EMBO/soda-nel huggingface dataset:
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment (2022)
Figure 4

Figure 4 Altered heat shock response in stress granule-deficient cells

A) Representative confocal images of immunofluorescent staining of and puromycin-labelled proteins and Hsp70 in parental and G3BP1/2 knock-out U2OS cells exposed to a heat shock at 43°C for 30 minutes (+ HS; left panel), and after 1 hour recovery (HS + 1h Recovery; right panel). Scale bar, 10 μm.

B) Representative confocal images of immunofluorescent staining of HSF1 in parental and G3BP1/2 knock-out U2OS cells directly after heat shock (+ HS) and 2 hours after 2 hours recovery (HS + 2h Recovery). Scale bar, 10 μm.

C) Quantification of the number of HSF1 foci per cell in images from B), shown as box plot with median and 5-95 percentiles (n=3 independent experiments, >300 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).

D) Western blot analysis of parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), heat shocked (HS), and followed for 1, 2, and 4 hours after heat shock (Rec). The blots were probed with antibodies against HSF1 and GAPDH (* indicated phosphorylated HSF1, and ** indicates unphosphorylated HSF1).

E) Analysis of HSP70 mRNA levels in parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), heat shocked (+ HS), and followed for 1, 2, and 4 hours after heat shock (Rec.). mRNA levels were normalized to untreated samples. Data represent the geometric mean with 95% confidence interval of triplicate samples(Student's unpaired t-test, ** P<0.01).
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment (2022)
Figure 5

Figure 5 Nuclear stress-induced SUMOylation is changed in stress granule-deficient cells

A) Representative confocal images of immunofluorescent staining of the nucleolar marker nucleolin and SUMO2/3 in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to a heat shock followed by 2 hours recovery. Scale bar, 10 μm.

B) Quantification of the nucleolus/nucleoplasm ratio of SUMO2/3 intensities in images from A). The frequency and distribution of the ratio per cell are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).

C) Representative confocal images of immunofluorescent staining of PML bodies (PML) and SUMO2/3 in parental and G3BP1/2 knock-out U2OS cells. Cells were heat shocked followed by 2 hours recovery. Arrows indicate dots positive for PML and SUMO2/3 staining. Scale bar, 10 μm.

D) Quantification of SUMO2/3 positive PML puncta in images from C). Data represent the mean ± SD (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).

E) Analysis of SUMO2/3 conjugates in parental and G3BP1/2 knock-out U2OS cells that were left untreated (- HS), exposed to a heat shock (+ HS), and followed for 2 hours (2h Rec.) with or without 100 nM proteasome inhibitor epoxomicin (EPX).

F) Quantification of the total SUMO2/3 conjugate band densities in E). Data represent the mean ± SD (n=3 independent experiments, Student's unpaired t-test, * P<0.05).
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment (2022)
Figure 6

Figure 6 Enhanced degradation of STUbL substrate TDP-43 in stress granule-deficient cells

A) Representative confocal images of immunofluorescent staining of SUMO2/3 and TDP43 in parental and G3BP1/2 knock-out U2OS cells. Cells were exposed to a heat shocked followed by 2 hours recovery. Arrows indicate dots positive for TDP-43 and SUMO2/3 staining. Scale bar, 10 μm.

B) Quantification of the percentage of SUMO2/3 positive TDP43 puncta in images from experiment shown in A)..Data represent the mean ± SD (n=3 independent experiments, >50 cells analyzed per condition, Mann-Whitney test, ** P<0.01).

C) Representative confocal images of immunofluorescent staining of SUMO2/3 and PML bodies (PML) in parental and G3BP1/2 knock-out U2OS cells transient transfected with GFP-TDP43.Cells were subjected to heat shock and followed by 2 hours recovery. Scale bar, 10 μm.

D) Analysis of detergent SDS and NP40 soluble fractions of TDP43 in parental and G3BP1/2 knock-out U2OS cells that were left untreated (-HS), exposed to 43°C (+HS) followed by 2 hours recovery (2h Rec.) in absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX).

E) Quantification of the TDP43 band densities in the SDS-soluble fraction. Data represent the mean ± SD (n=3 independent experiments, Student's unpaired t-test, * P<0.05).
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment (2022)
Figure 7

Figure 7 Inhibition of SUMO-targeted ubiquitylation prevents UPS impairment

A) Analysis of SUMO2/3 conjugates in control (siControl) or RNF4-depleted (siRNF4) G3BP1/2 knock-out U2OS cells. Cells were left untreated (-HS), exposed to 43°C (+ HS), followed by 2 hours recovery (HS + 2h Rec.) in the absence or presence of 100 nM proteasome inhibitor epoxomicin (EPX).

B) Quantification of the total SUMO2/3 band densities. Data represent the mean ± SD (n=3 independent experiments, *P<0.05).

C) Quantification of mean cellular Ub-YFP fluorescence intensities in control (siControl) and RNF4-depleted (siRNF4) parental U2OS cells. Cells were left untreated (- HS) or exposed to heat shock followed by 4 hours recovery (HS + 4h Rec.). Fluorescence intensities are normalized to untreated control cells (siControl - HS). Frequency and distribution of the relative fluorescence intensities are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ns: not significant).

D) Quantification of mean cellular Ub-YFP fluorescence intensities in control (siControl) and RNF4-depleted (siRNF4) G3BP1/2 knock-out U2OS cells. Cells were left untreated (- HS) or exposed to heat shock followed by 4 hours recovery (HS + 4h Rec.). Fluorescence intensities are normalized to untreated control cells (siControl - HS). Frequency and distribution of the relative fluorescence intensities are shown as violin plots. The solid lines in each distribution represent the median, and dash lines represent the upper and lower interquartile range limits. (n=3 independent experiments, >1000 cells analyzed per condition, Kruskal-Wallis test, ** P<0.01).

E) Representative confocal images of control (siControl), RNF4-depleted (siRNF4), G3BP1/2-depleted (siG3BP1/2) and G3BP1/2+RNF4-depleted (siG3BP1/2 + siRNF4) MelJuSo cells expressing the nuclear UPS reporter NLS-GFP-CL1. Cells were exposed to heat shock followed by 4 hours recovery in the absence or presence of the proteasome inhibitor epoxomicin (EPX). The nucleolar marker nucleolin was stained by immunofluorescence. Scale bar,10 μm.
Cytosolic stress granules relieve the ubiquitin-proteasome system in the nuclear compartment (2022)
Figure 8

Figure 8 Enhanced formation of ataxin-1 nuclear inclusions in stress granule-deficient cells

A) Representative confocal images of immunofluorescent staining of FLAG-tagged ubiquitin (FLAG-Ub) and mutant ataxin1 (ATX1) in parental and G3BP1/2 knock-out U2OS cells. Cells were subjected to heat shock followed by 4 hours recovery. Scale bar,10 μm.

B) Frequency distribution of ataxin-1 inclusions per cell from three independent experiments.

C) Schematic drawing of model. In the absence of stress granules, proteotoxic stress causes DRiPs to translocate from the cytosol to the nucleus where they accumulate in nucleoli and sequester Hsp70. This results in early activation of HSF1 and RNF4 and targeting of SUMOylated proteins for proteasomal degradation. These substrates overload the nuclear UPS impairing the degradation of other ubiquitin-dependent proteasome substrates.
Acetate Supplementation Restores Cognitive Deficits Caused by ARID1A Haploinsufficiency in Excitatory Neurons (2022)
Figure 1

Figure 1. Cognitive defects as a consequence of Arid1a haploinsufficiency in mice excitatory neurons

A. Schematic for the production of Arid1a knockout mice. By crossing them with Nex-Cre mouse lines, we specifically deleted Arid1a in neocortex and hippocampus pyramidal neurons and generated heterozygous Arid1afl/+ (WT), Arid1afl/+;Nex-Cre (cHet) mice and Arid1afl/fl;Nex-Cre (cKO) mice, respectively.

B. Left, representative images of Western blot for ARID1A expression in lysates extracted from the cortex of Arid1afl/+(WT), cHet, and cKO mice using the indicated antibodies. Right, quantification of protein levels normalized to β-actin (n = 3 mice per group).

C. Left, representative images of Western blot for ARID1A expression in lysates extracted from the hippocampi of Arid1afl/+, cHet, and cKO mice. Right, quantification of protein levels which were normalized to β-actin (n = 4 mice per group).

D. Immunostaining against ARID1A (red) in the dentate gyrus (DG), cortex, CA1, and CA3 regions of Arid1afl/+and cHet mice. Scale bar, 20 μm.

E. cHet mice spent more time reaching the platform during 5-day training in Morris water maze assay compared to the control group mice. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).

F. In the probe trial, cHet mice displayed spatial learning disabilities compared with Arid1afl/+ mice. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).

G. cHet mice undertook fewer platform crossings than Arid1afl/+ mice in the Morris water maze assay. Arid1afl/+ (n = 15 mice), cHet (n = 13 mice).

H-J. cHet mice were evaluated using the Barnes maze test. During the training phase, the cHet mice (n = 12 mice) failed to diminish the latency of the first entrance to the hiding box (H), indicating that Arid1a haploinsufficiency results in impaired spatial memory. Probe trials demonstrated that cHet mice also had spatial memory deficits (I), as they visited the target hole less often than the Arid1afl/+ mice (n = 12 mice) (J).

Data information: Data represent means ± SEM. In B, **P < 0.01 (cHet), * P < 0.05 (cKO), unpaired two-tailed t-test. In C, *P < 0.05 (cHet), ***P < 0.001 (cKO), unpaired two-tailed t-test. In E, P > 0.9999 (day 1), P = 0.8500 (day 2), **P < 0.01 (day 3), P = 0.0651 (day 4), *P < 0.05 (day 5), two-way ANOVA with Bonferroni post hoc test. In (F, G, I, J), *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. In H, **P < 0.01 (Trial 5), two-way ANOVA with Bonferroni post hoc test.
Acetate Supplementation Restores Cognitive Deficits Caused by ARID1A Haploinsufficiency in Excitatory Neurons (2022)
Figure 2

Figure 2. Deficient basal transmission and plasticity in hippocampal synapses with Arid1a haploinsufficiency

A. Representative traces of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in CA1 hippocampal acute slices from Arid1afl/+ and cHet mice.

B. Quantification of amplitude (B, left) and frequency (B, right) spontaneous miniature excitatory postsynaptic currents (mEPSCs) in Arid1afl/+ and cHet hippocampal CA1 neurons. Arid1afl/+ (17 neurons from n =3 mice) and cHet (15 neurons from n = 4 mice).

C. Representative recording of the paired-pulse ratio at the 50-ms interpulse interval ms from slices prepared from Arid1afl/+ (n = 4) and cHet (n = 4) mice.

D. Paired-pulse facilitation (PPF) studies across different interpulse intervals (20 ms, 40 ms, 50 ms, 100 ms, 200 ms, 300ms, and 500 ms) revealed a significant difference in paired-pulse ratios at 50-ms intervals.

E. Summary plots showing the time course of long-term potentiation (LTP) induced by frequency stimulation in the CA1 region from Arid1afl/+or cHet mice. Field excitatory postsynaptic potential (fEPSP) traces before (black) and after (black or red) are shown in the inset above.

F. Average amplitude of LTP measured at 55-60 min postinduction (n = 8 slices from 4 mice per group; 10 fEPSP slope (%) values were collected from 1 slice).

G. Representative Western blot images for the expression of several LTP-related proteins in the Arid1afl/+ and cHet hippocampi.

H. Quantification of the expression of each protein normalized to the β-actin level. Data information: Data represent means ± SEM. In (B, D, F, H), *P< 0.05, **P< 0.01, unpaired two-tailed t-test.
Acetate Supplementation Restores Cognitive Deficits Caused by ARID1A Haploinsufficiency in Excitatory Neurons (2022)
Figure 3

Figure 3. Haploinsufficiency of Arid1a in the excitatory neurons leads to impaired dendritic growth and synaptic loss in vivo

A. Golgi-stained coronal section in the hippocampal CA1 region of 2-month-old Arid1afl/+ and cHet mice. Scale bar, 100µm.

B-D. Partial loss of Arid1a significantly decreased dendritic length (B), dendritic nodes (C), and dendritic ends (D) in cHet neurons.

E, F. Sholl analysis showing that CA1 pyramidal neurons of cHet mice exhibited decreased basal (E) and apical (F) dendritic complexity compared with neurons of Arid1afl/+ mice. n = 6 mice.

G. Representative images of apical dendrites of CA1 pyramidal neurons are shown in (A). Scale bar, 5 μm.

H. Spine density per 10 μm was measured on secondary dendrites (at least 20 neurons from 4 mice for each group).

Data represent means ± SEM. In (B-F), *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Bonferroni post hoc test. In H, ***P < 0.001, unpaired two-tailed t-test.
Acetate Supplementation Restores Cognitive Deficits Caused by ARID1A Haploinsufficiency in Excitatory Neurons (2022)
Figure 4

Figure 4. Supplementation with acetate rescues cognitive deficits caused by Arid1a haploinsufficiency in excitatory neurons

A. Left, Western blot analysis of H3K27ac in forebrain tissues of Arid1afl/+and cHet mice. Right, relative quantitation of the intensity of H3K27ac normalized to the H3 level (n=3 mice per group).

B. Left, Western blot analysis of H3K27ac in forebrain tissues of vehicle- or acetate-treated cHet mice. Right, relative quantitation of the intensity of H3K27ac which was normalized to the H3 level (n=4 mice per group).

C. Experimental design showing the timeline for sodium acetate injection. cHet mice were treated with vehicle or acetate every day for 4 weeks.

D. Immunofluorescence (IF) staining of H3K27ac(green) and Vgult1(red) in forebrain tissues of Arid1afl/+ and cHet mice, respectively. IF staining was performed on 40-μm thick floating sections. Relative fluorescence intensities of H3K27ac decreased upon the haploinsufficiency of Arid1a in the cortex. Scale Bar, 20μm. Arid1afl/+ (n = 7) and cHet (n = 4) mice.

E. Immunofluorescence staining of H3K27ac (green) and Vgult1 (red) in cortex tissues of vehicle- or acetate-treated cHet mice, respectively. IF staining was performed on 40-μm thick floating sections. Relative fluorescence intensities of H3K27ac increased after treatment with acetate in the cortex of cHet mice. Scale Bar, 20μm (n=3 mice per group).

F-H. Morris water maze test of Arid1afl/+ and cHet mice treated with vehicle or acetate. Acquisition task, the latency to find the platform was used to assess learning ability. Latency to the platform was measured during training trials (F). Latency to the platform was measured during probe test (G). (H) Retention task, the times in the target crossings were used to assess memory retention. The times in the target crossings were measured in probe tests, n = 10-15 mice per group.

I-K. Barnes maze test of Arid1afl/+ and cHet mice treated with vehicle or acetate. (I) During the training phase, the time to find the target in Arid1afl/+ and cHet mice that were treated with vehicle or acetate for 28 days. (J) In probe test trials, the time of latency to the platform to find the target in Arid1afl/+ and cHet mice treated with vehicle or acetate. (K) In probe trials, the numbers of target crossings of Arid1afl/+ and cHet mice treated with vehicle or acetate (n = 12 mice)

Data are represented as mean ± SEM. In (A, B, D, E, G, H, J, K) *P < 0.05, **P <0.01, ***P < 0.001, unpaired two-tailed t-test.
Acetate Supplementation Restores Cognitive Deficits Caused by ARID1A Haploinsufficiency in Excitatory Neurons (2022)
Figure 7

Figure 7. Acetate supplementation rescues the decreased neurite complexity in hESCs-derived neurons caused by Arid1a deletion

A. Representative image of vehicle- and acetate-treated neurons derived from WT or ARID1A KO hESCs on day 40 of neural differentiation Scale Bar, 20μm

B. Quantification of total neurite length of hESC-derived neurons after treatment with acetate or vehicle. n = at least 34 neurons for each group from 3 independent experiments.

C. Sholl intersection profiles of neurons treated with vehicle or acetate on day 40 of neural differentiation (n = at least 34 neurons for each group from 3 independent experiments). n.s., non-significant.

D. Representative images of dendrites (MAP2, green) showing localization of foci of the pre- and postsynaptic protein complexes, synaptophysin (red), and PSD-95 (white) protein in control, acetate treatment of WT and ARID1A KO hESC-derived neurons on day 55 of neural differentiation. Scale Bar, 5μm.

E, F. Quantification of the spine numbers from synaptophysin (E) and PSD-95 protein (F) stained secondary dendrites of neurons on day 55 of neural differentiation (Data are representative of three independent experiments.)

G. qRT-PCR analysis of genes related to synaptic transmission in the WT and ARID1A KO hESC-derived neurons on day 55 of neural differentiation (Data are representative of three independent experiments.)

H. qRT-PCR analysis of genes related to synaptic transmission in the WT and ARID1A KO hESC-derived neurons treated with acetate or vehicle on day 55 of neural differentiation. (Data are representative of three independent experiments.)

Data are represented as mean ± SEM. In (B, E, F, G, H) *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed t-test. In C, *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA.

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RGI-GOLVEN signaling promotes cell surface immune receptor abundance to regulate plant immunity (2022)
Figure 1

Figure 1. GLVs are positive regulators of flg22-triggered PTI.

(A) ROS burst in wild type (WT) and 35S::GLV1 and 35S::GLV2 after elicitation with 100 nM flg22. The kinetic of ROS production is shown over 40 min -/+ SE, n=8 biological replicates. RLU = Relative light units. Similar results were obtained in 3 independent experiments.

(B) 5-day-old seedlings of WT, 35S::GLV1 16-1 and 35S::GLV2 8-5 were treated with 100 nM flg22 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=8 biological replicates (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05). Similar results were obtained in 3 independent experiments.

(C) Mean colony-forming units (cfu) of Pto DC3000 COR- 4 days after spray infection of WT, 35S::GLV1 16-1 and 35S::GLV2 8-5; n=12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test; a-b, p<0.01).

(D) 5-day-old seedlings of Col-0Cas9 WT and CRISPR glv were treated with 100 nM flg22 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=40 biological replicates from 5 pooled experiments (Students T-test, ***p<0.001).

(E) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22. Shown is the mean of n=12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b, p<0.001; a-c, p<0.001; b-c, p<0.001). Ethylene production was normalized to the average of WT responses to flg22 set as 1.

(F) 5-day-old Col-0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=30-32 biological replicates from 2 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001).

(G) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22 or co-treatment with 1 µM GLV2. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=11-12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.01).

(H) Mean cfu of Pto DC3000 COR- 4 days after spray inoculation of Col-0Cas9 WT and CRISPR glv; n=12 biological replicates from 3 pooled experiments-/+ SD (Students T-Test, *p<0.05).
RGI-GOLVEN signaling promotes cell surface immune receptor abundance to regulate plant immunity (2022)
Figure 2

Figure 2. GLV2 treatment enhances flg22-triggered responses.

(A) ROS burst in WT after elicitation with 100 nM flg22 or 1 μM GLV2. Shown is the kinetic of ROS production over 40 min -/+ SEM, n=8 biological replicates. RLU = Relative light units. Similar results were obtained in 3 independent experiments.

(B) ROS burst in WT after elicitation with 10 nM flg22, 1 μM GLV2, co-treatment or pre-treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22. Shown is the mean of total ROS production (Total RLU) over 40 minutes normalized to the flg22 response set as 100% -/+ SD, n=96-120 biological replicates from 15 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b/c/d, p<0.001; b-c, p<0.05; b-d, p<0.001).

(C) Ethylene production was measured in WT after elicitation with 500 nM flg22, 1 μM GLV2 or co-treatment for 3.5 h. Ethylene production was normalized to the average flg22 response set as 1. Shown is the mean of n=12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b/c p<0.001; b-c, p<0.01).

(D) PR1 expression was measured in 10-day-old WT seedlings treated for 24 h with 100 nM flg22, 1 μM GLV2 or co-treatment. Expression was normalized to the UBQ house-keeping gene. Shown is the mean of n=4. Symbol colors represent different biological replicates

(E) 5-day-old WT seedlings were treated with 10 nM flg22, 1 μM GLV2 or co-treated for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=12 biological replicates (Students T-test, ns = not significant, **p<0.01). Similar results were obtained in 3 independent experiments.

(F) Mean cfu of Pto DC3000 2 days after syringe inoculation and pre-treatment with 1 μM GLV2; n=20 biological replicates from 5 pooled experiments -/+ SD (Students T-test, *p <0.05).