EMBO/soda-nel huggingface dataset:
H2B monoubiquitination is a key epigenetic regulator of ferroptosis and is regulated by p53 (2019)
Figure 4

Figure 4. p53 regulates the nuclear translocation of USP7.

A, Coimmunoprecipitation (Co-IP) analysis was performed in A549 cells with antibodies against p53 or USP7.

B, H1299 cells were transfected with an empty vector (Cont.), a Flag-tagged p53, a USP7-specific shRNA, or the last two plasmids together for 48 h. Western blot analysis was then performed.

C, Nuclear and cytosolic extracts were differentially prepared from H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.). Western blot analysis was then performed.

D, H1299 cells transfected with a Myc-tagged USP7 or a Myc-tagged USP7 together with a Flag-tagged p53 were subjected to IF analysis (upper). A549 cells transfected with a p53-specific siRNA or a control siRNA (siCont.) were examined by IF analysis, and the RNAi efficiency is shown (lower). Representative images are shown (magnification, ×40).

E, Diagrams showing the interaction status of USP7 and its truncated mutants (USP7-△N, lacking the N-terminal region; USP7-△M, lacking the middle region; USP7-△C, lacking the C-terminal region) with p53, based on previous reports (56, 57).

F, IF analysis of A549 cells transfected with a Myc-tagged wild-type USP7 (Myc-USP7-WT) or three Myc-tagged truncated mutants. Representative images are shown (magnification, ×40).

G, H1299 cells were transfected with a Flag-tagged p53 or an empty vector together with a USP7-specific siRNA or a control siRNA as indicated for 48 h. Cells were then harvested and subjected to a MNase digestion assay.
H2B monoubiquitination is a key epigenetic regulator of ferroptosis and is regulated by p53 (2019)
Figure 5

Figure 5. p53 associates with H2Bub1 both in vivo and in vitro.

A, Co-IP analysis was performed in 293T cells with antibodies against p53 or H2Bub1.

B, peptide sequence synthesized according a previous report (16, 65) and used for peptide pull-down assay. C, Peptide pull-down assay was performed with synthesized peptides in (B).

D, Co-IP analysis for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) treated with a USP7-specific inhibitor P5091.

E, A proposed working model of the regulation of H2Bub1 by p53.
H2B monoubiquitination is a key epigenetic regulator of ferroptosis and is regulated by p53 (2019)
Figure 6

Figure 6. p53-USP7-H2Bub1 axis regulates SLC7A11 expression.

A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.

B, H1299 cells were transfected with Flag-tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h. Co-IP analysis was performed with antibodies against Flag.

C, H1299 cells transfected with Flag-tagged p53 were treated with erastin or untreated for 24 h. Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis.

D, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA, or a control siRNA (siCont.) were treated with 20 μM erastin or untreated for 24 h as indicated. Western blot analysis was then performed. E, qRT-PCR (above) and Western blot (below) analyses for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated.

F, ChIP analysis of H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated.

G, H1299 cells transfected with Flag-tagged p53 were treated with 12 μM erastin or untreated for 24 h. ChIP analysis with antibodies against Flag or USP7 was performed.

H, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h. ChIP analysis was performed with anti-H2Bub1 antibodies.

I, A proposed working model for the p53-USP7-H2Bub1 axis in the regulation of SLC7A11 expression.

Data information: Bars and error bars are mean±s.d., n=3 independent repeats.
Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease (2022)
Figure 1

Fig. 1 HSF1 associates with mitochondria in models of HD

Mitochondria-enriched fractions were isolated from HdhQ7 or HdhQ111 cells. mtHSF1 was examined by WB analysis. VDAC was analyzed as a loading control. (n=6 biological replicates).

HdhQ7 WT striatal cells were exposed to 5 mM 3-NP for 2 h. mtHSF1 levels were determined by immunoblotting. VDAC was used as a loading control. (n=5 biological replicates).

HSF1 was measured in the mitochondria-enriched fractions of the HD transgenic YAC128 mice (9 months old) or age-matched littermates. (n=5 biological replicates).

mtHSF1 was analyzed in HD patient fibroblasts (GM04208, GM04222, and GM21756) and fibroblasts from healthy individuals. (n = 5 for Con1/ GM04208 and Con2/ GM04222 groups; n = 3 for Con3/ GM21756 group).

Representative immunoblot of HSF1 levels in mitochondria isolated from patient-derived iPSCs and control iPSCs. (n=3 biological replicates).

Representative images of striatal organoids. Left, immunostaining for the MSN marker DARPP32 (green) and the radial glial marker SOX2 (red) in striatal organoids at D60. Middle, immunostaining for the LGE progenitor markers Ctip2 (green) and GSH2 (red) at D30. Right panel, immunostaining for the neuronal marker NeuN (green) and the proliferation marker Ki67 (red) at D30. The images are representative of 12 independent differentiation experiments of H9 and IMR90-4 cells. HO= Hoechst (blue). The scale bar represents 100 µm.

Left, schematic of the procedure by which the striatal organoids were dissociated for scRNA-seq. Right, UMAP visualization of single-cell RNA expression in striatal organoids at D30 (n=5,939 cells) and D60 (n=5,188 cells).

Representative images and scatterplot confirming the increased HSF1 (green) translocation to mitochondria (Tom20, red). The data were obtained from 3 independent biological experiments. Con: n=15 organoids; HD: n=14 organoids. Two-tailed unpaired t-test; Means ± SEMs. Images were taken using Structured Illumination Microscopy (SIM). Scale bar represents 5 μm.

The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy. The arrows (red) mark HSF1 in mitochondria. The negative control lacked the HSF1 antibody. The scale bar represents 200 nm.

HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection. The protein levels of HSF1 were detected in mitochondrial fractions. (n=3 biological replicates).

Data information: The data are the means ± SEMs from at least three independent biological experiments; unpaired Student's t-test was used in A-E, and H; one-way ANOVA followed by Tukey's multiple comparison test was used in J. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease (2022)
Figure 2

Fig. 2 mtHSF1 disturbs the dynamic balance by promoting Drp1 phosphorylation

Sketch of mitochondrial targeting by HSF1.

HdhQ7 cells were transfected with Flag-mtHSF1 and stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was examined by confocal microscopy. Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation. The scale bar represents 40 µm. (n=3 biological replicates, at least 100 cells per group were counted).

The total protein levels of p-Drp1 S616, p-Drp1S637, Drp1, OPA1, MFN2, Flag-mtHSF1 and actin (loading control) were determined by WB analysis. (n=3 biological replicates).

Timeline overview of mitochondrial analysis in mice. AAV-Con and AAV-mtHSF1 were injected into the striata of WT mice at 8 weeks of age. Mitochondrial morphology was analyzed after 3 weeks.

Brain sections were stained with an anti-Tom20 antibody (red), an anti-Flag antibody (green) and DAPI (blue). Mitochondrial morphology was analyzed in Flag+ or Flag- regions by confocal microscopy. The scale bar presents 2 mm, and the enlarged image scale bar is 40 µm. (n=6 mice/group).

Striata injected with AAVs were lysed. p-Drp1 S616 was tested by immunoblotting. (n=5 mice/group).

Mitochondrial morphology was analyzed by electron microscopy (EM) in mice injected with AAV-Con or AAV-mtHSF1. Scatterplot showed the length of mitochondria. The scale bar presents 4 µm, and the enlarged image scale bar is 400 nm. (n=3 mice/group). AAV-Con group, n=268 mitochondria were measured; AAV-Flag-mtHSF1 group, n=179 mitochondria were measured.

Timeline overview of mitochondrial analysis in striatal organoids. AAV-mtHSF1 were injected into the WT striatal organoids at 6 weeks. Mitochondrial analysis was performed after 2 weeks.

Representative images and scatterplot of mitochondrial length (Tom20, red) in Flag+ or Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=154 mitochondria; Flag+: n=132 mitochondria. The scale bar represents 5μm.

Representative images and scatterplot showing that the number of p-Drp1 S616 puncta in Flag+ cells was greater than that in Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=70 cells from 10 organoids; Flag+: n=55 cells from 10 organoids. The images were obtained using structured illumination microscopy (SIM). The scale bar represents 10 µm.

Data information: The data are the means ± SEMs from at least three independent biological experiments; unpaired Student's t-test was used. ***P < 0.001, ****P < 0.0001.
Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease (2022)
Figure 3

Fig. 3 mtHSF1 triggers mtDNA deletion and cell death.

The mtDNA content was examined with primers of D-loop, mtCO1, mtCO2 and mtND2 by qPCR in control or Flag-mtHSF1-expressing striatal cells. Scatterplot with bar showed the relative percentage of mtDNA content. (n=3 biological replicates).

Measurement of mitochondrial respiratory activity by Seahorse analysis. (n=9-10 biological replicates)

Cells were treated with EtBr for six days to delete mtDNA. mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr. (n=3 biological replicates).

D-E. Cell death was determined by detection of LDH release into the medium and cleavage of PARP. Scatterplot with bar showed the relative percentage of LDH release. (n=3 biological replicates).

Timeline overview of mtDNA and cell death analysis in mice.

qPCR analysis of the mtDNA content of AAV-Con-injected mice or AAV-mtHSF1-injected mice. Scatterplot with bar showed the relative percentage of mtDNA content (n=3 mice/group).

Cleaved-Caspase3 in AAV-Con- or AAV-mtHSF1-injected mice was detected by WB analysis. (n=5 mice/group).

Timeline overview of mtDNA and cell death analysis in striatal organoids.

Representative images and scatterplot showing the number of mtDNA (purple) colocalized with mitochondria (Tom20, green) in striatal organoids injected with AAV-Con or AAV-mtHSF1. The data were obtained from 3 independent biological experiments. Con: n=65 cells from 20 organoids; Flag-mtHSF1: n=70 cells from 20 organoids. Two-tailed unpaired t-test was used. Images were captured by Structured Illumination Microscopy (SIM). Scale bar represents 5 μm.

The typical nuclear fragmentation morphology of AAV-Con- (GFP) or AAV-mtHSF1-injected striatal organoids was measured. Scatterplot showed the quantitative result of nuclear fragmentation. The arrows (yellow) mark fragmented nuclei. Two-tailed unpaired t-test was used. The data were obtained from 3 independent biological experiments. Con: n=14 organoids; Flag-mtHSF1: n=15 organoids. The scale bar represents 20 µm.

Data information: The data are the means ± SEMs from at least three independent biological experiments; unpaired Student's t-test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease (2022)
Figure 4

Fig. 4 mtHSF1 suppresses SSBP1 oligomerization.

Mitochondria-enriched fractions were subjected to immunoprecipitation with IgG or an anti-HSF1 antibody. The immunoprecipitates were then analyzed by mass spectrometry.

Total cell lysates were subjected to immunoprecipitation with an anti-HSF1 antibody followed by WB analysis. (n=3 biological replicates).

HdhQ7 or HdhQ111 cell extracts were incubated with DSS (1 mM) for 30 min at RT. SSBP1 oligomers were detected by immunoblotting with an anti-SSBP1 antibody. (n=3 biological replicates).

SSBP1 oligomers in control and patient fibroblasts (GM04208 and GM21756) were analyzed. (n=3 biological replicates).

SSBP1 oligomers were measured in WT mice or YAC128 mice. (n=3 mice/group).

Cells were transfected with an empty vector or Flag-mtHSF1 for 48 h. The total cell lysates were harvested and crosslinked with DSS. SSBP1 oligomers were tested by immunoblotting. (n=4 biological replicates).

Flag-mtHSF1 was expressed in the striata of WT mice for three weeks. SSBP1 oligomers were determined by immunoblotting. (n=5 mice/group).

Flag-mtHSF1 or sh-SSBP1 was delivered into cells via lentiviral infection. The expression of SSBP1 and mtHSF1 was detected by WB analysis. The mtDNA content was examined by qPCR analysis. (n=3 biological replicates).

Data information: The data are the means ± SEMs from at least three independent biological experiments; unpaired Student's t-test was used in C-G, and one-way ANOVA followed by Tukey's multiple comparison test was used in H. *P < 0.05, ***P < 0.001, ****P < 0.0001, n.s., not significant.
Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease (2022)
Figure 5

Fig. 5 mtHSF1 causes neurodegeneration and HD-like behavior.

Primary neurons expressing Flag-mtHSF1 were stained with anti-DARPP32 (green) and anti-Flag (red) antibodies. The arrows (white) mark Flag+ or Flag- primary neurons. The neurite length of Flag+ or Flag- neurons was measured by Image J and showed by scatterplot. The scale bar represents 50 µm. (n = 155 for Flag- group and n = 154 for Flag+ group).

Schematic diagram illustrating the timeline of viral injection into striatal organoids.

Human striatal organoids were injected with AAV-Con (GFP) or AAV-mtHSF1 at D42. Scatterplot showed the percentage of NeuN+ cells (red) among Flag+ or GFP+ cells (green) after injection for 2 weeks. The data were obtained from 3 independent biological experiments. Con: n=11 organoids; Flag-mtHSF1: n=13 organoids. The scale bar represents 100 µm.

Timeline overview of AAV injection and tests. Immunohistochemistry and WB analysis were performed 3 weeks post injection. Behavior was tested after 16 weeks or 20 weeks of AAV injection.

Representative images of immunostaining with DARPP32 (red) and Flag (green). The DARPP32 fluorescence density was analyzed in Flag+ or Flag- sections by confocal microscopy and showed by scatterplot. Thickness: 30 µm. The scale bar is 2 mm, and the enlarged image scale bar is 40 µm. (n=12 mice/group).

The striatum of AAV-Con- or AAV-mtHSF1-injected mice were harvested. The DARPP32 protein levels were tested by WB analysis. (n=6 mice/group).

The percentage of NeuN+ cells in Flag+ or Flag- sections was determined by confocal microscopy and showed by scatterplot. Thickness: 10 µm. (n=9 mice/group).

H-K. Mouse weight and motor activity were recorded post AAV injection. (n=7-13 mice/group).

Data information: The data are the means ± SEMs; A-G are from at least three independent biological experiments; unpaired Student's t-test was used in A, C and E-K. *P < 0.05, **P < 0.01, ****P < 0.0001.
Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease (2022)
Figure 6

Fig. 6 DH1 reduces association of HSF1 with mitochondria by interfering with Drp1/HSF1 binding.

Total lysates of HdhQ7 or HdhQ111 cells were subjected to immunoprecipitation with an anti-HSF1 antibody followed by immunoblotting. (n=3 biological replicates).

Drp1 and HSF1 binding in vitro was analyzed by WB analysis. (n=3 biological replicates).

Mitochondrial fractions were isolated from WT or Drp1-KO MEFs. The protein levels of Drp1 and HSF1 were tested by immunoblotting. (n=3 biological replicates).

Sequence analysis of Drp1 and HSF1 was performed with L-ALIGN sequence alignment software.

Sketch of the domains of Drp1 and HSF1. DH1 in Drp1 and the corresponding region DH2 in HSF1 are marked in red.

GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1. (n=3 biological replicates).

-J G. HdhQ7 or HdhQ111 cells (1 μM/day, 3 days), H. YAC128 or age-matched littermates (3 mg/kg/day, 3 months), I. striatal organoids differentiated from patient iPSCs or control iPSCs (1 μM/day, 7 days) and J. patient fibroblasts (1 μM/day, 5 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).

-L K. HdhQ7 or HdhQ111 and L. patient fibroblasts were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).

Data information: The data are the means ± SEMs from at least three independent biological experiments; unpaired Student's t-test was used in C, and one-way ANOVA followed by Tukey's multiple comparison test was used in G-L. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Mitochondrial HSF1 triggers mitochondrial dysfunction and neurodegeneration in Huntington's disease (2022)
Figure 7

Fig. 7 DH1 reduces mitochondrial dysfunction and neurotoxicity.

Schematic showing the DH1 treatment procedure for striatal organoids.

Immunostaining for mitochondria (Tom20, green) and mtDNA (anti-DNA, red) in striatal organoids treated with DH1 or TAT at D60. Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria. The data were obtained from 3 independent biological experiments. Left, n=222-311 mitochondria per group; Right, n=14-20 organoids per group. Images were taken using Structured Illumination Microscopy (SIM). The scale bar represents 10 μm.

Immunostaining for mitochondria (Tom20, red) and p-Drp1 S616 (green) in striatal organoids treated with DH1 or TAT at D60. Scatterplot with bar showed the number of p-Drp1 S616 per cell in organoids. The data were obtained from 3 independent biological experiments. n=29-34 cells from at least 10 organoids. The scale bar represents 5 μm.

Scatterplot with bar showed the ratio of MSNs (DARPP32, green) in striatal organoids in the presence or absence of DH1. The data were obtained from 3 independent biological experiments. n=30 organoids per group. The scale bar represents 50 μm.

Representative tracing images of DARPP32+ neurons treated with DH1 or TAT at D45. Scatterplot with bar showed the results of longest neurites length. The data were obtained from 3 independent biological experiments. n=45-92 neurons per group. The scale bar represents 20 μm.

Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1. The arrows (white) mark fragmented nuclei. The data were obtained from 3 independent biological experiments. n=7-10 organoids per group. The scale bar represents 20 μm.

Schematic showing the DH1 treatment procedure for WT/HD YAC128 mice.

-I H. PGC1α and I. DARPP32 protein levels were determined by immunoblotting. (n=3 biological replicates).

Mouse behavior was tested after DH1 treatment. (n=9-12 mice/group).

Data information: The data are the means ± SEMs from at least three independent biological experiments; one-way ANOVA followed by Tukey's multiple comparison test was used. *P < 0.05, **P < 0.01, ****P < 0.0001.
Coupling spatial segregation with synthetic circuits to control bacterial survival (2016)
Figure 2

Figure 2. Programming collective survival using different circuits

(A-C) Mechanisms of three collective-survival circuits. The BlaM circuit consists of constitutive expression of BlaM to degrade carbenicillin upon cell lysis. BlaM works as an extracellular enzyme. The QS-CAT consists of QS-mediated expression of a chloramphenicol acetyl-transferase (CAT) that degrades chloramphenicol. The QS-BlaM circuit integrates QS control and extracellular carbenicillin degradation to establish collective survival.

(D-F) Regulation modes for the three circuits. The BlaM circuit depends on antibiotic-mediated release of an enzyme into the environment to realize the collective survival. The QS-CAT circuit depends on QS regulation. The QS-BlaM circuit depends on both QS regulation and enzyme release (by lysis and export).

(G-I) Experimental validation of collective survival. All populations grew to high cell densities in the absence of antibiotics (Blue curves). In the presence of antibiotics, only populations with initial densities greater than NCT grew. NCT was increased with the concentration of antibiotics. Engineered bacteria with the QS-BlaM circuit exhibited a significantly higher NCT than those with the BlaM or the QS-CAT circuits. Carbenicillin was used for the BlaM and QS-BlaM circuits; chloramphenicol for the QS-CAT circuit. Each error bar represents the standard deviation from triplicate measurements.
Coupling spatial segregation with synthetic circuits to control bacterial survival (2016)
Figure 3

Figure 3. Safeguard control in microbial swarmbot using the BlaM circuit.

Configuration of the two-compartment model. The microenvironment of microbial swarmbot is separated from that of the chamber by its permeable hydrogel shell (orange), resulting in two separate compartments, defined as swarmbot and environment (chamber). Four major components involved in the population dynamics are explored in the model, including cell density (n), nutrient (s), carbenicillin (a) and BlaM concentration (b). Each component has a different transport rate, depending on the molecule size.

Simulated microbial swarmbot dynamics under a static condition. a = 0.4, a microbial swarmbot with a low n0 cannot initiate growth (red lines) but one with a high n0 leads to growth both in the swarmbot and, after a delay, in the chamber (green lines). While (C) swarmbots with both low and high initial densities lead to eventual full growth in the chamber in the absence of the antibiotic (a = 0). Solid lines: cell density inside swarmbots; dashed lines: cell density in the chambers.

(D) A microfluidic device for controlling and monitoring population dynamics of microbial swarmbot. The device consists of an input channel leading to a culturing chamber and a trapping slot. The chamber is used for culturing engineered bacterial populations both inside and outside the swarmbot, while the trapping slot is for trapping the swarmbot due to its smaller size but allowing the medium to flow through. Input flow patterns (static or pulsing) are controlled by a programmable syringe pump.

(E) Experimental validation of model prediction. At 100µg/ml carbenicillin, a microbial swarmbot with a high initial density led to robust growth in the capsule and, after a delay, in the chamber (green lines). While a swarmbot with a low initial cell density did not exhibit growth either in the swarmbot or in the chamber at the same carbenicillin concentration (red lines). (F) In contrast, both the swarmbot and culturing chamber reached high cell densities in absence of carbenicillin, regardless of the initial cell densities.

(G) Time-lapse images of microbial swarmbot dynamics. The population initiated growth inside the swarmbot and subsequently in the chamber at a high initial density. But no growth occurred for a swarmbot with a low initial density at the same antibiotic concentration (rightmost). The concentration of carbenicillin was 100 µg/ml and the scale bar represents 200 µm.

(H) The control strain lacking BlaM circuit. The population could not survive at the same concentration of carbenicillin at the high initial density.