EMBO/soda-nel huggingface dataset:
Inhibition of O-GlcNAcylation protects from Shiga toxin-mediated cell injury and lethality in host (2021)
Figure 4

Figure 4. Stx2a-mediated apoptotic and pro-inflammatory responses in primary human renal cells are regulated through O-GlcNAcylation

A. Representative western blot showing the changes of O-GlcNAcylation in primary human renal proximal tubule epithelial (HRPTEpi) cells (originating from fetal or adult kidney) treated with different concentrations (0-10 ng/mL) of Stx2a for 24 h.

B. LDH cytotoxicity assay following Stx2a (10 ng/mL) treatment of HRPTEpi (adult) cells for 48 h in the presence or absence OSMI-1 (10 µM, final) (n = 3 biological replicates). The effects of Stx2a in vehicle controls were compared with culture medium prepared from HRPTEpi cells maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a.

C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on cytokine/chemokine production from HRPTEpi cells (adult) exposed to Stx2a (10 ng/mL) for 48 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) control.

D-F. Global transcriptome analysis of differentially expressed genes of HRPTEpi cells during Stx2a intoxication with vehicle or 10 µM of OSMI-1, respectively. (D) Venn diagram of overlapping differentially expressed genes (DEGs) in the presence or absence OSMI-1 treatment among Stx2a inducible genes compared to cells cultured without Stx2a. (E) Pie-chart showing genes significantly regulated by OSMI-1 in the Stx2a-exposed HRPTEpi cells as categorized by function. (F) Heat-maps representing the comparative expression levels for DEGs in the presence or absence of OSMI-1 treatment upon Stx2a intoxication related to the inflammatory response (left) and apoptotic process (right). The numbers within the tables are normalized gene expression level compared to HRPTEpi cells maintained in the absence of Stx2a. Expression values are represented with red (upregulation) or blue (downregulation) color using FPKM values by Cufflinks; the cutoffs used a fold change of at least 1.5 followed by pairwise comparison and Student's t test with a Benjamini and Hochberg correction. The FPKM values were normalized using EdgeR within R and visualized using ExDEGA. Data are the means from two independent replicates.
Data information: For graphs in (B and C), error bar represents mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
Inhibition of O-GlcNAcylation protects from Shiga toxin-mediated cell injury and lethality in host (2021)
Figure 5

Figure 5. Protection from Stx2a-induced apoptotic and inflammatory responses in 3D-renal spheroids by inhibition of O-GlcNAcylation

A. Representative images showing morphological changes in spheroids from 3D cultures of HRPTEpi cells following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final).

B. Quantification of estimated spheroid diameter in (A) [n = 11 independent spheroids (biological replicates)]. The effects of Stx2a in vehicle controls were compared with those of spheroids maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a.

C. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of HRPTEpi cells exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls.

D. Representative images showing morphological changes in spheroids from 3D cultures of human mini-kidneys following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final).

E. Quantification of estimated spheroid diameter in (D) [n = 11 independent spheroids (biological replicates)]. The effects of Stx2a in vehicle controls were compared with those of spheroids maintained in the absence of Stx2a, and OSMI-1 treatment was compared with that of the vehicle (DMSO) controls in the presence of Stx2a.

F. ELISAs were used to analyze the inhibitory effect of OSMI-1 on IL-8 and CCL-2 production from 3D cultures of human mini-kidneys exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls.

G, H. Human apoptosis antibody array analysis of multiple proteins using pooled lysate from 3D-human mini-kidney spheroids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (G) Antibody spots representing signal differences are indicated in red boxes. (H) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Raw values of fluorescence intensities are provided in the Source Data.

Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
Inhibition of O-GlcNAcylation protects from Shiga toxin-mediated cell injury and lethality in host (2021)
Figure 6

Figure 6. Downregulation of O-GlcNAcylation protects iPSC-derived human kidney organoids from injury caused by Stx2a exposure

A. Representative immunofluorescence images of the nephron structures within kidney organoids derived from hiPSCs on day 18 of the differentiation; proximal tubules (LTL, green), parietal epithelial cells (PAX8, purple), kidney glomerular podocytes (NPHS1, red), nephron progenitors (WT1, purple and SIX2, green). Cell nuclei were stained with DAPI. Scale bars: 50 µm.

B. Representative images showing morphological collapses in iPSC-derived human kidney organoids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final).

C, D. ELISAs were used to analyze the inhibitory effect of OSMI-1 (10 µM, final) on Kidney injury molecule-1 (KIM-1) secretion (C) and IL-8 and CCL-2 production (D) in culture supernatants of iPSC-derived human kidney organoids exposed to Stx2a (10 ng/mL) for 72 h (n = 3 biological replicates). The effects of OSMI-1 were compared with those of the vehicle (DMSO) controls.

E, F. Human apoptosis antibody array analysis of multiple proteins using pooled lysates from iPSC-derived human kidney organoids following Stx2a (10 ng/mL) treatment for 72 h in the presence or absence OSMI-1 (10 µM, final). (E) Antibody spots representing signal differences are indicated in red boxes. (F) The graph shows the average of relative spot intensities for each protein compared to those measured in the control in the absence of Stx2a exposure (n =2 biological replicates). Dashed line represents the reference point of the fold change. Raw values of fluorescence intensities are provided in the Source Data.

Data information: Error bars for bar graphs are presented as mean ± S.E.M. Statistical analysis was performed using two-tailed Student's t-test. *P < 0.05; **P < 0.01; and ***P < 0.001.
ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange (2021)
Figure 1

Figure 1. LDL enhances FA dynamics in a FAK-dependent manner.

A, Schematic outline of plasma membrane D4H labeling. CM and LPDS indicate complete medium (10 % FBS in DMEM) and 5 % LPDS in DMEM, respectively.

B, After 24 h LDL depletion in 5 % LPDS, cells were loaded with 50 μg/ml LDL for the indicated times. Plasma membrane cholesterol was labeled with D4H and imaged with widefield epifluorescence microscopy.

C, Analysis of B. Mean ± SD, n = 15 fields of cells as in B pooled from 2 independent experiments.

D, Representative FA lifetime in cells treated without or with 50 μg/ml LDL (-LDL or +LDL) for 1-2 h after 24 h in 5 % LPDS. Time-lapse imaging of GFP-paxillin dynamics in cells was acquired by TIRF microscopy and maximum intensity during FA lifetime was normalized to 1.

E, Analysis of FA assembly and disassembly rate of cells incubated +/-LDL as in D. Where indicated, 10 μM PF228 was added after 30 min of LDL loading. Assembly rate: n = 290 FAs (-LDL); 410 (+LDL); 192 (+LDL+PF228), disassembly rate: n = 397 (-LDL); 502 (+LDL); 214 (+LDL+PF228) pooled from 4 independent experiments. The Tukey box plots represent the median and 25th and 75th percentiles (interquartile range). The whiskers show the hightest and lowest observations. Non-parametric Kruskal-Wallis analysis of variance with Bonferroni error correction.

F, Representative immunoblot and quantification of pFAK levels in 5% LPDS (-LDL) and upon increasing LDL loading times. Mean ± SD, n = 3, paired student's t-test (*p < 0.05).

G, Representative confocal images of NPC1, internalized integrin β1 and pFAK localization. Stably NPC1-GFP expressing cells were loaded with 2 μg/mL integrin β1 antibody for 30 min before fixation and stained with pFAK antibody followed by secondary antibodies. Dashed line indicates the cell edge.

H, Quantification of G using Mander's colocalization for the indicated markers. Mean ± SD, n = 5 cells. Cells were treated with 5 % LPDS for 1 day and then without (-) or with (+) 50 μg/ml LDL for 1h.
ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange (2021)
Figure 2

Figure 2. LDL-cholesterol is rapidly delivered to FAK/integrin endosomes.

A, Outline of live-cell BODIPY-cholesteryl linoleate/LDL labeling and chase combined with AF680-integrin β1 antibody labeling. Cells were pre-treated for 1 day in 5 % LPDS.

B, Quantification of the fraction of BODIPY-cholesterol (BC) fluorescence in integrin β1 positive organelles at the indicated chase times. Examplary images are shown in Fig. EV2B. Mean ± SEM, n = 8 (0 min); 6 (15 min); 5 (30 min); 6 (45 min); 7 (60 min) cells from 3 independent experiments.

C, Schematic outline of intracellular D4H labeling.

D, After 1 day LDL depletion in 5 % LPDS, cells were loaded with 50 μg/ml LDL for the indicated times and intracellular cholesterol was detected with D4H as in C by confocal microscopy. CM, complete medium. Dashed lines indicate cell outlines.

E, Quantification of D. Mean ± SD, n = 25-26 cells pooled from 2 independent experiments.

F, Cells were treated for 1 day in 5 % LPDS followed by 50 μg/ml LDL loading for the indicated times. Representative confocal images of cells triple labeled with intracellular accessible cholesterol (D4H), NPC1-GFP and internalized integrin β1. Dashed lines indicate cell outlines.

G, Quantification of D4H area positive for NPC1 or internalized integrin β1 as in F.

H, Fraction of D4H area overlapping with NPC1 or internalized integrin β1 in a peripheral region toward the leading edge (exemplary box shown as inset in F). Mean ± SD, n = 27-30 cells pooled from 2 independent experiments. Student's t-test.
ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange (2021)
Figure 3

Figure 3. FAK activity regulates cholesterol delivery to FAK/integrin endosomes and endomembrane PI(4,5)P2 levels.

A, After 1 day in 5 % LPDS, NPC1-GFP expressing cells were loaded with 50 μg/ml LDL with or without 10 μM PF228 for the indicated times, and stained for intracellular D4H. Representative confocal images of intracellular D4H, NPC1-GFP and internalized integrin β1 labeling. Dashed lines indicate cell outlines.

B, Fraction of D4H positive area in the cell as in A.

C, Fraction of D4H area overlapping with NPC1 in a peripheral region toward the leading edge (exemplary box shown as inset in A).

D, Quantification of D4H area positive for integrin β1 in a peripheral region toward the leading edge. Mean ± SD, n = 26-30 cells pooled from 2 independent experiments. Student's t-test.

E-G, Control and FAK siRNA treated cells were LDL depleted for 1 day, loaded with LDL for the indicated times, and stained for plasma membrane D4H or for intracellular D4H with lamp1 or integrin β1 antibodies. Quantification of plasma membrane D4H (E) and intracellular D4H area positive for lamp1 (F) or integrin β1 (G); corresponding images in Fig EV3D, E. Mean ± SD, n = 20-35 cells from 2 independent experiments. Student's t-test.

H, Schematic of FAK-regulated PI(4,5)P2 generation on endosomes via PIPKIγ, modified from (Nader et al., 2016).

I, After 1 day 5 % LPDS, cells were loaded with 50 μg/ml LDL with or without 10 μM PF228 for the indicated times and endomembrane PI(4,5)P2 was stained with antibody and imaged by confocal microscopy.

J, Quantification of mean PI(4,5)P2 immunoreactivity in cells. Mean ± SD, n = 16-26 cells pooled from 2 independent experiments. Student's t-test.
ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange (2021)
Figure 4

Figure 4. Endogenous ORP2 localization and acute depletion via auxin-inducible degradation. A, Schematic of endogenous ORP2 tagging and auxin-inducible degron system.

B, Control (endogenously GFP-tagged ORP2 without AtAFB2) and degron ORP2 cells were co-plated and treated with IAA for 1 h followed by live-cell confocal imaging. Dotted line separates GFP-ORP2 containing and depleted cells, asterisk indicates the area of ORP2-depleted cells, inset shows no prominent PM enrichment of endo-GFP-ORP2.

C, Confocal image of endo-GFP-ORP2 and pFAK immunofluorescence staining in saponin pre-permeabilized cells loaded with LDL for 2 h before fixation. GFP antibody was used to enhance endo-GFP-ORP2 signal. Insets show examples of "no contact", "contact" and "overlap" of organelles. Dashed line indicates cell edge.

D, Quantification of pFAK, integrin β1, and NPC1 association with endo-GFP-ORP2 in C. n = 380 pFAK; 353 integrin β1; 232 NPC1 organelles in 2 cells.

E, Representative confocal images of triple labeling for endogenous ORP2, NPC1-mCherry and integrin β1 after 2 h of LDL loading, and saponin permeabilization prior to fixation. Dashed line indicates cell edge.

F, Schematic outline for plasma membrane D4H labeling in combination with ORP2 degron system, and representative widefield epifluorescent images of co-plated cells after 4 h of LDL loading. Asterisk indicates ORP2-depleted degron cells. Dashed lines separate ORP2 expressing and depleted cells.

G, Quantification of F. Mean ± SD, n = 20 fields of cells pooled from 2 independent experiments. Student's t-test.

H-J, Control and degron-GFP-ORP2 cells were incubated in 5 % LPDS for 1 day, loaded with LDL + IAA for the indicated times, and LDLR (H) and hydroxymethylglutaryl- CoA reductase (HMGCR) (I) mRNA levels were quantified with qPCR or [3H]-oleic acid (OA) incorporation into cholesteryl esters (CE) was analyzed (J). Mean ± SD, n = 2-3 RNA samples from independent experiments with triplicate measurements (H,I); n = 3 (-LDL and 6 h); n = 6 (all other time points) pooled from 2 independent experiments (J).
ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange (2021)
Figure 5

Figure 5. Acute removal of endogenous ORP2 impairs LDL-cholesterol transfer from late endosomes to FAK/integrin endosomes.

A, Control and degron ORP2 cells were co-plated, and after 1 day incubation in 5 % LPDS, cells were loaded with LDL (+LDL) and IAA (+IAA; ORP2 depletion) for the indicated times. Intracellular cholesterol was labeled with D4H and imaged by widefield epifluorescence microscopy. Asterisk indicates region of ORP2-depleted cells. CM, complete medium. Dashed lines separate ORP2 expressing and depleted cells.

B, Quantification of A. Mean ± SD, n = 15-27 cells pooled from 2 independent experiments. Student's t-test.

C, E, Control and degron ORP2 cells were co-plated, and after 1 day in 5 % LPDS, treated with 50 μg/ml LDL and IAA for the indicated times. Asterisk indicates an ORP2-depleted cell. Double labeling of intracellular D4H and Lamp1 (C) or intracellular D4H and internalized integrin β1 (E). Images were acquired by confocal microscopy. Red box indicates a peripheral region toward the leading edge in the control cell, and blue box a corresponding region in the ORP2-depleted cell.

D, F, Fraction of D4H area overlapping with lamp1 (D) and D4H area positive for internalized integrin β1 (F) in a peripheral region toward the leading edge (exemplary box as inset). Mean ± SD, n = 20-30 cells pooled from 2 independent experiments. Student's t-test.
ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange (2021)
Figure 6

Figure 6. ORP2 regulates PI(4,5)P2 and NPC1 distribution.

A, Endo-GFP-ORP2 cells were treated with control or OCRL siRNAs for 2 days, stained with PI(4,5)P2 antibody and imaged by confocal microscopy.

B, Cells stably expressing NPC1-mCherry were treated with control or OCRL siRNAs for 2 days, or transfected with GFP-ORP2 or -ORP2-mHHK (PI(4,5)P2 binding-deficient mutant) for 1 day, stained with PI(4,5)P2 antibody and imaged by confocal microscopy. For FAK inhibition, GFP-ORP2 transfected cells were treated with 10 μM PF228 for 4 h. Dashed lines indicate cell edges. Arrowheads indicate colocalization of PI(4,5)P2 and NPC1.

C, Quantification of B. Values from Ctrl siRNA and Vector control were pooled and are shown as Ctrl siRNA/Vector ctrl. Mean ± SD, n = 20-43 cells pooled from 2 independent experiments. Student's t-test.

D, E, Cells stably expressing NPC1-mCherry were transfected with GFP-ORP2 or GFP-ORP2-mHHK for 1 day and were indicated, treated with 10 μM PF228 for 4 h. For degron ORP2 cells, NPC1-mCherry was transiently transfected for 2 days and cells incubated without (-IAA; ORP2 present) or with IAA (+IAA; ORP2 depleted) for 1 h. Arrowheads indicate tubular NPC1 organelles. Live cell images were acquired by widefield epifluorescence miroscopy and the longest NPC1 tubule per cell was measured. Mean ± SD, n = 102-122 cells. Students's t-test. Please see also ctrl (MovieEV4) and PF228 treated cell (MovieEV5), both videos 1 min recordings with 1 s frame rate.
ORP2 couples LDL-cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange (2021)
Figure 7

Figure 7. Cholesterol dependent activation of FAK.

A, B, Control and degron ORP2 cells were co-plated, and after 1 day 5 % LPDS, treated with 50 μg/ml LDL and IAA (+IAA; ORP2 depletion) for the indicated times and immunostained for pFAK to quantify its intensity. For rescue experiments, degron ORP2 cells were plated and transfected with GFP-ORP2 or -ORP2-mHHK or -ORP2-∆ELSK for 6 h. Cells expressing GFP-ORP2 constructs at levels similar to endo-GFP-ORP2 were used for quantifying pFAK intensity. Dashed lines indicate cell outlines. See also Fig EV6C. Asterisk indicates cells depleted of endogenous ORP2. Images were acquired by confocal microscopy. Mean ± SD, n = 20-23 cells pooled from 2 independent experiments. Student's t-test.

C, Quantification of FAK protein in cytosolic and membrane fractions in degron ORP2 cells with or without IAA. Cells were starved overnight in LPDS and loaded with 50 µg/mL LDL +/- IAA for 2 h. The proportion of FAK signal in the membrane fraction (of total FAK in cytosol+membranes) is presented ± SD. For no IAA n=13 and for IAA n= 11, 2 independent experiments. Student's t-test.

D-F, Liposome-co-sedimentation of FAK FERM with increasing concentrations of PI(4,5)P2 and cholesterol. "S" in D indicates the supernatant and "P" pellet containing the FAK FERM-bound to liposomes. The total lipid concentration was kept at 50 μM, and purified FAK FERM concentration at 1 μM. The initial lipid composition was POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5)P2:cholesterol (70:19:10:1:0:0, mol/mol). The increasing PI(4,5)P2 and cholesterol concentrations were compensated by decreasing the amount of POPC. Mean ± SD, n = 5. Student's t-test (*p < 0.05; **p < 0.002). Numbers under the blots indicate fraction of FAK FERM bound to liposomes (pellet) of total FAK FERM (supernatant + pellet).

G, H, Binding of Alexa Fluor 488-labeled FAK FERM domain to GUVs containing PI(4,5)P2 and cholesterol. The lipid compositions were POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5)P2:cholesterol (50:19:10:1:20:0) and POPC:POPE:POPS:Rhodamine-DHPE:PI(4,5)P2:cholesterol (20:19:10:1:20:30, mol/mol). Images were acquired by widefield epifluorescence microscopy. Scale bar, 5 μm. Mean ± SD, n = 68-76 pooled from 2 independent experiments. Student's t-test.
SPATA2 promotes CYLD activity and regulates TNFinduced NF-κB signaling and cell death (2016)
Figure 1

Figure 1. Interaction of CYLD and SPATA2

(A) Differentially SILAC labelled CYLD-/- cells and CYLD-/- cells expressing FLAG-CYLD were treated with mTNF (10ng/ml). Purified FLAG-CYLD protein complexes were combined 1:1 and analyzed by LC-MS/MS. (B) Heavy/light ratio for approx. 500 proteins identified in the screen. SPATA2 stands out with a substantially elevated H/L ratio. Each dot represents a protein. (C) 293T cells were transfected with empty vector (EV), a vector encoding FLAG-tagged full length CYLD (wt), or constructs encoding FLAG-tagged CYLD fragments 1-581 (F1) lacking the C-terminus, or 581-956 (F2) lacking the N-terminus, as indicated, followed by FLAG-IP. The blot was probed with antibodies recognizing SPATA2, FLAG and tubulin. Endogenous SPATA2 was co-immunoprecipitated to comparable levels by CYLD and the C-terminal CYLD protein fragment, containing the USP domain. (D) CYLD-/- MEF were infected with retrovirus encoding FLAG-CYLD as indicated and CYLD was purified by FLAG-IP. The blot was probed with antibodies recognizing CYLD, SPATA2 and actin. (E) 293T cells were transfected with empty vector (EV), a vector encoding FLAG-tagged full length SPATA2 or a construct encoding the FLAG-tagged N-terminal part of SPATA2 (NT), as indicated, followed by FLAG-IP. The blot was probed with antibodies recognizing CYLD, SPATA2 and tubulin. M, marker lane.
SPATA2 promotes CYLD activity and regulates TNFinduced NF-κB signaling and cell death (2016)
Figure 2

Figure 2. Interaction of HOIP and SPATA2 and recruitment of SPATA2 to the TNF-RSC

(A) 293T cells were transfected with a vector encoding V5-tagged full length SPATA2, along with empty vector (EV) a vector encoding FLAG-tagged HOIP wild-type (wt) or a vector encoding a FLAG-tagged PUB domain mutant of HOIP (N102A). After FLAG-IP, the blot was probed with antibodies recognizing HOIP, CYLD, V5 and actin. (B) 293T cells were transfected with empty vector or a vector encoding FLAG-tagged HOIP along with either V5-tagged SPATA2 (wt) or a V5-tagged PIM mutant (PIMmut) of SPATA2. After V5-IP, the blot was probed with antibodies recognizing FLAG, CYLD, V5 and tubulin. (C) 293T cells were transfected with a vector encoding FLAG-tagged HOIP and increasing amounts of V5-tagged SPATA2. After FLAG-IP, the blot was sequentially probed with antibodies recognizing OTULIN, HOIP, V5 and tubulin. (D) 293T cells were transfected with empty vector, FLAG-SPATA2 or FLAG-HOIP. After FLAG-IP, the blot was probed with antibodies recognizing OTULIN, CYLD, FLAG and actin. (E) 293T cells were transfected with empty vector, V5-tagged SPATA2 (wt) or a V5-tagged PUB domain mutant PUBmut of SPATA2. After V5-IP, the blot was probed with antibodies recognizing CYLD, FLAG, V5 and tubulin. (F) A549 cells were treated with FLAG-TNF (2µg/ml) for the indicated time. The purified TNF-RSC was sequentially probed with antibodies recognizing SPATA2, CYLD, RIPK1, TRADD, SHARPIN, TNF and tubulin as indicated.