheral and internal pools of lysosomal cholesterol in NPC1-deficient cells. Human NPC1 patient-derived fibroblasts depleted of STARD3 or OSBP via shRNA or deleted of ORP1L via CRISPR/Cas9 genome editing were subjected to cholesterol labeling by GST-D4H*-mCherry and filipin, and stained for endogenous LAMP2. Scale bar, 10 μm. g , Quantitation of co-localization of D4H*-mCherry with filipin-labeled cholesterol deposits in NPC1-null cells that were silenced of STARD3, OSBP, and ORP1L, respectively (box plots showing the min, 1 st quartile, median, 3 rd quartile, and max, 10 fields of view per genotype; n represents cell number: shLuc ( n = 12), shSTARD3 ( n = 10), shOSBP ( n = 10), sgORP1L ( n = 11), ANOVA with Dunnett’s multiple comparison test. ****Adjusted P = 0.0001 vs. NPC1 mut -shLuc). Experiments in c – e performed independently two times with similar results. See unprocessed blots in . Statistics source data are provided in .

Article Snippet: In cases where indicated, a doxycycline-inducible shRNA system via Tet-pLKO-puro (Addgene #21915) was used to silence OSBP using doxycycline.

Techniques: shRNA, Quantitation Assay, Control, Comparison, Activation Assay, Western Blot, Knock-Out, Derivative Assay, CRISPR, Labeling, Staining

Journal: Cell systems

Article Title: tRNA Methylation Is a Global Determinant of Bacterial Multi-Drug Resistance

doi: 10.1016/j.cels.2019.03.008

Figure Lengend Snippet: A) Quantitative mass spectrometry analysis of membrane proteins in E. coli trmD-KD cells isolated from Ara− and Ara+ conditions. The label-free quantification intensity is compared to the signal of log2 (fold-change) (Ara−/Ara+). OM proteins are plotted in black with a vertical line indicating the median of −0.33 (equivalent to a decrease of 21%), while non-OM proteins are plotted in blue with a vertical line showing the median of 0.22 (equivalent to an increase of 16%). p < 0.001 by a Kolmogorov-Smirnov analysis.

Article Snippet: Escherichia coli strain K-12 substrain MG1655 , ATCC , 700926.

Techniques: Mass Spectrometry, Membrane, Isolation, Quantitative Proteomics

Journal: Cell systems

Article Title: tRNA Methylation Is a Global Determinant of Bacterial Multi-Drug Resistance

doi: 10.1016/j.cels.2019.03.008

Figure Lengend Snippet: A,B) E. coli (A) and Salmonella (B) trmD-KD cells in m1G37 deficiency (m1G37−) show increased membrane permeability relative to m1G37+ cells. Cells were grown as in Figure 2C and the intracellular accumulation of AlamarBlue in m1G37+ (Ara+, blue) and m1G37-deficient (Ara−, red) conditions was monitored in the presence of CCCP. Levels of intracellular dye accumulation were normalized by OD600. Data and error bars are mean ± SD, n = 3.

Article Snippet: Escherichia coli strain K-12 substrain MG1655 , ATCC , 700926.

Techniques: Membrane, Permeability

Journal: Cell systems

Article Title: tRNA Methylation Is a Global Determinant of Bacterial Multi-Drug Resistance

doi: 10.1016/j.cels.2019.03.008

Figure Lengend Snippet: A,B) m1G37− cells had at least 2-fold lower MICs than m1G37+ cells. The fold-decrease in MIC of each antibiotic was calculated for E. coli (A) and Salmonella (B) trmD-KD cells as the ratio of the MIC in m1G37+ and m1G37− cells (red) and was compared with the relative decrease of m1G37+ cells upon treatment with polymyxin B (PMB) at 0.25X MIC (blue). Overnight cultures were inoculated into fresh LB at 106 CFUs/mL and incubated with an antibiotic in serial dilutions. After 18 h of incubation at 37 °C, cell densities lower than OD600 = 0.15 were scored as no growth. Fold-changes are taken from Figure S6A, where data and errors are mean ± SD, n > 4. Amp, ampicillin; Cbc, carbenicillin; Rif, rifampicin; Kan, kanamycin; Gen, gentamicin; Par, paromomycin; Cip, ciprofloxacin; Van, vancomycin.

Article Snippet: Escherichia coli strain K-12 substrain MG1655 , ATCC , 700926.

Techniques: Incubation

Journal: Cell systems

Article Title: tRNA Methylation Is a Global Determinant of Bacterial Multi-Drug Resistance

doi: 10.1016/j.cels.2019.03.008

Figure Lengend Snippet: A-C) Resistance arises less frequently in m1G37− (red) E. coli (A) and Salmonella (B) trmDKD cells than in m1G37+ (blue) cells. An overnight culture of cells at 105 CFUs was plated onto an LB agar plate containing the indicated concentration of gentamicin (Gen), kanamycin (Kan), ampicillin (Amp), or vancomycin (Van). Each concentration was near 1X MIC for m1G37+ cells. Resistant colonies were counted after incubation at 37 °C for 3 days. Mutants were verified to have an increase in MIC to the respective antibiotic (C). Data and error bars are mean ± SD, n = 3. Welch’s t-test: *p < 0.1, **p < 0.05, ***p < 0.01.

Article Snippet: Escherichia coli strain K-12 substrain MG1655 , ATCC , 700926.

Techniques: Concentration Assay, Incubation

Journal: Cell systems

Article Title: tRNA Methylation Is a Global Determinant of Bacterial Multi-Drug Resistance

doi: 10.1016/j.cels.2019.03.008

Figure Lengend Snippet: A) Western blot analysis showed that m1G37− (red) E. coli trmD-KD cells had lower lolB expression relative to cysS from the native gene than m1G37+ (blue) cells, but higher expression from the codon-engineered gene. Data and error bars are mean ± SD, n = 6. Welch’s t-test: **p < 0.05.

Article Snippet: Escherichia coli strain K-12 substrain MG1655 , ATCC , 700926.

Techniques: Western Blot, Expressing

Journal: Cell systems

Article Title: tRNA Methylation Is a Global Determinant of Bacterial Multi-Drug Resistance

doi: 10.1016/j.cels.2019.03.008

Figure Lengend Snippet: Key Resources Table

Article Snippet: Escherichia coli strain K-12 substrain MG1655 , ATCC , 700926.

Techniques: Virus, Recombinant, Plasmid Preparation, Purification, Software, Mass Spectrometry, Targeted Proteomics, Membrane, Imaging, Fluorescence, Microscopy

Journal:

Article Title: Isolated left ventricular non-compaction: cardiomyopathy with homogeneous transmural and heterogeneous segmental perfusion

doi:

Figure Lengend Snippet: Quantitative contrast echocardiography showing segmental heterogeneous segmental perfusion (higher perfusion in the middle septal segment shown by blue and yellow curves in comparison with the lateral wall shown by the green curve; ventricular cavum is indicated by the red curve) and transmural homogeneous perfusion (no significant differences between the septal positions: endocardial layer indicated by the blue curve and outer layer by the yellow curve).

Article Snippet: Myocardial contrast echocardiography (Vived FiVe, GE Vingmed Ultrasound, Horten, Norway; echo contrast: infusion of 3 ml Optison over four minutes; real time perfusion imaging: quantitative measurement, mechanical index 0.12) showed segmental heterogeneous segmental perfusion (higher perfusion in the middle septal segment than in the lateral wall) and transmural homogeneous perfusion (fig 2 ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 Quantitative contrast echocardiography showing segmental heterogeneous segmental perfusion (higher perfusion in the middle septal segment shown by blue and yellow curves in comparison with the lateral wall shown by the green curve; ventricular cavum is indicated by the red curve) and transmural homogeneous perfusion (no significant differences between the septal positions: endocardial layer indicated by the blue curve and outer layer by the yellow curve).

Techniques: Comparison

Journal: Journal of Molecular Cell Biology

Article Title: Vertebrate Dynein-f depends on Wdr78 for axonemal localization and is essential for ciliary beat

doi: 10.1093/jmcb/mjy043

Figure Lengend Snippet: Wdr78 is located in motile cilia. ( A ) Wdr78 was highly expressed in motile cilia-containing tissues, including trachea, lung, oviduct, and testis. Cultured IMCD3 and NIH3T3 cells were serum-starved for 48 h to induce primary cilia. Gapdh served as loading control. ( B ) Expression profile of Wdr78 during multiciliogenesis. qRT-PCR was performed using mRNAs extracted from mTECs cultured at an ALI for the indicated days (d). Plk4 and Deup1 served as markers for centriole amplification, whereas Wdr63 and Dnai2 served as markers for multiciliogenesis. The results were representative of two independent sets of experiments. ( C ) Protein levels of Wdr78 during the multiciliogenesis of mTECs. The increased levels of Ift80 and acetylated tubulin (AC-tub) indicate multiciliogenesis. Plk4 and Gapdh served as marker for centriole amplification and loading control, respectively. ( D ) Wdr78 localized in the axonemes of mTEC cilia. mTECs were fixed at ALI d7 and stained with anti-Wdr78 antibody preincubated with or without the Wdr78 peptide that was used for antibody generation. AC-tub marks ciliary axonemes. Nuclear DNA was stained with DAPI. ( E ) Wdr78 was not a primary ciliary protein. NIH3T3 cells were serum-starved for 48 h to induce primary ciliary formation. AC-tub marks ciliary axonemes. Nuclear DNA was stained with DAPI.

Article Snippet: Commercial rabbit antibodies used were Flag (Sigma, F7425), Ift80 (GeneTex, GTX109393), Ift52 (Proteintech, 17534-1-AP), Rsph3 (Proteintech, 17603-1-AP), GFP (Life Technologies, A6455), Wdr63 (GeneTex, GTX45697), Lamin B1 (Proteintech,12987-1-AP), and Gapdh (Proteintech, 10494-1-AP).

Techniques: Cell Culture, Control, Expressing, Quantitative RT-PCR, Amplification, Marker, Staining

Journal: Journal of Molecular Cell Biology

Article Title: Vertebrate Dynein-f depends on Wdr78 for axonemal localization and is essential for ciliary beat

doi: 10.1093/jmcb/mjy043

Figure Lengend Snippet: Vertebrate Dynein-f is critical for ciliary beat. ( A ) Experimental scheme for RNAi in mEPCs. Glial cells from dissected mouse telencephalon tissues were induced to differentiate into multiciliated mEPCs through serum starvation at day 0 (SS d0). The cells were transfected three times with siRNA. ( B and C ) Efficient knockdown of Wdr78 by RNAi. Gapdh and AC-tub served as loading control and ciliary marker, respectively. ( D and E ) Depletion of Wdr78 paralyzed ependymal cilia. Ciliary movements were monitored by bright-field microscopy. ( D ) Trajectories of four cilia during the first 80 ms of imaging are shown for each mEPC. Please also refer to . ( F–H ) GFP-Wdr78, but not Centrin1-GFP, restored ciliary motility in 78-i2-treated mEPCs. In addition to the transfections with 78-i2 in A , the cells were also infected with lentivirus at SS d–2 to express an siRNA-resistant GFP-Wdr78 or Centrin1-GFP. ( F ) The ciliary localization of GFP-Wdr78 was obvious in fixed mEPCs. ( G ) Due to the difficulty to clearly capture the fluorescent images of rapidly beating cilia, live GFP-positive cells were identified by the fluorescence in their cell bodies. Please refer to . ( I–K ) Depletion of Dnah2 also caused immobile cilia in mEPCs. Please refer to . Quantification results in E , H , and K were from three independent experiments and presented as mean ± SD. Cell numbers analyzed are listed over each histogram. Student’s t- test: n.s., not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Commercial rabbit antibodies used were Flag (Sigma, F7425), Ift80 (GeneTex, GTX109393), Ift52 (Proteintech, 17534-1-AP), Rsph3 (Proteintech, 17603-1-AP), GFP (Life Technologies, A6455), Wdr63 (GeneTex, GTX45697), Lamin B1 (Proteintech,12987-1-AP), and Gapdh (Proteintech, 10494-1-AP).

Techniques: Transfection, Knockdown, Control, Marker, Microscopy, Imaging, Infection, Fluorescence