EMBO/soda-nel huggingface dataset:
Ethanolamine-phosphate on the second mannose is a preferential bridge for some GPI-anchored proteins (2022)
Figure 7

Figure 7

Expression levels of various GPI-APs in PIGG-KO cells compared to levels in the wild type HEK293 cells, determined by FACS analysis.

A. Expression levels of various GPI-APs in different clones of PIGG-KO HEK293 cells. Fluorescence intensities of NT5E in PIGG-KO clones #4, #10, #17, #19 and wild type cells are 575, 910, 696, 530 and 1548 while those of EphrinA5 in clones #4, #17, #19 and wild type cells are 1431, 2080, 1868 and 3336, respectively.

B. Expression levels of GPI-APs in PIGG-KO clone #19 cells stably transfected with PIGG cDNA compared to levels in empty vector transfected cells. Fluorescence intensities of NT5E in PIGG cDNA and in vector transfected cells are 1332 and 886, respectively.

Data information: Each FACS analysis was performed at least three times.
Ethanolamine-phosphate on the second mannose is a preferential bridge for some GPI-anchored proteins (2022)
Figure 8

Figure 8

PIGG dependent expression of GPI-APs (FACS analysis).

A. Expression levels of different GPI-APs in PIGG-KO HEK293 cells transiently co-transfected with PIGG cDNA or empty vector. Vertical bars represent % of levels of GPI-APs expression in non-rescued PIGG-KO cells relative to those in PIGG cDNA rescued PIGG-KO cells. Analysis was repeated 3 to 6 times and SD bars were shown.

B. HA tagged NTNG2 and FLAG tagged DAF were transiently co-transfected together with PIGG cDNA or empty vector to PIGG-KO cells and were analyzed by FACS. Fluorescence intensity levels of NTNG2 in PIGG-KO cells and in PIGG rescued cells are 639 and 3426, while those of DAF in PIGG-KO cells and in PIGG rescued cells are 562 and 599, respectively. Each FACS analysis was performed at least three times.
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 1

Figure 1. Incisor CLE has distinct ciliary dynamics in the stem cells and transit amplifying cells

A. Representative IF staining of Sox2 (green) and Ki67 (red) on the P7 CLE stem cell and transit amplifying cell regions and counterstained with DAPI (blue) on a sagittal section. Dotted lines, basement membrane; yellow arrowheads mark approximate stem cell boundaries. SCs, stem cells; TACs, transit amplifying cells; Ant, Anterior; Post, Posterior.

B, C. The mRNA expression profiling on specific markers of stem cells (B) and transit amplifying cells captured on P7 incisors CLE followed by analysis using real-time RT-PCR (C). qRT- PCR results are in arbitrary values after normalization for GapDH. Statistics was performed on triplicates using one-way ANOVA followed by Bonferroni's test. *, p < 0.05; **, p < 0.01. Data are represented as mean and standard deviation.

D. Representative IF double staining of E-Cadherin (ECad, green, for cell border) and AcTub (red, for the cores of primary cilia) on a sagittal section. Sample is counterstained with DAPI (blue).

E. 3D reconstructions of ciliary cores of the stem cells and transit amplifying cells based on AcTub immunostaining.

F, G. Quantification of cilia length (F) and volume (G) based on 3D reconstruction (E) and comparison between those in stem cell and transit amplifying cell regions. Numbers of quantified cilia were indicated (n). Statistics was performed using one-way ANOVA: All error bars were defined in Statistical Analysis section: All quantification and real-time RT-PCR results were presented using style of mean and standard deviation. **, p < 0.01.

H. Tamoxifen induced IFT88 deletion strategy (top panel) and representative images of Sox2 (green) and Ki67 (red) immunostaining on CLE regions of the indicated mouse strains (bottom panels). Dotted lines, basement membrane. Note the significant reduction of both Sox2 and Ki67 immunolabeling upon deletion of IFT88. For Cre staining on the same set of samples please see Appendix Fig S1F.

I. J. 3D reconstruction of Sox2-positive regions based on its immunolabeling of the indicated mouse strains (I) and their volume comparison between mouse strains as indicated (J). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.

K, L. Representative IF double staining of ADP-ribosylation factor-like protein 13B (Arl13b, red, K) or histone deacetylase 6 (Hdac6, red, L) with AcTub (green) as a marker of the primary cilium core following stem cell to transit amplifying cell transition.

M. Summary of the immunolabeling of Arl13b or Hdac6 with AcTub (K, L) along stem cell to transit amplifying cell transition.
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 2

Figure 2. Prom1 has a dynamic expression in the incisor CLE primary cilia and nuclei

A. Representative IF staining of Prom1 using specific antibody clone 13A4 targeting extracellular loop (green) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Sample is counterstained with DAPI (blue). Dotted lines, basement membrane. SCs, stem cells; TACs, transit amplifying cells; Ams, ameloblasts.

B. 3D reconstruction showing the association of Prom1 (green) with AcTub-labeled (red) primary cilia in stem cell and transit amplifying cell regions. Note that the expression of Prom1 is not limited primary cilium but also to microvilli.

C. A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region. Green channel transparency was set up to 70%.

D. Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice.

E, F. The mRNA (E) and protein (F) profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. qRT- PCR results are in arbitrary values after normalization for GapDH. The immunoblotting was performed with anti-Prom1 C-terminal antibody. β-actin was used as loading control (F). Molecular mass markers are indicated. Statistics was performed using one-way ANOVA followed by Bonferroni's test: **, p < 0.01. Data are represented as mean and standard deviation.

G. Representative IF staining of Prom1 (red) and Sox2 (green) in CLESCs in vitro. Sample is counterstained with DAPI (blue). Arrows indicate colocalisation of Prom1 with low-medium level of Sox2 in the cell nuclei.

H, I. Subcellular fractionation of CLESCs into cytoplasm/membrane (C+M) and nuclear (N) fractions. Materials were analyzed by immunoblotting for Prom1 (H) and Arl13b (I). As controls, membranes were blotted for anti-Lamin A/C (nuclei markers) and GapDH (cytoplasm marker). Molecular mass markers are indicated. Note the presence of full-length Prom1 as well as Arl13b in nuclear fraction.
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 3

Figure 3. Epithelial Prom1 regulates CLESCs maintenance and activation

A, B. Representative images (A) and quantitative analysis (B) of IF staining and 3D reconstruction on Sox2-positive region for WT vs. Prom1 KO mice. Ant: Anterior; Post: Posterior (A). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.

C. Representative IF double staining of Sox 2 (green) and Ki67 (red) on the and transit amplifying cell regions of WT vs. Prom1 KO mice on frontal sections. Samples are counterstained with DAPI (blue).

D. Quantification of Ki67+ cell number in Sox2-positive region based on IF data from WT and Prom1 KO mice (C). Statistics was performed using Student's t-test:: **, p < 0.01.

E. Illustration of CLE tissue recombination assay. P7 CLE were microdissected then dissociated with Dispase II to achieve epithelium and mesenchyme. The Prom1 KO epithelium then reassociated with WT mesenchyme or vice versa.

F. Stereo images of three explants cultured with indicated recombination conditions before harvesting at day 3. Dotted line indicates the CLE.
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 4

Figure 4. CLESCs renewal is compromised in the absence of Prom1

A. Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice. Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage. Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage.

B, C. Clonogenicity assay for colony number (B) and size (C) after the first the passage of CLESCs derived from WT vs. Prom1 KO mice in the absence or presence of SHH (100ng/ml). Cells were seeded in triplicate on 24-well plates as 5,000 cell/per well. Statistics was performed using Student's t-test: *, p < 0.05; **, p < 0.01. Data are represented as mean and standard deviation of triplicated samples.

D. Sphenoid analyses. For these assays, epithelial and mesenchymal cells were mixed at ratio of 5,000:5,000 per well in triplicates on 96-well plates and grown for 3 days on Matrigel. Representative micrographs of entire well under indicated conditions are shown.

E. Quantification of sphere diameter on samples presented in (D). Statistics was performed using Dunnett's test: **, p < 0.01. N.S., non-significant. Number of quantified spheres is indicated (n).
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 5

Figure 5. Prom1 is important for quiescent cell re-enter cell cycle

A. Designing strategy of the Ki67-Fucci cell cycle indicator system.

B. Representative image of CLESCs harbouring the Ki67-Fucci system showing colored cells at different cell cycle phases.

C. Flow cytometry analysis of Ki67-Fucci CLESCs at different cell cycle stages in cells transfected with empty vector or full length Prom1. As a result, >95% of the events have been included in the analysis. The accuracy of the strategy has been validated by two independent researchers for IF quantification using Fiji 1.0 software.

D. Representative IF staining of AcTub (blue) on Ki67-Fucci CLESCs without and with the presence of SHH. Note that the cilium could be visualised (arrows) solely when SHH is present.

E, F. Representative IF double staining of either AcTub (D, red) or Arl13b (E, red) with Sox2 (green) or Ki67 (green) in CLESCs in the presence of SHH. Note the expression levels of Sox2 and Ki67 distinguished stem cells and transit amplifying cells, respectively.
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 6

Figure 6. Prom1 regulates ciliary dynamics by directly interacting with Hdac6 and Arl13b

A, B. Size of individual cilia in stem cell and transit amplifying cell regions of WT and Prom1 KO mice (A) and their density in the respective regions (B). 3 animals were analyzed for each genotype. The number of quantified cilia is indicated (n). Statistics was performed using Dunnett's test: *, p < 0.05; **, p < 0.01. Data are represented as mean and standard deviation. SCs, stem cells; TACs, transit amplifying cells.

C, D. Clonogenicity analysis of CLESCs treated with either Hdac6 inhibitor (Tubacin) and appropriate controls (Nitubacin and DMSO, C) or siArl13b and its siControl. Colonies were stained with crystal violet (D).

E, F. siRNA knockdown efficiency of Arl13b (E) and Hdac6 (F) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH Statistics was performed in triplicates using one-way ANOVA followed by Bonferroni's test: **, p < 0.01. Data are represented as mean and standard deviation.

G. Flow cytometry analysis and quantification of samples indicated in (E and F).

H. Mutation strategy of Hdac6-binding site on Prom1. The lysine (K) 138 in the first cytoplasmic loop of Prom1 was mutated to glutamine residue (Q). TM, transmembrane domain.

I. Representative IF double staining and 3D reconstruction of the Prom1 (green) and AcTub (red) at the apical domain of polarized MDCK cells expressing Prom1 and its K138Q mutant (top panels). Black-white inserts show Prom1 signals only. Same cells were analyzed by SEM (bottom panels). Note that Prom1 is asymmetrically distributed along the primary cilium (white arrows) as observed in vivo (see Fig 2C) and mutation K138Q significantly impacts on the primary cilium length. MV, microvilli.

J. IF staining and 3D reconstruction of AcTub-labeled (red) primary cilium at the apical membrane of parental MDCK cells (MDCK) and those cells transfected with Prom1 or its K138Q mutant. The images were used to quantify the primary cilium length (Appendix Figs S6B and C). Arrows indicate primary cilia with more than 4 μm in length.

K-M. Co-immunoisolation (IS) analyses were performed with paramagnetic beads using antibodies against either Arl13b (K) or Prom1 (AC133) or with anti-FITC antibody as control (L). Recovered materials were analyzed by immunoblotting against the indicated proteins. Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum-associated forms of Prom1, respectively, while arrows show Arl13b. Arrowhead, immunoglobulin. The ratio Arl13b/Prom1 immunoreactivities was quantified (n = 3) (M). Mann-Whitney test: **, p < 0.01. Data are represented as mean and standard deviation.
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 7

Figure 7. Prom1 regulates stem cell homeostasis and differentiation through lysine 138

A-D. Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc (A-C) or as indicated (D). Cells were observed by phase-contrast microscopy (A), SEM (B, top panels) or confocal laser scanning microscopy after DAPI staining (B, bottom panels, C, D). Box-whisker plots show the number of domes per 600 mm2 (A). Three images were analyzed per experiment (n = 4). Individual cells within a given dome are indicated (B, top panels) and were quantified using three different planes (bottom, centre and top) of DAPI-labeled nuclei (B, bottom panels). Domes of similar size were evaluated (C). Means and standard deviations are displayed. 15 domes per independent experiment were examined (n = 3). Cell density at a given time of culture (7, 10 and 14 days) was determined by counting DAPI-labeled nuclei per 0.1 mm2 (D, n = 4). 4X magnification (inset in panel A). Two-tailed unpaired student's t-test: *, p < 0.05; **, p < 0.01. ***, p < 0.005.

E. Cilia with a length of >4 µm were rarely observed in K138Q mutant and displayed an irregular shape with constrictions along their distal part as observed by SEM (arrows).

F, G. Mutation in the Hdac6-binding site of Prom1 increases the release of Prom1+ vesicles. 16 h-conditioned medium was subjected to differential centrifugation for 5 min at 300 x g (P1); 20 min at 1,200 x g (P2); 30 min at 10,000 x g (P3) and 1 h at 200,000 x g (P4). In parallel, detergent cell lysates were prepared. The entire pellets and 1/40 of cell lysates were analyzed by immunoblotting using antibodies against human Prom1, AcTub and β-actin (F). Molecular mass markers are indicated. All three proteins were detected in the vesicle fractions (P3 and P4) suggesting Prom1+ vesicles are released from primary cilia and microvilli. The release of Prom1+ vesicles was quantified using its immunoreactivities associated with cell and vesicle fractions (G) (n = 3). Two-tailed unpaired student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 8

Figure 8. Prom1 meditates SHH activation through Glis2

A. The mRNA expression of SHH effector genes (Gli1/2 and Glis1-3) on laser microdissection captured samples were evaluated by real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH. Each sample was isolated from distinct mice (n = 3). Statistics was performed using Student's t-test: **, p < 0.01. SCs, stem cells; TACs, transit amplifying cells.

B. Representative IF staining of Gli1 (left panel, red) and Glis2 (right panel, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Dotted lines, basement membrane.

C. 3D reconstruction of Glis2 staining (see B, red) and its association with DAPI-labeled nuclei (blue). Nuclear transparency was set at 0 or 70% as indicated.

D. Representative IF double staining of Glis2 (red, bottom panel) and AcTub (green, top and bottom panels) along stem cell-transit amplifying cell axis in lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions.

E. Volume of Sox2-positive region on the lower incisor CLE at P7 was evaluated in WT vs. Glis2 KO mice upon IF staining. Number of animals analyzed is indicated (n). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.

F. Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop (top panels, green) or cytoplasmic C-terminal end (bottom panels, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice. Samples are counterstained with DAPI (blue). Dotted lines, basement membrane. Arrowheads marks approximate boundary of stem cell regions.

G. Representative IF double staining of Glis2 (red) and AcTub (green) as a marker of primary cilia in stem cell region of WT vs. Prom1 KO mice. Note the reduction of Glis2 expression in Prom1-deficient animals.
Prominin-1 controls stem cell activation by orchestrating ciliary dynamics (2018)
Figure 9

Figure 9. Stat3 is downstream target of Glis2

A. Proximity ligation assay analysis of Prom1 and Glis2 association (red) in stem cell and transit amplifying cell regions of lower incisor CLE at P7. As negative control, one conjugated primary antibody (anti-Glis2) was omitted. SCs, stem cells; TACs, transit amplifying cells.

B. Time dependent Prom1 and Glis2 co-expression (proximity ligation assay) in the cultured CLESCs starved for 4 days, and then treated with SHH (100 ng/ml) and importazole (40 µM), an inhibitor of importin β1function. DMSO was used as vehicle control. Data are represented triplicated samples as mean and standard deviation.

C. PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). Arrows indicated genes with expression changed for more than 2 folder increase or decrease.

D, E. The mRNA expression of gene significantly changed in the PCR array (in (C)) (D) and Stat pathway members (E) was evaluated in WT, Prom1 and Glis2 KO mice by real-time RT-PCR analysis. Each sample was isolated from distinct mice (n = 3). The results are in arbitrary values after normalization for GapDH. Statistics was performed using one-way ANOVA followed by Bonferroni's test: *, p < 0.05; **, p < 0.01. Data are represented as mean and standard deviation.

F. Chromatin IP analysis of binding of Glis2 to Stat3 promoter (see Appendix Fig S9D) in the absence or presence of SHH at different concentration as indicated on triplicated samples, using specific anti-Glis2 antibodies and rabbit IgG as control. Statistics was performed using one-way ANOVA followed by Bonferroni's test: *, p < 0.05; **, p < 0.01.
Infancy-onset diabetes caused by de-regulated AMPylation of the endoplasmic reticulum chaperone BiP (2022)
Figure 3

Figure 3:

The FICD Arg371Ser mutation compromises BiP AMPylation and eliminates all detectable BiP deAMPylation in vitro.

A Plot of the principal melting temperatures (Tms) of wild-type FICD (WT) and FICDR371S in the presence or absence of nucleotide ligands and/or FICD's gatekeeper Glu234 residue, derived from differential scanning fluorimetry (DSF). The mean Tm ± 95% confidence interval (CI) is displayed, from n = 3 independent experiments. Arg371Ser mutation slightly destabilises the apo enzyme and significantly reduces the ability of ATP and ADP to stabilise FICD. Glu234Gly mutation partially restores ATP (but not ADP) stabilisation of FICDE234G-R371S (see Figure EV2A).

B Size exclusion chromatography confirms that the oligomerisation tendency of FICDR371S is unperturbed relative to wild-type FICD. Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species. The elution profile of a monomeric FICDL258D-H363A mutant is also shown for reference, alongside the elution times of molecular weight standards.

C Representative immunoblot comparing the ability of wild-type and Arg371Ser FICD (at a wide range of enzyme concentrations) to catalyse the accumulation of BiP-AMP in the presence of physiological ATP concentrations (5 mM). At low enzyme concentrations (and early time points) the AMPylation defect imposed by Arg371Ser is manifest. At higher enzyme concentrations where the deAMPylation activity of the wild-type enzyme begins to dominate, the residual AMPylation activity of FICDR371S results in a greater accumulation of BiP-AMP (relative to the same concentration of wild-type enzyme).

D Quantification of the AMPylated BiP signals relative to total BiP of experiments as in C (mean values ± SEM, n = 4, biological replicates). Figure EV2B displays the values from all four repetitions.

E A fluorescence polarisation-based deAMPylation assay highlighting the lack of activity catalysed by FICDR371S. Enzyme-saturating concentrations of BiP-AMP was provided as deAMPylation substrate. No enzymatic activity of 10 µM (grey trace, shown) or 20 µM enzyme FICDR371S was detectable. The estimated fluorescence anisotropy value of a fully deAMPylated BiP sample is derived from a heuristic fitting of a single exponential decay curve. The kcat value for wild-type FICD was calculated from n = 3 biological replicates (mean ± SEM).

F Representative immunoblot comparing the accumulation of BiP-AMP in reactions constituted with the indicated concentrations of wild-type and Arg371Ser FICD, performed as in A (upper panel). The lower panel is a Coomassie-stained gel of the reaction constituents. An N-terminally extended version of the wild-type enzyme (residues 45-458was used in lanes 2 and 5-7, to distinguish its migration on the gel from the mutant (residues 104-445). The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F (from n = 3, biological replicates, mean values ± SEM).

Source data are available online for this figure.
Infancy-onset diabetes caused by de-regulated AMPylation of the endoplasmic reticulum chaperone BiP (2022)
Figure 4

Figure 4:

FICDR371S promotes accumulation of AMPylated BiP in CHO cells lacking endogenous FICD

A Immunoblots of AMPylated proteins detected with a pan-AMP antibody, BiP, FICD and eIF2α (a loading control) in lysates of FICD∆ CHO cells transiently transfected with plasmids encoding the indicated derivatives of FICD. Signals corresponding to AMPylated BiP, auto-AMPylated FICDE234G, total BiP, FICD and eIF2α are indicated. The novel species reactive with the anti-FICD antiserum in lysates of cells transfected with plasmids expressing FICDR371S (*) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369.

B Histograms of Integrated Stress Response reporter CHOP::GFP and XBP::Turquoise (XBP::Turq, a reporter of the IRE1 branch of the UPR) fluorescence in FICD∆ CHO dual reporter cells transfected with expression vectors as in A. Transfected cells were gated for mCherry positivity, a marker carried on the expression plasmid in trans to the indicated FICD effector.

C Quantification of the data from repeats of the experiment as shown in B. The upper two graphs show the percent of cells with high CHOP::GFP or high XBP::Turq for each transfection. The lower panel provides quantitation of the median fluorescence intensity for each reporter. Mean ± SD, n=4, biological replicates.

Source data are available online for this figure.