EMBO/soda-nel huggingface dataset:
SCHENGEN receptor module drives localized ROS production and lignification in plant roots (2020)
Figure 6

Fig. 6 CIF2 induces large-scale transcriptional changes for cell wall remodeling

(A) CASP1-GFP and lignin deposition in WT, sgn3, rbohD, rbohF and rbohDF. CASP1-GFP and lignin (fuchsin) are presented in green and magenta respectively. Pictures were obtained from more than 10 roots from each background with similar results. Scale bar = 10 µm.

(B) Time lapse imaging of single- or cotreatment of 10 nM CIF2 with 25 µM cycloheximide (CHX) on CASP1-GFP in cif1 cif2. Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium. Scale bar = 10 µm (See also movie EV1).

(C) Quantification of (B). Relative numbers of holes in CASP1-GFP domain after single- or co-treatment with CIF2 or CHX from the pictures in (B). Bars are S.D. * indicates statistical significance from all other conditions (p<0.01) after one-way ANOVA and Tukey test. 6 roots in total for each condition were observed during two independent tests.

(D) Fold-change of 930 genes (p<0.05 and log2(Fold change) ≥1 or ≤ -1 at least one time point in one genotype) after CIF2 treatment at indicated time points in WT, cif1,2 and sgn3. Degree of the fold changes are shown in color code as indicated.

(E) Relative expression levels of PER15 to CLATHRIN control in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).

(F)Fold changes of PER15 in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistical significance differences (p < 0.01, ANOVA and Tukey test).

(G) Relative expression levels of PER49 to CLATHRIN control in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).

(H) Fold changes of PER49 in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).

(I)Relative fold changes of PER15 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).

(J)Relative fold changes of PER49 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).
ORP5/ORP8 localise to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function (2016)
Figure 1

Figure 1: ORP5 and ORP8 localize at ER-PM and ER-mitochondria contact sites.

(A) Domain structures of human ORP5 and ORP8. PH, Pleckstrin homology domain; ORD, OSBP-related lipid binding domain; TM, transmembrane domain. (B) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 showing variable expression levels. Arrows indicate ORP5 and ORP8 localization to cortical ER (cER). Scale bar, 10 μm. (C-D) Electron micrographs of ultrathin cryosections of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 and immunogold stained with anti-GFP (15 nm gold) to detect ORP5 or ORP8 and anti-PDI (10 nm gold) to label the ER lumen. ORP5 and ORP8 localize at ER-PM (arrows) and ER-mitochondria contact sites (arrowheads). m, mitochondria; cER, cortical ER. Scale bar 200nm. (E-F) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 (E) and GFP-Sec61β (F) in transfected HeLa cells. Results are presented as the percentage of the total number of gold particles (800 per condition, n=35-45 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (non-cER, non-cortical ER; ER-PM, ER-PM contact sites; ER-mito, ER-mitochondria contact sites. % gold particles ±SEM. Data are the mean of three independent replicates. *P<0,001 compared to non-cER and ER-mito [EGFP-ORP5] and to ER-PM and ER-mito [EGFP-ORP8 and GFP-Sec61β], #P<0,001 compared to ER-PM and ER-mito [GFP-Sec61β]). (G) Representative electron micrographs of mitochondria and (H) quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells expressing EGFP-ORP5, EGFP-ORP8 together with HRP-KDEL, to visualize the ER. Control (Ctrl) consisted of HeLa cells overexpressing only HRP-KDEL. (I) Crude mitochondria, mitochondria and MAM fractions were purified from HeLa cells and equal amount of protein (25g) from each fraction were loaded on a 4-20% gradient SDS-PAGE gel and immunoblotted using anti-ORP5, anti-ORP8 and anti-IP3R-3 (MAM protein), anti-PDI (ER protein) and anti- cytochrome c (mitochondrial protein). TC,Total cell; MAM, mitochondria-associated ER membrane .
ORP5/ORP8 localise to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function (2016)
Figure 2

Figure 2: ORP5 and ORP8 targeting to ER-mitochondria contact sites and interaction with the mitochondrial protein PTPIP51

(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP8, or GFP-Sec61β together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm. (B) Electron micrograph of ultrathin cryosections of HeLa cells co-transfected with EGFP-ORP5 and PTPIP51-HA and immunogold stained with anti-GFP (15 nm gold) and anti-HA (10 nm gold) showing EGFP-ORP5 localization at ER-mitochondria contacts (arrows). Arrowheads indicate the highly expanded ER membrane juxtaposed to mitochondria. Scale bar 200 nm. (C) Quantification of the IEM labelling for EGFP-ORP5, EGFP-ORP8 and GFP-Sec61β in HeLa cells co-transfected with PTPIP51-HA. Results are presented as the percentage of the total number of gold particles (800 per condition, n=30-35 cells) for ORP5, ORP8 and Sec61β in the indicated compartments (ER, reticular ER; ER-mito MCS, ER-mitochondria membrane contact sites; ER-PM MCS, ER-PM membrane contact sites). % gold particles ±SEM. Data are representative of three independent replicates. **P<0,001. (D) Confocal images of HeLa cells transfected with EGFP-ORP5 or EGFP-ORP8 together with PTPIP51-HA and RFP-Sec61β and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm. (E) Representative EM images of ER-mitochondria contacts in the indicated overexpression conditions. (F) Quantification of the percentage of ER-mitochondria contact sites (ER-mito contact) per mitochondria of HeLa cells transfected with HRP-KDEL (Ctrl) or co-transfected with PTPIP51-HA and EGFP or PTPIP51-HA and EGFP-ORP5 or PTPIP51-HA and EGFP-ORP8 (n=30 cells and 1082-1223 mitochondria). % ER-mitochondria contact sites ±SEM. Data are representative of three independent replicates. **P<0,001, *P<0,05. (G) EGFP, EGFP-ORP5 or EGFP-ORP8 were immuno-precipitated from lysates of HeLa cells co-expressing PTPIP51-HA and treated with control (siCtrl) or VAPA and VAPB (SiVAPA/B) siRNAs and analyzed by western blot using antibodies against GFP (ORPs), HA (PTPIP51), VAPA, VAPB and tubulin.
ORP5/ORP8 localise to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function (2016)
Figure 3

Figure 3: Recruitment of ORP5 and ORP8 to ER-mitochondria contact sites requires their lipid-binding domain.

(A) Confocal images of HeLa cells transfected with EGFP-ORP5, EGFP-ORP5PH or EGFP-ORP5ORD and immunostained using anti-TOM20 antibody to visualize mitochondria. Scale bar, 10 μm. (B) Confocal images of HeLa cells transfected with the EGFP-tagged ORD domain of ORP5 (EGFP-ORD ORP5) and immunostained using anti-TOM20 antibody (upper panels) or co-transfected with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm. (C-D) Confocal images of HeLa cells transfected with EGFP-ORP5PH or EGFP-ORP5ORD (C) or EGFP-ORP5, EGFP-ORP5L389D or EGFP-ORP8L425D (D) together with PTPIP51-HA and immunostained using HA antibody to detect PTPIP51. Scale bar, 10 μm. (E) EGFP-ORP5, EGFP-ORP8, EGFP-ORP5L389D, EGFP-ORP8L425D, GFP-Sec61β or EGFP alone were immunoprecipitated from lysates of HeLa cells co-expressing PTPIP51-HA and analyzed by western blot with antibodies against GFP and HA.
ORP5/ORP8 localise to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function (2016)
Figure 4

Figure 4: Depletion of ORP5 affects mitochondria morphology and function.

(A) ORP5, ORP8 and actin levels of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs (siCtrl) or siRNAs against ORP5 (siORP5) or ORP8 (siORP8). (B) Electron micrographs of HRP-KDEL-expressing HeLa cells treated with Ctrl siRNAs or siRNAs against ORP5 or ORP8. Red arrows indicate ER-PM contact sites. Scale bar, 2 μm. Insets show representative mitochondria and red arrows in the insets indicate ER-mitochondria contacts. Inset scale bar, 500nm. (C) Representative EM micrographs showing the morphology of mitochondria in siCtrl, siORP5 and siORP8-treated cells. Scale bar, 1 μm. (D) Quantifications of the number of mitochondria with altered morphology in the indicated siRNA conditions. % altered mitochondria ±SEM, n=25 cells and 632-710 mitochondria. *P<0,001 compared to siCtrl. (E) Mitochondrial oxygen consumption rate (OCR) measured in Ctrl and ORP5 siRNA transfected cells. OCR trace was obtained by sequential measurement of basal OCR (OCRBAS), OCR after the addition of oligomycin (OM) and OCR after addition of antimycin A (AA). Note the reduced basal OCR (OCRBAS) compared to ctrl siRNA cells. Error bars denote ±SEM. Data are the mean of 4 independent repeats (n=4), *P<0,05.
GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum (2022)
Figure 1

Figure 1. The Endo H-resistant glycosylated form of SMO is decreased by PTCH1 but increased by Hh.

(A) Three glycosylated forms of endogenous SMO protein are shown. NIH3T3 cells were harvested and treated with indicated glycosidases. Black bracket indicates mature glycosylated form and grey bracket indicates core glycosylated form. The representative results of three independent experiments are shown.

(B) Densitometric analysis of three glycosylated forms of SMO shown in (A) was quantified by Image J, expressed as a percentage of the total SMO protein.

(C) PTCH1 inhibited the Endo H-resistant glycosylation of SMO. HEK293T cells were co-transfected with the SMO and PTCH1 expression plasmids. After 48 h, the cells were harvested, lysed, and treated with indicated glycosidases. Results shown are representative of three independent experiments.

(D) Quantification of Endo H-resistant form of SMO shown in (C). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein. Data are expressed as mean ± SEM (***p < 0.001, student's t-test).

(E) Shh increased the Endo H-resistant form of SMO. Cells were transfected as indicated for 24 h, and incubated with ShhN conditioned medium for an additional 24 h. Cells were harvested and analyzed as in (C). Results shown are representative of three independent experiments.

(F) Quantification of Endo H-resistant form of SMO shown in (E). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein. Data are expressed as mean ± SEM (***p < 0.001; **p < 0.01, two-way ANOVA with Sidaks' multiple comparison test).

(G) Shh increased the Endo H-resistant form of endogenous SMO in MEF cells. Results shown are representative of three independent experiments.

(H) Densitometric analysis of Endo H-resistant form of SMO shown in (G). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein. Data are expressed as mean ± SEM (*p < 0.05, one-way ANOVA with Dunnett's multiple comparison test).
GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum (2022)
Figure 2

Figure 2. Recognition of SMO by SAR1B is regulated by Hh and PTCH1.

(A) Biotinylation of SMO by SAR1B-BirA* (asterisk denotes the R118G mutation in BirA) in a proximity-dependent biotinylation assay. The cells were transfected with plasmids encoding SMO and SAR1B-BirA*-FLAG. After 48 h, cells were treated with different doses of biotin for 4 h. Cells were harvested and lysates were dialyzed to remove free biotin. Biotinylated proteins were pulled down by streptavidin beads and subjected to immunoblotting analysis.

(B) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.

(C) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A.

(D) PTCH1 decreased SAR1B-BirA*-mediated biotinylation of SMO, but not LMAN1. The experiments were done similarly in Fig. 2A.

(E) The Shh increased the binding of SAR1B to SMO. After transfection for 24 h, cells were incubated with the ShhN conditioned medium for an additional 24 h. Then 15 μM biotin was added into the culture medium for 4 h. The cells were harvested and analyzed as described in Fig. 2A.

Data information: All of the experiments were done in the SMO-KO HEK293T cells, except B, in which the HEK293T cells stably expressing SAR1B-BirA* were used.
GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum (2022)
Figure 3

Figure 3. The RRxWxR motif in the cytoplasmic tail is required for ER exit and SMO activity.

(A) Topology of human SMO and the alignment of SMO partial sequences of different species are shown. The RRxWxR (x stands for any amino acid) motif is highlighted in red.

(B) Treatment with glycosidase demonstrated that SMO (4A, in which RRTWCR was mutated to AATACA) was sensitive to Endo H. The experiments were done similarly in Fig. 2A. Black bracket indicates mature glycosylated form.

(C) SAR1B-BirA*-mediated biotinylation of the SMO variants. The experiments were done similarly in Fig. 2A.

(D) In vitro budding of SMO. Membranes were prepared and incubated in vitro in the presence of cytosol, ATP, GTP, and an ATP-regenerating system as described in experimental procedures. Then vesicle and membrane fractions were separated by centrifugation and subjected to immunoblotting analysis.

(E) In vitro budding of the SMO variants. The experiments were done as same as in (D).

(F) The ciliary localization of SMO variants. Smo-KO NIH3T3 cells were transfected with the EGFP fused SMO variants, incubated with serum starvation medium for 24 h to fully induce cilia formation, and treated with or without ShhN conditioned medium for 4 h. Cells were immunostained with the acetylated tubulin (red). Results shown are representative of three independent experiments. Scale bars: 5μm.

(G) Quantitative assessment of the intensity of ciliary SMO in (F) by Image J. More than 45 cells were randomly selected and counted for each group. Data are presented as median with interquartile. (****p < 0.0001; ns, not significant, two-way ANOVA with Sidak's multiple comparisons test).

(H) Quantification of the percentage of cells with ciliary SMO. Data are presented as mean ± SEM from three biological replicates (****p < 0.0001; ns, not significant, two-way ANOVA with Tukey's multiple comparisons test). 80-90 cilia were analyzed in each group in each experiment.

(I) Gli-luciferase reporter assay in Smo-KO NIH3T3 cells. The cells were transfected with plasmids encoding SMO or SMO variants, GLI-promoted firefly luciferase and renilla luciferase. After 24 h, cells were incubated with a medium containing different amounts of ShhN for another 24 h. Data are presented as mean ± SEM from three technical replicates (****p < 0.0001; ***p < 0.001, two-way ANOVA with Dunnett's multiple comparison test).

Data information: In B, C, D, E, the SMO-KO HEK293T cells were used.
GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum (2022)
Figure 4

Figure 4. Cholesterylation of SMO accelerates its ER exit.

(A) Shh increased cholesterylation of SMO. HEK293T cells were transfected, harvested, and analyzed as depicted in Fig. EV4D.

(B) The SMO cholesterylation of Ptch1-KO NIH3T3 cells were increased comparing with WT cells.

(C) The CP-modified SMO was mainly resistant to Endo H. The CP-modified SMO proteins were pulled down by streptavidin beads and digested with Endo H or PNGase F at 37 ℃. Then proteins were eluted with SDS-PAGE sample buffer and were subjected to immunoblotting analysis. Results shown are representative of two independent experiments.

(D) Cholesterylation of SMO(4A) was inhibited by PTCH1 and increased by ShhN.

(E) The CP-modified SMO(4A) was sensitive to Endo H treatment.

(F) The levels of CP-modified WT SMO and SMO(4A) were compared. 1.39% of SMO(WT) pellet samples and 11.1% of SMO(4A) pellet samples were loaded. Results shown are representative of three independent experiments.

(G) Quantification of CP-modified SMO shown in (F). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO. Data are expressed as mean ± SEM (****p < 0.0001, unpaired two-tailed Student's t-test).

(H, I) The stimulative /inhibitory effect of Shh/PTCH1 on SMO and SAR1B interaction was compromised by D95E mutation. The experiments were done the same as Fig. 2E.

Data information: In C, D, E, F, HEK293T cells were used.
GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum (2022)
Figure 5

Figure 5. Restraint of cholesterol transport from the PM to the ER blocks the Hh signaling.

(A) ALOD4 decreases ER cholesterol levels by binding and trapping cholesterol in the outer leaflet of plasma membranes.

(B, C) ALOD4 inhibited the cholesterol modification of SMO and SMO(4A). Cells were treated with ALOD4 for 1 h, and then incubated with a medium containing ShhN and CP for 2 and 3 h. In C, ShhN and CP added for 2 h. Cells were harvested and analyzed as depicted in Fig. EV4D.

(D) Ciliary localization of endogenous SMO treated with or without ALOD4. Cells were treated with ALOD4 for 1 h, and then incubated with ShhN conditioned medium for 2 h. Results shown are representative of three independent experiments. Scale bars: 5μm.

(E) Quantitative assessment of the intensity of ciliary SMO in (D). More than 80 cells were randomly selected and counted for each group. Data are presented as median with interquartile. (****p < 0.0001, one-way ANOVA with Dunnett's multiple comparison test).

(F) Quantification of the percentage of cells with ciliary SMO in (D). Data are presented as mean ± SEM from three biological replicates (****p < 0.0001, one-way ANOVA with Dunnett's multiple comparison test). 90-100 cilia were analyzed in each group in each experiment.

(G) Effect of ALOD4 on the expression of Gli1 was measured by qRT-PCR. The cells were treated with ALOD4 for 1 h, and then incubated with ShhN conditioned medium for 5 h. Data are expressed as mean ± SEM from three technical replicates (****p < 0.0001, two-way ANOVA with Dunnett's multiple comparison test).

Data information: All of the experiments were done in NIH3T3 cells.
GRAMD1/ASTER-mediated cholesterol transport promotes Smoothened cholesterylation at the endoplasmic reticulum (2022)
Figure 6

Figure 6. Overexpression of GRAMD proteins promote the maturation of SMO and Hh signaling.

(A, B) GRAMD1B increased cholesterylation of SMO. HEK293T cells were co-transfected with the SMO or SMO (4A) and GRAMD1B protein expression plasmids for 24 h, and then incubated with a cholesterol depletion medium (DMEM supplemented with 5% lipoprotein-deficient serum, 1 μM lovastatin and 10 μM mevalonate) for 12 h, followed by 2 μg/mL of CP treatment. The cells were harvested and analyzed as depicted in Fig. EV4D. Results shown are representative of two independent experiments.

(C, D) GRAMD1B promoted SMO maturation. The expression plasmids of SMO or SMO(D95E) and GRAMD1B were transfected into SMO-KO HEK293T cells. After 12 h, cells were switched to a cholesterol depletion medium for 16 h and then added 50 μM MβCD-cholesterol for 8 h. Black bracket indicates mature glycosylated form.

(E) Expression of Gli1 mRNAs measured by qRT-PCR in NIH3T3 cells stably expressing GRAMD1B. After incubating in serum starvation medium for 16 h, cells were depleted of cholesterol by treatment with 1.5% HPCD for 0.5 h and then incubated in serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 10 μM MβCD-cholesterol, ShhN as indicated for 24 h. Data are expressed as mean ± SEM from three technical replicates (****p < 0.0001; *p < 0.05; ns, not significant, unpaired two-tailed Student's t-test).

(F) Confocal images of NIH3T3 cells stably expressing mCherry-GRAMD1B. PM was labeled with wheat germ agglutinin (WGA)-CF®488A conjugate. Cells were incubated in a cholesterol depletion medium for 16 h and switched to the same medium containing 50 μM MβCD-cholesterol for 1 h. Cells were then treated with or without ShhN conditioned medium for another 2 h. Results shown are representative of three independent experiments. Scale bars: 5μm.

(G) The percentage of cells with GRAMD1B localization on PM. Data are represented as mean ± SEM from three biological replicates (**p < 0.01, two-way ANOVA with sidak's multiple comparison test). 60-70 cells were analyzed in each group in each experiment.