EMBO/soda-nel huggingface dataset:
Infantile spasms-linked Nedd4-2 mediates hippocampal plasticity and learning via cofilin signaling (2021)
Figure 6

Figure 6. Inhibition of cofilin partially restores learning and memory behavior in Nedd4-2 cKO mice.

(A) Experimental scheme for contextual fear conditioning, Barnes maze, and novel object recognition tests with intraperitoneal injections of TAT or TAT-pS3 peptide (2 mg/kg) in Nedd4-2 WT or Nedd4-2 cKO mice at 6-8 weeks of age.

(B) Quantification of freezing behavior in a fear conditioning test after intraperitoneally injecting TAT or TAT-pS3 peptides in Nedd4-2 WT mice (left) or Nedd4-2 cKO mice (right) (n = 8).

(C) Barnes maze test after intraperitoneally injecting TAT (n = 8) or TAT-pS3 (n = 8) peptide in Nedd4-2 WT mice (top), or TAT (n = 9) or TAT-pS3 (n = 8) peptides in Nedd4-2 cKO mice (bottom). Learning curve during 4 training days of Barnes maze task showing the escape latency, number of errors before escaping, and the quadrant occupancy on probe trial at day 5 and at day 12 were assessed.

(D) Novel object recognition test after intraperitoneally injecting TAT or TAT-pS3 peptides in Nedd4-2 WT mice (left) or Nedd4-2 cKO mice (right) (n = 8). Exploration time on familiar and novel objects and preference index for the novel object on testing session were shown. The dash line represents the 50% chance of exploring either of the objects.

Data information: Student's t-test was used. Data are represented as mean ± SEM with *p < 0.05, **p < 0.01 and NS: non-significant.
Vitamin D receptor enhances the NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association (2022)
Figure 1

Figure 1. VDR deficiency impairs NLRC4-dependent inflammasome activation

A-H LPS-primed WT and Vdr-/- BMDMs were infected with 50 MOI S. Typhimurium for 2 h. Culture supernatants (SN) and cell lysates(Lysate) were collected and immunoblotted with the indicated antibodies（A）. (B-E) IL-1β secretion(B), IL-18 secretion (C) and TNF-ɑ secretion (D) and LDH release(E) in supernatants. (F) HBVLV-Vdr expression in Vdr-/- BMDMs by lentivirus-mediated transduction primed with LPS and infected with 50 MOI S. Typhimurium for 2 h, cell lysates, and culture supernatants (SN) were collected and immunoblotted with the indicated antibodies. (G) LPS-primed WT and Vdr-/- BMDMs were treated with 1 µg/ml FLIC (LFn-Flagellin and PA) or PrgJ （1 µg/ml LFn- PrgJ and PA）for 1 h, total mixtures( culture supernatants and cell lysates) were collected and immunoblotted with the indicated antibodies. (H) LPS-primed WT, Vdr-/-, NLRC4-/- and Vdr-/-NLRC4-/- BMDMs were treated with FLIC for 1 h, Culture supernatants (SN) and cell lysates were collected and immunoblotted with the indicated antibodies. (I-L) IL-1β secretion(I), IL-18 secretion (J) and TNF-ɑ secretion (K) and LDH release(L) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; *, P < 0.05; **, P < 0.01 In each panel, data are representative of at least three independent experiments.
Vitamin D receptor enhances the NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association (2022)
Figure 3

Figure 3. NLRP3 is not involved in the protective role of VDR in the defense against S. Typhimurium infection.

A-F LPS-primed WT, Vdr-/-, NLRP3-/- and NLRP3-/- Vdr-/- BMDMs were infected with different MOI (0,10,50) S. Typhimurium for 2 h.Culture supernatants (SN) and Cell lysates were collected and immunoblotted with the indicated antibodies(A).ELISA detected TNF-ɑ(B)andIL-1β (C) in supernatants. Bacterial burden was detected in spleens(D), PCF(E), and blood (F)of WT, Vdr-/-, NLRP3-/-, and NLRP3-/- Vdr-/- mice 24 h after S. Typhimurium infection. Data information:n ≥ 3 biological replicates. Data are presented as the mean ± SEM; determined by Student's t-test ; **, P < 0.01.
Vitamin D receptor enhances the NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association (2022)
Figure 4

Figure 4. VDR deficiency reduces ASC speck formation upon NLRC4 inflammasome activation

A LPS-primed WT and Vdr-/- BMDMs were infected with S. Typhimurium at an MOI of 50 for 2 h. Endogenous ASC specks (arrows) were represented and quantified by immunofluorescence images. The data show representative results from three independent experiments: scale bar, 20 μm. Data are presented as the mean ± SEM; determined by Student's t-test ; ***,p < 0.001.

B ASC oligomerization induced by the indicated stimuli in WT and Vdr −/− BMDMs primed with LPS.
Vitamin D receptor enhances the NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association (2022)
Figure 5

Figure 5. VDR interacts with NLRC4

A-F LPS-primed BMDMs were infected with S. Typhimurium at an MOI of 50 for 2h. Lysates were immunoprecipitated (IP) using an anti-VDR antibody and were immunoblotted with the indicated antibodies(A). Whole-cell lysates were shown as the input. (B-C) Flag-VDR was co-expressed with Myc-NLRC4 in HEK293T cells; proteins were immunoprecipitated and analyzed by immunoblotting with indicated antibodies. Whole-cell lysates were shown as the input. (D) Purified GST-VDR was incubated with purified His-NLRC4 for 2 h. His-NLRC4- bound to GST-VDR was pulled down by glutathione beads and subjected to immunoblot analysis. (E) WT or mutant NLRC4 (CARD, NACHT, or LRR (leucine-rich repeat)) and HA-VDR were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies.*no specific band. (F) WT or mutant VDR (DBD or LBD) and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated, and analyzed by immunoblotting with indicated antibodies. Data are representative of three independent experiments.
Vitamin D receptor enhances the NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association (2022)
Figure 6

Figure 6. VDR enhances NAIP interaction with NLRC4

A-B HEK293T cells were transfected with Flag-NLRC4, VDR, Myc-FLagellin, and HA-NAIP5 (A) or Flag-NLRC4, VDR, Myc- PrgJ and HA-NAIP2(B), or Flag-NLRC4, LBD-VDR, Myc-FLagellin, and HA-NAIP5 (C) or Flag-NLRC4, LBD-VDR, Myc-PrgJ, and HA-NAIP2 (D). Samples were immunoprecipitated with the anti-Flag and analyzed by immunoblotting with indicated antibodies.
Vitamin D receptor enhances the NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association (2022)
Figure 7

Figure7. VDR K123A mutation attenuates VDR regulation of NLRC4 inflammasome activation

A Predict possibility of interaction amino acids between lys123 of VDR and NLRC4.

B VDR or mutant VDR(K123A)and Myc-NLRC4 were expressed in HEK293T cells, immunoprecipitated with the anti-Flag antibody, and analyzed by immunoblotting with indicated antibodies.

C Transfected VDR or mutant VDR(K123A) and Flag-NLRC4, Myc- PrgJ, and HA-NAIP2 in HEK293T cells, immunoprecipitated with the anti-HA antibody, and analyzed by immunoblotting with indicated antibodies.

D Vdr-/- BMDMs were infected with lentivirus, which expressed Vector(NC), VDR, and VDR-K123A, infected with S. Typhimurium at an MOI of 50 for 2 h, cell lysates(Lysate) and culture supernatants(SN) were collected and immunoblotted with the indicated antibodies.

E-H LDH release(E), IL-1β secretion(F), IL-18 secretion (G) and TNF-ɑ secretion (H) in supernatants. Data are shown as means ± SEM; determined by Student's t-test; **, P < 0.01; ***, P <0.001; In each panel, data are representative of at least three independent experiments.

Expanded View Figure Legends
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 3

Determination of accumulation of autophagosomes using GFP-ATG8a.

(A) Four-weeks old transgenic wild-type Col-0 (WT) and atg7-2 mutant plants expressing GFP-ATG8a were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (B) Numbers of punctate GFP-ATG8a spots representing autophagosomes per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 4

Increased formation of punctate structures containing GFP-NBR1 under heat stress.

(Top panel) Four-weeks old transgenic wild-type Col-0 (WT), atg7-2 and nbr1-1 mutant plants expressing GFP-NBR1 were treated with (45°C) or without (22°C) heat shock for 3 h and then placed at room temperature for 0.5 h. The leaves were visualized by fluorescence confocal microscopy of GFP signal. (Bottom panel) Numbers of punctate GFP-NBR1 spots per 10,000 µm2 section. Means and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter. Bar = 20 µm.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 5

Enhanced sensitivity of <italic>atg5</italic>, <italic>atg7</italic> and <italic>nbr1</italic> mutants to heat and oxidative stresses.

(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth for 10 hours and then moved to room temperature for 3-day recovery or (B) sprayed with 20 µM methyl viologen (MV) and kept under light for two days before the picture of representative plants was taken. The experiments were repeated three times with similar results.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 6

Enhanced sensitivity of <italic>atg5</italic>, <italic>atg7</italic>, and <italic>nbr1</italic> mutants to heat stress.

(A) Electrolyte leakage. (B) Fv/Fm images. (C) Average values for the Fv/Fm images. Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed in 22°C and 45°C growth chambers. Fully expanded leaves were sampled after 10 hours in the indicated temperatures and immediately measured for electrolyte leakage and Fv/Fm. Error bars in (A) and (C) indicate SE (n = 5). According to Duncan's multiple range test (P = 0.05), means of EL or Fv/Fm do not differ significantly if they are indicated with the same letter. The experiments were repeated twice with similar results.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 7

Enhanced sensitivity of <italic>atg5</italic>, <italic>atg7</italic>, and <italic>nbr1</italic> mutants to drought and salt stresses.

(A) Five weeks-old Arabidopsis Col-0 wild type (WT), atg5, atg7 and nbr1 mutant plants were placed into a growth chamber with approximately 50% humidity. The photograph of representative plants was taken 10 days after withholding watering. The experiment was repeated twice with similar results. (B) Seven days-old seedlings Col-0 WT, atg5, atg7 and nbr1 grown on solid MS medium were transferred to the same medium (control) or the same medium containing 0.16 M NaCl and photographed 5 days later. The survived seedlings were scored 5 days after the transfer and the average values and SE were calculated from three experiments. According to Duncan's multiple range test (P = 0.05), means do not differ significantly if they are indicated with the same letter.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 8

Normal phenotypes of the <italic>nbr1</italic> mutant plants in age- and darkness-induced senescence.

(A) Eight weeks old wild type (WT), atg5, atg7 and nbr1 plants grown under normal growth conditions in a growth chamber. (B) Four weeks old WT, atg5, atg7 and nbr1 plants after being kept in the dark for 6 days. (C) Percentage of plants that survived the various lengths in dark as determined by resumption of growth. Each point represents the average of 10 plants. The experiments were repeated twice with similar results.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 9

Normal phenotypes of the <italic>nbr1</italic> mutant plants in resistance to <italic>Botrytis</italic>.

(A) Responses of Col-0 wild type (WT), atg5, atg7 and nbr1 plants to Botrytis. Wild-type and mutant plants were inoculated by spraying with spore suspension at a density of 2.5×105 spores ml−1, and kept at high humidity. Pictures of representative plants were taken at 4 and 5 days post inoculation (dpi). (B) Quantitative real-time PCR analysis of the B. cinerea ActA (BcActA) transcript levels in infected Arabidopsis plants at 5 dpi. The experiments were repeated twice with similar results.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 10

Requirement of the LIR motif for the critical role of NBR1 in heat tolerance.

(A) Diagrams of wild-type and mutant NBR1 proteins. The conserved W and I residues in the LIR motif between the two UBA domains were changed to A residues in the NBR1W661A/I664A (mNBR1) mutant protein. (B) Five weeks-old Arabidopsis Col-0 wild type (WT), nbr1 and transgenic nbr1 plants expressing the wild-type NBR1 (lines 3 and 6; see Figure S2) or the NBR1W661A/I664A (mNBR1) (lines 2 and 6; see Figure S2) mutant gene were placed in a 45°C growth chambers for 10 hours and then moved to room temperature for 3-day recovery before the picture as taken. The experiment was repeated three times with similar results.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 11

Accumulation of NBR1 and its increased enrichment in the insoluble protein fraction in the <italic>atg7</italic> mutant under heat stress.

Leaf tissues from wild type (WT) and atg7 expressing NBR1-TAP were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with a peroxidase-conjugated anti-peroxidase antibody for detection of NBR1-TAP. Protein samples prepared from non-transgenic Col-0 wild-type plants were included as controls.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 12

Increased accumulation of insoluble protein aggregates in the <italic>atg7</italic> and <italic>nbr1</italic> mutants under heat stress.

(A) Accumulation of insoluble proteins. Leaf tissues from wild-type (WT), atg7 and nbr1 mutants collected at indicated hours (h) under 45°C for preparation of total, soluble and insoluble proteins as described in Materials and Methods. Total proteins in the starting homogenates and insoluble proteins in the last pellets were determined the percentages of insoluble proteins to total proteins were calculated. (B) Profiles of soluble and insoluble proteins. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and stained with Coomassie brilliant blue. Major proteins accumulated in the pellets from heat-stressed atg7 mutant plants were indicated by arrows.
NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses. (2013)
Figure 13

Ubiquitination of insoluble protein aggregates in the <italic>atg7</italic> and <italic>nbr1</italic> mutants under heat stress.

Leaf tissues from wild type (WT), atg7 and nbr1 mutants were collected at indicated hours (h) under 45°C and prepared for soluble and insoluble proteins as described in Materials and Methods. Proteins from the first supernatants (S) and last pellets (P) were subjected to SDS-PAGES and probed with anti-ubiquitin monoclonal antibody. The experiment was repeated three times with similar results.
Methamphetamine inhibits antigen processing, presentation, and phagocytosis. (2008)
Figure 1

Meth Induces Rapid Alkalization of Acidic Organelles, and Changes in Endosomal Structure in Dendritic Cells

(A) Morphometric analysis of the decrease in acridine orange staining after 10 min treatment. Similar results were obtained in three independent experiments. (Statistics n = 25–35, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)

(B) Progressive quenching of cytosolic acridine orange staining over 4 h in the absence () or presence (▴) of 50 μM Meth. p < 0.001 for all time points versus control (n = 25–35, mean ± SEM, Student's two-tailed t-Test).

(C) Acidic organelle pH in cells exposed to Meth for 10 min measured by ratiometric measurement. (Statistics n = 30–90 cells, mean ± SEM, **p < 0.001, ***p < 0.0001, two-tailed ANOVA.)

(D) Representative electron micrographs showing multivesicular endosomal compartments (arrows) in control, Clq- and Meth-treated cells at 4 h. Scale bar, 200 nm. (E) Ultra-thin cryosections of control, Clq- and Meth-treated dendritic cells as in (D), immunolabeled for LAMP-1 (15 nm gold particles) and MHC class II (10 nm gold particles). Scale bar, 200 nm.
Methamphetamine inhibits antigen processing, presentation, and phagocytosis. (2008)
Figure 3

Meth Induces Autophagosome Accumulation in Dendritic Cells

(A) Accumulation of GFP-LC3 labeled autophagosomes in Meth- or Clq-treated dendritic cells at 2 h and 24 h. Fisher's exact test P values versus control (n =300–400 cells, mean ± SEM, *p < 0.05, ***p < 0.0001).

(B) GFP-LC3 labeled autophagosome formation in different concentrations of Meth after 24 h treatment. Similar results were obtained in three independent experiments. Fisher's exact test versus control (n = 300–400 cells, mean ± SEM, ***p < 0.0001).

(C) Representative fluorescence micrographs of GFP-LC3 decorated autophagosomes in cells treated with 50 μM Meth or 10 μM Clq for 24 h. Arrow denotes examples of autophagosomes. Scale bar, 10 μm.
Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport (2020)
Figure 5

Fig. 5 | Impact of Ser439 on PIN2 localization in roots supplemented with ammonium or nitrate.

a - c. Pseudo-colored, optical longitudinal cross sections of 5 DAG roots expressing (a) PIN2-GFP (PIN2::PIN2-GFP, P2wt) (b) PIN2S439D-GFP (PIN2::PIN2S439D-GFP, P2D) and (c) PIN2S439A (PIN2::PIN2S439A-GFP, P2A) - all in eir1-4 background - 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=50 µm.

d. Box plots display the distribution of the cell membrane derived PIN2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) in roots transferred to ammonium ((grey, epidermis (ep) and red, cortex (co) and to nitrate (blue, epidermis (ep) and green, cortex (co)). 5 cells per roots were analyzed in at least 9 roots per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.

e and f. Microscopic images of 5 DAG old roots expressing (e) PIN2::PIN2S439D-GFP and (f) PIN2::PIN2S439A-GFP 12 HAT to ammonium or nitrate amended media. "e" denotes epidermis and "c" cortex, respectively. White arrows point to PIN2-GFP protein localization on the lateral membranes.

g. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes vs inner/outer membranes) of P2wt, P2D and P2A roots transferred to ammonium (grey) or nitrate (red) supplemented medium. At least 24 cells from 5 roots were analyzed per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.
Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion. (2019)
Figure 1

Figure 1. Assessment of endosome-associated PI4P pools.

(A) Cartoon depicting the principle of the endosomal PI4P BRET measurement. The Venus fluorescent protein is targeted to the respective endosomes using Rab proteins, while Renilla luciferase [termed Super luciferase (Sluc)] is fused to the P4M2x PI4P reporter. This reporter binds to PI4P the majority of which is in the PM. The PI4KA inhibitor, GSK-A1 (A1) reduces PI4P in the PM allowing more of the reporter to find the endosomal PI4P that is produced by PI4Ks that are insensitive to GSK-A1. Relocation of the P4M2x reporter tagged with Sluc to the PI4P-rich endosomes will increase the energy transfer between the enzyme and Venus in the presence of the coelenterazine H substrate.

(B) Representative confocal images show the distribution of the P4M2x reporter in HEK293-AT1 cells before and after the addition of GSK-A1 (30 nM). Note the signal disappearing from the PM and strongly highlighting the endosomes and Golgi. Scale bars are 20 µm.

(C) Representative result from a BRET experiment detecting PI4P in various endosomes (Rab7, Rab5 and Rab11) following addition of GSK-A1. The graph shows normalized BRET values where the averages of triplicate measurements of A1-treated cells were divided by the averages of triplicates obtained in DMSO-treated controls. Note that these signals originate from thousands of cells.

(D) Similar BRET experiment showing the Rab7-pool of PI4P both in control HEK293-AT1 cells and in two clones of cells with PI4K2A deletion (#20 and #26). Means ± S.E.M. are shown from three experiments performed in triplicates.
Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKMH1 membrane cycling during autophagosome-lysosome fusion. (2019)
Figure 2

Figure 2. Restoration of endosomal PI4P by re-expression of PI4K2A and distribution of Rab7 in PI4K2A K/O cells.

(A) BRET measurement of PI4P in Rab7 compartment in parental and PI4K2A K/O cells after expression of HA-tagged PI4K2A or its kinase-dead mutant. Means ± S.E.M. are shown from three experiments performed in triplicates.

(B) Western blot analysis shows that the level of PI4K2A expression was closely matched with that of wild-type cells.

(C) Distribution of GFP-Rab7 in parental (left panels) and two clones of PI4K2A K/O cells. Representative pictures show tubulation in the K/O cells reminiscent of those seen in wild-type cells expressing the GTP-locked Rab7Q67L mutant. Scale bars are 10 µm.