Journal: Scientific Reports

Article Title: A digital plating platform for robust and versatile microbial detection and analysis

doi: 10.1038/s41598-025-11525-6

Figure Lengend Snippet: Isolation and cultivation of bacterial cells from a microbial mixture consisting of S. aureus and GFP-expressing E. coli with the DP platform. (A) Typical bright-field, fluorescence, and merged microimages of microcolonies grown in a PicoArray device after 8 h of incubation at 37 °C. Initial concentrations of S. aureus and E. coli ~ 6 × 10 4 CFU/mL. (B) Enumeration of S. aureus and GFP-expressing E. coli in the microbial mixture using the DP platform vs. the agar plating.

Article Snippet: The bacterial species used in this work were Escherichia coli ( E. coli ) JM109, green fluorescent protein (GFP)-tagged E. coli BL21, Staphylococcus aureus ( S. aureus ) ATCC 43,300, and Salmonella enterica 14,028, which were purchased from China General Microbiological Culture Collection Center.

Techniques: Isolation, Expressing, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: A digital plating platform for robust and versatile microbial detection and analysis

doi: 10.1038/s41598-025-11525-6

Figure Lengend Snippet: Enrichment and identification of bacteria in complex samples by coupling the DP with selective and differential medium. ( A ) Enrichment of S. aureus from a microbial mixture consisting of S. aureus and GFP-expressing E. coli by coupling the DP platform with a NaCl-based selective medium. (i) Representative bright-field, fluorescence, and merged microimages of microcolonies formed in two PicoArray devices after being covered with LB medium agar sheet and selective medium agar sheet (incubated at 37 ºC for 8 h), respectively. Initial concentrations of S. aureus and E. coli ~ 3 × 10 5 CFU/mL and ~ 5 × 10 4 CFU/mL (ii) Enumeration of the fluorescent and non-fluorescent microcolonies formed in the PicoArray devices with LB medium agar sheet and selective medium agar sheet, respectively. ( B ) Representative bright-field microimages of microcolonies formed in three PicoArray devices after being loaded with S. aureus , E. coli , and S. aureus / E. coli mixture and incubated with MCA sheets at 37 ºC for 8 h.

Article Snippet: The bacterial species used in this work were Escherichia coli ( E. coli ) JM109, green fluorescent protein (GFP)-tagged E. coli BL21, Staphylococcus aureus ( S. aureus ) ATCC 43,300, and Salmonella enterica 14,028, which were purchased from China General Microbiological Culture Collection Center.

Techniques: Bacteria, Expressing, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: A digital plating platform for robust and versatile microbial detection and analysis

doi: 10.1038/s41598-025-11525-6

Figure Lengend Snippet: Phenotyping antibiotic resistance using the DP platform. ( A ) Representative bright-field microimages showing E. coli microcolonies grown in the PicoArray chips under different concentrations of ampicillin sodium after 6 h of incubation at 37 ºC. Initial concentration of E. coli ~ 1 × 10 5 CFU/mL. ( B ) Dose response curve for E. coli viability after treatment with different concentrations of ampicillin sodium. Calculation of MIC using a Gompertz function fit. Blue vertical dashed line shows the position of the MIC. Each test was performed in triplicate ( n = 3). Error bars represent standard deviation (SD). ( C ) Representative bright-field microimages showing the morphology changes of E. coli under antibiotic stress.

Article Snippet: The bacterial species used in this work were Escherichia coli ( E. coli ) JM109, green fluorescent protein (GFP)-tagged E. coli BL21, Staphylococcus aureus ( S. aureus ) ATCC 43,300, and Salmonella enterica 14,028, which were purchased from China General Microbiological Culture Collection Center.

Techniques: Incubation, Concentration Assay, Standard Deviation

Journal: eLife

Article Title: Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

doi: 10.7554/eLife.70090

Figure Lengend Snippet: ( A ) ( Top ) Design of experiment monitoring promoter unwinding in real time. Grey, RNAP; orange, RNAP clamp; purple dot, RNAP active-centre; black, ds-DNA; blue, ss-DNA; light green, Cy3 on ds-DNA; dark green, Cy3 on ss-DNA. ( Bottom ) A cropped area (0.94 μm × 1.034 μm) of the field of view, showing appearance and enhancement of fluorescence signal from binding of single Cy3-labelled promoter fragment to an immobilised RNAP molecule. ( B ) ( Left ) Time trajectories of intensity from Cy3 on downstream segment of promoter bubble. Black, raw intensity; dark blue, idealised intensity; hidden Markov model (HMM)-assigned states: no promoter (black bars), closed promoter (light yellow bars) and open promoter (green bars). Frame rates: 50 ms, top and middle; 200 ms, bottom. Laser powers: 0.60 mW, top and middle; 0.15 mW, bottom. ( Right ) Dwell-time histograms of promoter state before unwinding, t UNWIND . ( C ) ( Left ) Time trajectories of intensity from Cy3 on upstream segment of promoter bubble. Colours as in B. Frame rates: 50 ms. Laser powers: 0.6 mW. ( Right ) Dwell-time histograms of promoter state before unwinding, t UNWIND . ( D ) Table comparing unwinding times for different promoter constructs. Figure 1—source data 1. Data for single-molecule UIFE experiments in .

Article Snippet: For experiments in and , hexahistidine-tagged E. coli RNAP holoenzyme was prepared using co-expression of genes encoding RNAP β', β, α, ω, and σ 70 subunits to afford an RNAP σ 70 holoenzyme derivative as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pV10 ( ) and plasmid pRSFduet-sigma ( ) were used to inoculate 20 ml LB broth ( ) containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultures were incubated 16 hr at 37°C with shaking.

Techniques: Fluorescence, Binding Assay, Construct

Journal: eLife

Article Title: Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

doi: 10.7554/eLife.70090

Figure Lengend Snippet: ( A ) Raw images (field of view: 24 μm × 24 μm) of immobilised hexahistidine-tagged RNAP holoenzyme ( left ), immobilised hexahistidine-tagged RNAP holoenzyme bound to lacCONS-[+ 2Cy3] promoter fragment before ( middle ) and after ( right ) addition of heparin. ( B ) Mean number of localisations per field of view for single lacCONS-[+ 2Cy3] promoter fragments bound to immobilised hexahistidine-tagged RNAP holoenzyme in absence and presence of heparin. ( C ) Mean number of localisations per field of view for single non-promoter-[Cy3] fragments bound to immobilised hexahistidine-tagged RNAP holoenzyme in absence and presence of heparin. ( D ) Mean number of localisations per field of view for single Cy3-labelled promoter fragments bound to immobilised hexahistidine-tagged RNAP core enzyme in absence and presence of heparin. Mean number of localisations are average of three measurements. Errors bars represent the standard deviation from the mean.

Article Snippet: For experiments in and , hexahistidine-tagged E. coli RNAP holoenzyme was prepared using co-expression of genes encoding RNAP β', β, α, ω, and σ 70 subunits to afford an RNAP σ 70 holoenzyme derivative as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pV10 ( ) and plasmid pRSFduet-sigma ( ) were used to inoculate 20 ml LB broth ( ) containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultures were incubated 16 hr at 37°C with shaking.

Techniques: Standard Deviation

Journal: eLife

Article Title: Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

doi: 10.7554/eLife.70090

Figure Lengend Snippet: ( A ) Binding of lacCONS-[+ 2 Cy3] promoter fragment to immobilised RNAP holoenzyme. ( B ) ( Top ) Stalled initial transcribing complexes (RPitc <11) formed with RNAP holoenzyme, lacCONS-[+ 2 Cy3] and subset of NTPs (ATP, GTP, and UTP). (Bottom) Real-time addition of NTPs (GTP and CTP) to stalled initial transcribing complexes (Rpitc <11). ( C ) Binding of non-promoter-[Cy3] fragment to immobilised RNAP holoenzyme. ( D ) Binding of lacCONS-[+ 2 Cy3] promoter fragment to immobilised RNAP core enzyme. Black, raw intensity; dark blue, idealised intensity; hidden Markov model (HMM)-assigned states: no promoter (black bars), closed promoter (light yellow bars), and open promoter (green bars). Frame duration: 50 ms; laser power: 0.60 mW.

Article Snippet: For experiments in and , hexahistidine-tagged E. coli RNAP holoenzyme was prepared using co-expression of genes encoding RNAP β', β, α, ω, and σ 70 subunits to afford an RNAP σ 70 holoenzyme derivative as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pV10 ( ) and plasmid pRSFduet-sigma ( ) were used to inoculate 20 ml LB broth ( ) containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultures were incubated 16 hr at 37°C with shaking.

Techniques: Binding Assay

Journal: eLife

Article Title: Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

doi: 10.7554/eLife.70090

Figure Lengend Snippet: ( A ) Raw images showing field of view (24 μm × 24 μm) with immobilised biotin promoter fragment bound to labelled RNAP complexes formed in presence of Myx ( left ), and with remaining immobilised hexahistidine-tagged RNAP holoenzyme complexes bound to Cy3-labelled promoter fragment, formed in presence of Myx, after addition of heparin ( right ). ( B ) Mean number of localisations per field of view for single Cy3-labelled promoter fragments bound to immobilised hexahistidine-tagged RNAP holoenzyme, formed in presence of Myx, before and after addition of heparin. Mean number of localisations are average of three measurements. Error bars represent the standard deviation from the mean. ( C ) Hidden Markov model (HMM)-assigned histograms and Gaussian fits of fluorescence intensities corresponding to pre-unwinding (yellow) and post-unwinding (green) states for promoter fragments with Cy3 on +2 position of the non-template strand ( top ) or –9 position of the template strand ( bottom ) of the promoter bubble. Mean intensities for the two states and fold-increase of intensities are shown ( inset ). ( D ) Histogram and Gaussian fit of E* showing mean E* values for clamp conformational state for clamp-labelled RNAP molecules bound to immobilised lacCONS-promoter fragments in presence of Myx.

Article Snippet: For experiments in and , hexahistidine-tagged E. coli RNAP holoenzyme was prepared using co-expression of genes encoding RNAP β', β, α, ω, and σ 70 subunits to afford an RNAP σ 70 holoenzyme derivative as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pV10 ( ) and plasmid pRSFduet-sigma ( ) were used to inoculate 20 ml LB broth ( ) containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultures were incubated 16 hr at 37°C with shaking.

Techniques: Standard Deviation, Fluorescence

Journal: eLife

Article Title: Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

doi: 10.7554/eLife.70090

Figure Lengend Snippet: ( A ) Design of experiment monitoring clamp status in real time. Black, ds-DNA; orange, RNAP clamp; grey, rest of RNAP; purple dot, RNAP active-centre; blue, ss-DNA; green, Cy3B; and red, Alexa647. ( B ) Representative time trajectories of E* for experiments with a lacCONS promoter fragment, showing hidden Markov model (HMM)-assigned closed-clamp state (orange), locked-clamp state (red), and interstate transition (dark-blue). Expected range of E* values for an open-clamp state is highlighted in light blue. Frame rate: 100 ms. Laser powers: 200 μW in red and 500 μW in green. ( C ) HMM-assigned histograms and Gaussian fits of E* for full-time trajectories from experiments with a lacCONS promoter fragment. ( D ) Dwell-time histograms of time before transition to the locked-clamp state, t LOCK , for experiments with a lacCONS promoter fragment. ( E ) Table showing mean E*; difference in E* (ΔE*) between closed-clamp or locked-clamp states and time to transition to a locked-clamp state after initial binding for the lacCONS and lacCONS-GC promoter fragments. Figure 3—source data 1. Data for single-molecule FRET experiments in .

Article Snippet: For experiments in and , hexahistidine-tagged E. coli RNAP holoenzyme was prepared using co-expression of genes encoding RNAP β', β, α, ω, and σ 70 subunits to afford an RNAP σ 70 holoenzyme derivative as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pV10 ( ) and plasmid pRSFduet-sigma ( ) were used to inoculate 20 ml LB broth ( ) containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultures were incubated 16 hr at 37°C with shaking.

Techniques: Binding Assay

Journal: eLife

Article Title: Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

doi: 10.7554/eLife.70090

Figure Lengend Snippet: ( A ) Raw images of the green emission channel showing field of view (24 mm × 24 mm) with immobilised biotin-lacCONS promoter fragments ( left ), with immobilised biotin-lacCONS promoter fragments bound to clamp-labelled RNAP ( middle ), and with remaining immobilised biotin-lacCONS promoter fragments bound to clamp-labelled RNAP, after addition of heparin ( right ). ( B ) Mean number of localisations per field of view for single clamp-labelled RNAP molecules with both green (Cy3B) and red (Alexa647) probes, bound to immobilised biotin-lacCONS promoter fragments, before and after addition of heparin. Mean number of localisations are average of three measurements. Error bars represent the standard deviation from the mean.

Article Snippet: For experiments in and , hexahistidine-tagged E. coli RNAP holoenzyme was prepared using co-expression of genes encoding RNAP β', β, α, ω, and σ 70 subunits to afford an RNAP σ 70 holoenzyme derivative as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pV10 ( ) and plasmid pRSFduet-sigma ( ) were used to inoculate 20 ml LB broth ( ) containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultures were incubated 16 hr at 37°C with shaking.

Techniques: Standard Deviation

Journal: eLife

Article Title: Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

doi: 10.7554/eLife.70090

Figure Lengend Snippet: ( A ) Fluorescence intensity vs. time trajectories showing simultaneous appearance (at ~8 s) of donor and acceptor signals from labelled RNAP bound to biotin-lacCONS promoter fragment ( top ) and FRET efficiency, E* ( bottom ). Range for expected E* values corresponding to an open-clamp conformation is highlighted in cyan. ( B ) Histogram and Gaussian fit of E* values for first five frames after binding define the mean E* for initial binding. Frame duration: 100 ms. Laser powers: 200 μW in red and 500 μW in green. ( C ) Fluorescence intensity vs. time trajectories showing simultaneous appearance of donor and acceptor signals from labelled RNAP bound to biotin-non-promoter fragment ( top ) and FRET efficiency, E* ( bottom ). Range for expected E* values corresponding to an open-clamp conformation is highlighted in cyan.

Article Snippet: For experiments in and , hexahistidine-tagged E. coli RNAP holoenzyme was prepared using co-expression of genes encoding RNAP β', β, α, ω, and σ 70 subunits to afford an RNAP σ 70 holoenzyme derivative as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pV10 ( ) and plasmid pRSFduet-sigma ( ) were used to inoculate 20 ml LB broth ( ) containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultures were incubated 16 hr at 37°C with shaking.

Techniques: Fluorescence, Binding Assay

Journal: eLife

Article Title: Transcription initiation at a consensus bacterial promoter proceeds via a ‘bind-unwind-load-and-lock’ mechanism

doi: 10.7554/eLife.70090

Figure Lengend Snippet: Orange, closed clamp; red, locked clamp; grey, rest of RNAP; purple dot, RNAP active-centre; black, ds-DNA; magenta, partly unwound bubble and blue, fully unwound bubble.

Article Snippet: For experiments in and , hexahistidine-tagged E. coli RNAP holoenzyme was prepared using co-expression of genes encoding RNAP β', β, α, ω, and σ 70 subunits to afford an RNAP σ 70 holoenzyme derivative as follows: single colonies of E. coli strain BL21(DE3) (Millipore) co-transformed with plasmid pV10 ( ) and plasmid pRSFduet-sigma ( ) were used to inoculate 20 ml LB broth ( ) containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and cultures were incubated 16 hr at 37°C with shaking.

Techniques:

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Representative image of celastrol-induced mitophagy in HeLa, Nur77 −/− HeLa, and Nur77 −/− HeLa cells transfected with Myc-Nur77 by EGFP-mCherry-COX8 assay as described in Methods. Scale bar, 10 μm. b Representative images of celastrol-induced mitophagy in MEFs and p62 −/− MEFs by EGFP-mCherry-COX8 assay as described in Methods. Scale bar, 10 μm. c Colocalization of Nur77, LC3, and p62 with mitochondria within mitophagosome/autolysosome. Upper panel: Electron micrographs of HeLa cells stained with 15 nm immunogold-conjugated Nur77 antibody to detect Nur77 (red), and 10 nm immunogold-conjugated p62 antibody to detect p62 (green). Bottom panel: Electron micrographs of HeLa cells stained with 15 nm immunogold-conjugated LC3 antibody to detect LC3, and 10 nm immunogold-conjugated p62 antibody to detect p62. Cells were treated for 1 h with celastrol. The blue dotted line indicates mitophagosome/autolysosome. Mito mitochondrion, Scale bar, 200 nm. d Representative images showing Hsp60, a mitochondrial marker, in the liver tissue from wild-type and Nur77 −/− mice in aging model. Young mice, 8 weeks old. Aged mice, 2 years old. Scale bar, 10 μm. e Statistical analysis of mitochondrial size was represented from liver tissue. Left graph, n = 316, 253, 267, and 287, respectively; Right graph, n = 3 biologically independent samples. A two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM. f The expression of Nur77 protein in the liver tissue from wild-type and Nur77 −/− mice in the aging model. g Representative images of EGFP-mCherry-COX8 in the liver from wild-type or Nur77 −/− mice in the aging model. Purple arrows indicate mitophagy. Scale bar, 2 μm. h Quantification of cells showing mCherry-COX8 accumulation on liver tissue. Two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM ( n = 5 mice per group). Data represent at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The N-terminal 6xHis tagged GFP, GFP-Nur77, GFP-Nur77-IDR, and GFP-Nur77-LBD and C-terminal 6xHis tagged mCherry, mCherry-Nur77, mCherry-Nur77-IDR, and mCherry-Nur77-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen) using pET-28a-GFP or pET-21a-mCherry vectors.

Techniques: Transfection, Staining, Marker, Two Tailed Test, Expressing

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Representative images showing the time-dependent effect on celastrol induction of cytoplasmic Nur77 body formation. Bottom panels: quantitative analysis of the number and size of Nur77/p62 body formation. Bottom left graph, n = 4 biologically independent samples; Bottom right graph, n = 20, 23, 25, and 19, respectively. Data were presented as mean values ± SEM. Scale bar, 10 μm. b Real-time images showing the formation and fusion of GFP-Nur77 and mCherry-p62 droplets in HeLa cells after treatment with celastrol (2 μM) for 1 h. White arrows indicate droplets formation and fusion (see also Supplementary Movie ). Scale bar, 10 μm. c Representative images illustrating the role of celastrol in promoting p62 body formation in a Nur77-dependent manner immunostaining. Nur77 −/− HeLa cells were also transfected with GFP-Nur77 to determine its effect on p62 body formation. The diameter of the biggest p62 puncta in each cell was measured. The number of p62 puncta >0.5 μm in each cell was assessed. A two-tailed unpaired Student’s t -test was used for statistical analysis, and data are presented as mean values ± SEM ( n = 5 biologically independent samples). d FRAP analysis of the effect of Nur77 in regulating p62 mobility in HeLa cells. Data were presented as means ± SEM ( n = 3 independent experiments). Scale bar, 1.5 μm. Data represent at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The N-terminal 6xHis tagged GFP, GFP-Nur77, GFP-Nur77-IDR, and GFP-Nur77-LBD and C-terminal 6xHis tagged mCherry, mCherry-Nur77, mCherry-Nur77-IDR, and mCherry-Nur77-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen) using pET-28a-GFP or pET-21a-mCherry vectors.

Techniques: Immunostaining, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a GFP-Nur77 (2 μM) undergoes phase separation. The size of Nur77 droplets was analyzed. Data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 10 μm. b Top, changes in fluorescence intensity of GFP-Nur77 droplets after photobleaching were plotted over time. Bottom, representative images of fluorescence recovery. Data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 1.5 μm. c Fusion of GFP-Nur77 droplets in 10% PEG-3.35 K. Scale bar, 20 μm. d Live imaging of GFP-Nur77 in HeLa cells. Scale bar, 5 μm. e Time course analysis of GFP-Nur77 nuclear body recovery after photobleaching in HeLa cells. Representative images of fluorescence recovery are shown. Data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 1.5 μm. f Three-dimensional (3D) images of Nur77 nuclear assemblies. An enlarged view of inset is also shown. Scale bar, 5 μm. g Fixed imaging of Myc-Nur77 in HeLa cells. Scale bar, 5 μm. h Endogenous Nur77 displays nuclear puncta in HeLa cells revealed by immunostaining with anti-Nur77. Scale bar, 5 μm. Data represent at least three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The N-terminal 6xHis tagged GFP, GFP-Nur77, GFP-Nur77-IDR, and GFP-Nur77-LBD and C-terminal 6xHis tagged mCherry, mCherry-Nur77, mCherry-Nur77-IDR, and mCherry-Nur77-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen) using pET-28a-GFP or pET-21a-mCherry vectors.

Techniques: Fluorescence, Imaging, Immunostaining

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Intrinsic disorder tendency of Nur77. IDR intrinsically disordered region, DBD DNA-binding domain, LBD ligand-binding domain. b Schematic representation of Nur77 and its mutants. c In vitro phase separation of GFP-Nur77-IDR and GFP-Nur77-LBD (2 μM). Scale bar, 10 μm. d Quantification of Nur77 mutant droplets formation in absence of celastrol in HeLa cells. Data were presented as mean values ± SEM ( n = 3 independent experiments). e Representative images of Nur77 mutant droplets formation in absence of celastrol in HeLa cells. Scale bar, 10 μm. f Representative real-time images showing the formation and fusion of cytoplasmic GFP-Nur77 droplets after treatment with celastrol (2 μM) for 1 h in HeLa cells (see also Supplementary Movie 4). Right: quantification of the cytoplasmic retention of GFP-Nur77 protein. A two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 10 μm. g Droplet formation of GFP-Nur77 and mutants in HeLa cells treated with the indicated compounds (2 μM). Left: Representative droplet images of transfected GFP-Nur77 and mutants. An enlarged view of the inset is also shown. Scale bar, 10 μm. Right: Quantification of droplet formation of GFP-Nur77 and mutants. Data represent at least three independent experiments. Source data are provided as Source Data file.

Article Snippet: The N-terminal 6xHis tagged GFP, GFP-Nur77, GFP-Nur77-IDR, and GFP-Nur77-LBD and C-terminal 6xHis tagged mCherry, mCherry-Nur77, mCherry-Nur77-IDR, and mCherry-Nur77-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen) using pET-28a-GFP or pET-21a-mCherry vectors.

Techniques: Binding Assay, Ligand Binding Assay, In Vitro, Mutagenesis, Two Tailed Test, Transfection

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Immunofluorescence images of GFP-Nur77, mCherry-p62, and HA-Ub transfected in HeLa cells treated with or without celastrol. Data illustrate the colocalization of Nur77 with p62 and Ub in the presence of celastrol. Scale bar, 10 μm. b , c Interaction of indicated Nur77 or deubiquitinated mutant (K536R) and p62 was analyzed in HeLa cells treated with or without celastrol by co-immunoprecipitation (co-IP) assay. d , e Immunofluorescence images showing the effect of celastrol-induced Nur77 ubiquitination on mCherry-p62 droplet formation. Scale bar, 10 μm. f Representative images showing ubiquitination-dependent colocalization of Nur77 with p62 and mitochondria in HeLa cells treated with celastrol. Scale bar, 10 μm. Data represent at least three independent experiments. Source data are provided as Source Data file.

Article Snippet: The N-terminal 6xHis tagged GFP, GFP-Nur77, GFP-Nur77-IDR, and GFP-Nur77-LBD and C-terminal 6xHis tagged mCherry, mCherry-Nur77, mCherry-Nur77-IDR, and mCherry-Nur77-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen) using pET-28a-GFP or pET-21a-mCherry vectors.

Techniques: Immunofluorescence, Transfection, Mutagenesis, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a –d Representative images showing colocalization of Nur77 or mutants with p62, mitochondria, and lysosome in HeLa cells after celastrol treatment. The blue arrow indicates line profiles of fluorescence intensities including Pearson’s correlation coefficients shown in b and d . A two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM ( n = 20 biologically independent samples). Dotted box: higher magnification of indicated region. Scale bar, 10 μm. e Mutating K536 in Nur77 inhibits celastrol-induced interaction between p62 and LC3. HeLa cells transfected with the indicated expression plasmids were treated with or without 2 μM celastrol and 20 ng/mL TNFα. Interaction of Flag-p62 with GFP-LC3 was examined by co-IP assay. f Characterization of domain requirement of Nur77 for promoting celastrol-induced p62 interaction with LC3. HeLa cells transfected with the indicated Flag-p62, GFP-LC3, and GFP-Nur77 or mutant were treated with celastrol and TNFα for 1 h and analyzed for Flag-p62 interaction with GFP-LC3 by co-IP assay. Data represent at least three independent experiments. Source data are provided as Source Data file.

Article Snippet: The N-terminal 6xHis tagged GFP, GFP-Nur77, GFP-Nur77-IDR, and GFP-Nur77-LBD and C-terminal 6xHis tagged mCherry, mCherry-Nur77, mCherry-Nur77-IDR, and mCherry-Nur77-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen) using pET-28a-GFP or pET-21a-mCherry vectors.

Techniques: Fluorescence, Two Tailed Test, Transfection, Expressing, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: a Schematic representation of p62 and mutants and their interaction with Nur77. PB1 Phox/Bem1p protein-protein binding domain. ZZ zinc-finger domain, TB TRAF6 binding domain, UBA ubiquitin-associated domain. b – d Interaction of Nur77 and p62, as well as their mutants, was analyzed in HeLa cells treated with or without celastrol by co-IP assay. e Multivalent interaction between Nur77 and p62. The interaction between the IDR of Nur77 and PB1 of P62 is ligand-independent (red), whereas the interaction between LBD of Nur77 and UBA of p62 depends on celastrol that triggers Nur77-LBD ubiquitination (pink). f Immunofluorescence images showing colocalization of GFP-Nur77-IDR with mCherry-p62 or mCherry-p62-PB1 after treatment with or without celastrol. Scale bar, 10 μm. g FRAP analysis of the effect of Nur77-IDR in regulating p62 mobility in HeLa cells. Data were presented as mean values ± SEM ( n = 3 independent experiments). Scale bar, 1.5 μm. h Representative images showing the effect of GFP-Nur77-IDR on the filamentous structures of mCherry-p62-PB1 when mCherry-p62-PB1 was incubated with GFP or GFP-Nur77-IDR at intermediate molar ratio (1:2). Scale bar, 10 μm. i FRAP analysis of the effect of GFP-Nur77-IDR on mCherry-p62-PB1 mobility in vitro. Two-tailed unpaired Student’s t -test was used for statistical analysis, and data were presented as mean values ± SEM ( n = 3 independent experiments). Data represent at least three independent experiments. Source data are provided as Source Data file.

Article Snippet: The N-terminal 6xHis tagged GFP, GFP-Nur77, GFP-Nur77-IDR, and GFP-Nur77-LBD and C-terminal 6xHis tagged mCherry, mCherry-Nur77, mCherry-Nur77-IDR, and mCherry-Nur77-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen) using pET-28a-GFP or pET-21a-mCherry vectors.

Techniques: Protein Binding, Binding Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Incubation, In Vitro, Two Tailed Test

Journal: Nature Communications

Article Title: Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

doi: 10.1038/s41467-021-26295-8

Figure Lengend Snippet: The phase separation of Nur77 and p62/SQSTM1 triggered by their multivalent interaction sequesters damaged mitochondria and directs cargo mitochondria to the autophagic machinery. DBD DNA-binding domain, LBD ligand-binding domain, IDR intrinsically disordered region, PB1 Phor and Bem1p, UBA ubiquitin-associating, Ub ubiquitin.

Article Snippet: The N-terminal 6xHis tagged GFP, GFP-Nur77, GFP-Nur77-IDR, and GFP-Nur77-LBD and C-terminal 6xHis tagged mCherry, mCherry-Nur77, mCherry-Nur77-IDR, and mCherry-Nur77-LBD were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen) using pET-28a-GFP or pET-21a-mCherry vectors.

Techniques: Binding Assay, Ligand Binding Assay