EMBO/soda-nel huggingface dataset:
The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation (2021)
Figure 3

Figure 3. OFD1 is a component of the TBC1D31/praja2 complex.

A. Protein-protein interaction network of physical interactors of OFD1, praja2 and TBC1D31. Node size represents the degree of association of the proteins (grey nodes) associated with the input proteins set (black nodes). Edge size is representative of the strength of the mapped interaction between the two connecting nodes.

B. The bar plot reports the statistical significance of selected sets of Gene Ontology functional annotations enriched by the nodes in the network. Statistical significance is reported as -log10[p-value] on the X axis, enriched functional annotations are reported on the Y axis.

C. Lysates from HEK293 cells expressing GFP-TBC1D31 and flag-OFD1 were immunoprecipitated with anti-flag or control IgG. Endogenous praja2 and the overexpressed transgenes were revealed by immunoblot analysis.

D. Total lysates from mouse brain were immunoprecipitated with anti-TBC1D31 antibodies or with control IgG. The precipitates and an aliquot of lysates were immunoblotted with the indicated antibodies.

E. Schematic picture of the assembled TBC1D31/OFD1/praja2/PKA complex.

F. HEK293 cells were transiently co-transfected with GFP-TBC1D31 and flag-OFD1. Cells were fixed and stained for flag and praja2.
The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation (2021)
Figure 4

Figure 4. OFD1 is phosphorylated by PKA.

A. Schematic representation of OFD1 domain organization, with the predicted PKA phosphorylation sites and the position of S735 and S899 in the human variant.

B. GST-fused, recombinant OFD1 polypeptides were used as substrates for in vitro phosphorylation assays using purified PKAc. GST-immobilized fusions were immunoblotted with an anti-phospho-(K/R)(K/R)X(S*/T*) antibody. A representative set of 3 independent experiments is shown. A Ponceau S staining of recombinant GST-OFD1 polypeptides is shown on the right panel. *Degradation products.

C. HEK293 cells transiently expressing flag-OFD1 or flag-S735A mutant were starved for 24 hours and then treated with forskolin (FSK, 40μM/1 hour). Where indicated, cells were pre-treated with H89 (10μM). Lysates were subjected to flag immunoprecipitation and immunoblot analysis with anti-phospho-PKA substrates and anti-flag antibodies. Anti-GFP IgG were used as control.

D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05, **p<0,01.

E. Same as in C, with the exception that cells were co-transfected with flag-OFD1 vector and with control siRNA (siCNT) or siRNA targeting praja2.

F. Quantitative analysis of the experiments shown in E. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05.

G. Schematic picture of the assembled TBC1D31 complex showing that following cAMP stimulation PKA phosphorylates the co-assembled OFD1.
The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation (2021)
Figure 5

Figure 5. cAMP targets OFD1 to UPS.

A. Immunoprecipitation of OFD1 from HEK293 cell lysates expressing myc-OFD1, ubiquitin-HA and flag-praja2 (or flag-praja2rm). The precipitates were immunoblotted with anti-HA (ubiquitinated OFD1) and anti-myc antibodies. Flag-praja2 expression was evaluated in total lysates.

B. Flag-OFD1 and ubiquitin-HA vectors were co-transfected with control siRNA or siRNA targeting praja2 (sipraja2). Serum deprived cells were left untreated or stimulated with FSK for 1 hour. Ubiquitinated OFD1 and the levels of endogenous praja2 were detected as in A.

C. HEK293 cells transfected with siRNAs were serum deprived, pre-treated with cycloheximide and stimulated with FSK at different time point. Lysates were immunoblotted for endogenous praja2 and OFD1. Hsp90 was used as loading control.

D. Quantitative analysis of the experiments shown in C. A mean value ± SD of three independent experiments is shown. Student's t test * p<0,05; **<0,01 versus sipraja2 (+FSK) and basal values (0).

E. Ubiquitination of flag-OFD1 or flag-S735A in HEK293 cells treated with FSK for 1 hour.

F. Immunoblot analysis of flag-OFD1 or flag-S735A in HEK293 cells stimulated with FSK as in C. The levels of α-tubulin were used as loading control.

G. Quantitative analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Student's t test *p<0,05 versus S735A mutant (3 hours and 4 hours, +FSK) and basal values (-FSK).

H. Immunoblot analysis of flag-OFD1 or flag-E97G mutant in HEK293 cells stimulated with FSK as in C.

I. Quantitative analysis of the experiments shown in H. A mean value ± SD of three independent experiments is shown. Student's t test, *p<0,05 versus E97G mutant (+FSK) and basal values (0).
The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation (2021)
Figure 6

Figure 6. The TBC1D31 signalling circuitry controls ciliogenesis.

A. siRNA transfected HEK293 cells were serum deprived and stained for acetylated-tubulin, TBC1D31 and DRAQ5.

B. Quantitative analysis of the experiments shown in A. A mean value ± SD of four independent experiments is shown. Student's t test *p< 0,05.

C. HEK293 cells expressing flag-OFD1 or flag-S735A were serum deprived and subjected to staining analysis for acetylated α-tubulin, flag and DRAQ5. Images were collected by super-resolution microscopy (upper panels). Where indicated, flag-OFD1 or flag-S735A was co-transfected with cilia-APEX-GFP vector (middle panels). NIH3T3 expressing flag-OFD1 or flag-S735A were stained with anti-ARL13B and anti-flag antibodies.

D, E. Statistical analysis of the experiments shown in D. A mean value ± SD of three independent experiments is shown. For each experimental group, a minimum of 40 cilia were analyzed. Student's t test p***<0,001.

F. HEK293 cells transiently transfected with flag-OFD1 or flag-S735A were serum deprived, treated with FSK (6 hours) and stained for flag, acetylated-tubulin and DRAQ5.

G. Statistical analysis of the experiments shown in F. A mean value ± SD of three independent experiments is shown. Cells analysed for each experiment: 300 for NT, 80 for flag-OFD1 and 80 for flag-S735A. Student's t test, p*<0,05, p**<0,01.

H. Quantitative RT-PCR analysis showing levels of Gli2 mRNA in NIH3T3 fibroblasts transfected with vectors for flag-OFD1 and flag-S735A. An empty vector (CMV) was used as control. Serum deprived cells were stimulated with the SHH ligand purmorphamine (1μM) for 24 hours. Where indicated, cells were treated with FSK for 6 hours before harvesting. The data represent a mean value ± SD of three independent experiments. Student's t test, p**<0,01.
The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation (2021)
Figure 7

Figure 7. TBC1D1 and PKA/OFD1 pathway controls medaka fish development.

A. Stereo-microscopic images of wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735D and Ol-TBC1D31 KD + hOFD1S735D + hpraja2rm injected Medaka larvae, at stage 40.

B. Confocal images of cilia of the neural tube cells in the wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, wild-type hOFD1, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735D and Ol-TBC1D31 KD + hOFD1S735D + hpraja2rm stained with anti-acetylated α-tubulin antibody (green) and DAPI (blue).

C. In the graph is reported the cilia length in wild-type, Ol-TBC1D31 KD, Ol-TBC1D31 KD + hTBC1D31, wild-type hOFD1, Ol-TBC1D31 KD + wild-type hOFD1, hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735A, Ol-TBC1D31 KD + hOFD1S735D and Ol-TBC1D31 KD + hOFD1S735D + hpraja2rm. The data are expressed as mean value ± SE of twelve independent experiments. Student's t test , ***p≤0.001).

D. TBC1D31 assembled complex at centrosome positively regulates ciliogenesis and signalling. Ligand (L)-induced stimulation of G-protein coupled receptors (GPCRs) activates the adenylate cyclase (AC) and elevates cAMP levels, which in turn activates PKA. PKA phosphorylates OFD1 at S735, priming the ciliopathy protein to praja2-dependent ubiquitylation and proteasomal degradation. Decreasing the levels of OFD1 negatively impacts on cilium biogenesis and downstream signalling.

Extended View Figures.
A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage (2022)
Figure 1

Figure 1. Identification of CBASS-associated genes capH and capP and role of CapH and CapP in the CBASS antiviral response

(A) Operon schematics of four representative CBASS systems with associated capH (yellow) and capP (pink) genes. See Table EV1 for all identified systems. For each system, core CBASS genes are colored as in the key: CD-NTases orange, putative regulator(s) blue, and effector(s) green. Dotted outlines indicate unknown theoretical genes.

(B) Distribution of capH+capP-associated CBASS systems, sorted by system type as defined by Millman et al. (Millman et al., 2020). Type III (1H) and Type III (2H) refer to Type III systems with one or two HORMA domain proteins, respectively.

(C) Distribution of capH+capP -associated CBASS systems, sorted by effector type as defined by Millman et al. (Millman et al., 2020). 1TM, 2TM, and 4TM refer to effectors with one, two, or four predicted transmembrane segments, respectively.

(D) Top: Schematic of GFP expression reporter system, with the CBASS promoter, capH, and capP genes from E. coli MS115-1 and the CBASS core genes replaced with GFP. Bottom: Western blot showing GFP expression in cells with the wild-type GFP reporter or constructs lacking either capP or capH genes. RNAP: RNA polymerase loading control. See full blot in Fig EV1A.

(E) Western blots of the CBASS expression reporter system with FLAG-NucC (see Fig EV1B), showing FLAG-NucC expression after infection with phage λ cI- (multiplicity of infection: 10). MPI: minutes post infection. α-RNAP: anti-RNA polymerase loading control. Low RNAP expression at later time points is due to cell death.

(F) Quantitative plaque assay showing infectivity of λ cI- against cells containing no CBASS system (EV: empty vector), the wild-type E. coli MS115-1 CBASS system (WT), or a mutant system lacking capP (∆capP). Bars represent average of plaque forming units per mL of purified phage (PFU/mL), from duplicate experiments.

(G) Size of phage plaques for λ cI- infecting cells containing no CBASS system (EV: empty vector; n=52), the wild-type E. coli MS115-1 CBASS system (WT; n=20), or a mutant system lacking capP (∆capP; n=23). Data are shown as average and standard deviation of all plaques counted in panel F.
A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage (2022)
Figure 5

Figure 5. CapP cleaves CapH and is stimulated by single-stranded DNA

(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type or catalytic-dead (E98Q) CapP. Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. RNAP: RNA polymerase loading control.

(B) In vitro cleavage of purified MBP-CapH-GFP (yellow arrowhead) into N-terminal and C-terminal products (white arrowheads) by CapP is stimulated by DNA. For both ssDNA and dsDNA, the highest concentration is 350 ng/μL, followed by three 5-fold dilutions. Red asterisk indicates the band that was analyzed by Edman degradation. See Fig EV5 for analysis of DNA binding and sequence-specificity for cleavage activation.

(C) Results of Edman degradation of CapH C-terminal cleavage product (red asterisk from panel (B), showing cleavage at residue R83. See Appendix Fig S3 for full data.

(D) Cartoon view of the CapH67-107 dimer, with R83 colored white and shown as sticks.

(E) In vitro cleavage of MBP-CapH-GFP (wild-type or R83A mutant) by CapP, in the presence of 10 μM ssDNA.

(F) GFP reporter assay showing effect of a CapP E98Q catalytic-dead mutant, CapH R83A mutant, or removal of CapH residues 83-107 (capH 1-82) on GFP expression. RNAP: RNA polymerase loading control. See full blot in Fig EV1A.

(G) CBASS expression reporter system with FLAG-NucC, showing effect of a CapH R83A mutant, or removal of CapH residues 83-107 (capH 1-82) on FLAG-NucC expression. α-RNAP: anti-RNA polymerase loading control. See full blot in Fig EV1B.
A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage (2022)
Figure 6

Figure 6. CapH and CapP induce CBASS expression in response to DNA damage

(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type CapP after exposure to zeocin (100 μg/mL). Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. α-RNAP: anti-RNA polymerase loading control.

(B) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to zeocin (100 μg/mL). α-RNAP: anti-RNA polymerase loading control.

(C) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to mitomycin C (1 μg/mL). α-RNAP: anti-RNA polymerase loading control.

(D) Proposed signaling pathway for DNA damage-responsive transcriptional control systems in bacteria.

(E) Diverse systems in bacteria that include an HTH transcriptional repressor (yellow) and a DNA damage-activated Zn2+ metallopeptidase (pink) that targets the transcriptional repressor for cleavage.

(F) Known or likely bacterial defense systems associated with CapH (yellow) and CapP (pink)-like genes. See Materials and Methods for accession numbers for each gene.
Altered bioenergetics and mitochondrial dysfunction of monocytes in patients with COVID-19 pneumonia (2020)
Figure 2

Figure 2. Unsupervised analysis of monocyte subsets in CTR and COVID-19 patients.

A. Uniform Manifold Approximation and Projection (UMAP) representation of monocyte subpopulations. B. Heatmaps representing different monocyte clusters identified by FlowSOM, with percentages in healthy donors (CTR) and COVID-19 patients. The colors in the heat map represent the median of the arcsinh, 0-1 transformed marker expression calculated over cells from all the samples, varying from blue for lower expression to red for higher expression. Each cluster has a unique color assigned (bar on the left which colors correspond to those used in panel A). C. Differential analysis between healthy donors (CTR, n=8) and COVID-19 (COVID, n=7). The heatmap represents arcsine-square-root transformed cell frequencies that were subsequently normalized per cluster (rows) to mean of zero and standard deviation of one. The color of the heat varies from blue, indicating relative under-representation, to red indicating relative over-representation. Bar and numbers at the right indicate significant differentially abundant clusters (green) and adjusted p values (Differential analysis performed by diffcyt package present in the Catalyst package). The statistical analyses was done using GLMM (according to the actual guidelines) and all p-values were Bonferroni corrected. Clusters were sorted according to the adjusted p values, so that the cluster at the top shows the most significant abundance changes between the two conditions. D. Boxes and whiskers plots representing maximum, minimum, median value, 25 and 75 percentile of different subpopulations of monocytes in healthy donors (CTR, n=8) and COVID-19 patients (COVID, n=7). E. Scatter plots showing the frequency of PD-1+ cells, PD-L1+ cells among total monocytes in healthy controls (CTR, n=8) and COVID-19 patients (COVID, n=7). A scatter plot reporting the median fluorescence intensity (MFI) of PD-L1 in total monocytes is also shown. Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.
CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma (2022)
Figure 1

Figure 1. CRIP1 is upregulated in HCC and associated with poor survival.

(A) Western blot analysis of CRIP1 protein expression in 16 paired HCC tumor tissues (T) and adjacent nontumor tissues (N). The graph represents CRIP1 protein expression normalized to GAPDH levels (right).

(B) Representative IHC images of CRIP1 protein expression in 108 paired HCC tumor tissues and adjacent nontumor tissues. Scale bars: 20 μm. Statistical analysis of immunohistochemical score of CRIP1 expression in HCC tumor tissues and paired adjacent tissues (right).

(C) CRIP1 mRNA expression in the TCGA-LIHC dataset.

(D) mRNA levels of CRIP1 in the GSE36376 dataset from the GEO database.

(E-F) The mRNA (E) and protein (F) levels of CRIP1 in HCC cell lines and normal LO2 hepatocytes.

(G) Relative CRIP1 protein level compared to normal LO2 hepatocytes.

(H-I) Kaplan-Meier survival analyses of OS (H) and PFS (I) based on CRIP1 expression levels in 108 HCC patients.

Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Differences were tested using a two-tailed, paired (A, B) and unpaired (C-E, G) Student's t-test and the log-rank test (H-I).
CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma (2022)
Figure 2

Figure 2. CRIP1 promotes HCC stemness features.

(A) GSEA from TCGA data mining.

(B) Tumor sphere formation assay in CRIP1-overexpressing Huh7 and Hep3B cells. The data are shown as the relative fold change spheres compared with the vector group (right). Scale bar: 50 μm.

(C) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 and Hep3B cells.

(D) Flow cytometry analysis of the proportion of CD133+ cells in CRIP1-overexpressing Huh7 and Hep3B cells.

(E-G) In vivo limiting dilution xenograft formation of Hep3B cells with CRIP1-overexpressing or control cells (n = 5). In vivo bioluminescent images (E), bright-field images (F), and the statistical analysis (G) of xenograft formation of Hep3B cells are presented.

(H) Tumor sphere formation assay in CRIP1-knockdown MHCC-97H and HepG2 cells. The data are shown as the relative fold change spheres compared with the siCtrl group (right). Scale bar: 50 μm.

(I) Western blot analysis of HCC cancer stem cell markers in CRIP1-knockdown MHCC-97H and HepG2 cells.

(J) Flow cytometry analysis of the proportion of CD133+ cells in CRIP1-knockdown MHCC-97H and HepG2 cells.

(K) In vivo limiting dilution xenograft formation of CRIP1-knockdown or control MHCC-97H cells (n = 5). Bright-field images (top), and the statistical analysis (bottom) of xenograft formation are presented.

Data information: Data represent the mean ± SD at least three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test (B, D, H, J). Limiting dilution assay calculations were performed using the ELDA software (G, K)
CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma (2022)
Figure 4

Figure 4. CRIP1 promotes STUB1-mediated degradation of BBOX1.

(A) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells.

(B) Western blot analysis of BBOX1 protein levels in CRIP1 knockdown MHCC-97H and HepG2 cells.

(C) ELISA showing carnitine levels in CRIP1-overexpressing Huh7 and Hep3B cells transfected with BBOX1.

(D) Western blot analysis of BBOX1 in CRIP1-overexpressing Huh7 cells treated with CHX (100 μg/ml) for the indicated durations (lfte). The graph shows quantification of relative BBOX1 levels (right).

(E) Western blot analysis of BBOX1 protein levels in CRIP1-overexpressing Huh7 and Hep3B cells treated with MG132 (10 μM) for 10 h.

(F) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells.

(G) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in CRIP1-overexpressing Huh7 and Hep3B cells transfected with STUB1 siRNAs.

(H) Co-immunoprecipitation and immunoblot analyses of the interaction among endogenous CRIP1, STUB1, and BBOX1 in Huh7 and Hep3B cells.

(I) GST pulldown assay analysis of the association of CRIP1 with BBOX1 and STUB1.

(J) Co-immunoprecipitation and immunoblot analyses of the interaction between STUB1 and BBOX1 and BBOX1 ubiquitination in Huh7 cells transfected with STUB1-Myc and CRIP1 siRNAs.

Data information: Data represent the mean ± SD of at least three independent experiments. *P < 0.05 and **P < 0.01. Differences were tested using an unpaired 2-tailed Student's t-test (C, D).
CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma (2022)
Figure 5

Figure 5. BBOX1 ubiquitination at K240 mediated by STUB1 promotes cancer stem-like properties induced by CRIP1.

(A) BBOX1 ubiquitination modification at Lys240 by MS/MS.

(B) Co-immunoprecipitation and immunoblot analyses of BBOX1 ubiquitination in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant.

(C) Western blot analysis of BBOX1-WT or K240R mutant protein expression in Huh7 and Hep3B cells transfected with increasing amounts of STUB1.

(D-G) Carnitine levels (D), tumor sphere formation ability and quantification of spheres (E), flow cytometric analysis of the proportion of CD133+ cells (F), and protein levels of HCC cancer stem cell markers (G) in STUB1-overexpressing Huh7 and Hep3B cells transfected with BBOX1-WT or BBOX1 K240R mutant. Scale bar: 50 μm.

Data information: Data represent the mean ± SD of at least three independent experiments. **P < 0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test (D-F).
CRIP1 suppresses BBOX1-mediated carnitine metabolism to promote stemness in hepatocellular carcinoma (2022)
Figure 6

Figure 6. CRIP1 activates the Wnt/β-catenin pathway by decreasing β-catenin acetylation.

(A) ELISA analysis of acetylcarnitine levels in CRIP1-knockdown cells treated with mildronate (5 mM) for 24 h.

(B) GSEA based on CRIP1 expression in the TCGA-LIHC database.

(C-D) Representative images of tumor sphere formation and quantification of spheres (C) and flow cytometric analysis of the proportion of CD133+ cells (D) in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h. Scale bar: 50 μm.

(E) Western blot analysis of HCC cancer stem cell markers in CRIP1-overexpressing Huh7 cells with or without iCRT3 (50 μM) for 24 h.

(F) Coimmunoprecipitation and immunoblot analyses of β-catenin acetylation in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24 h.

(G) Western blot analysis of cytoplasmic and nuclear fractions of β-catenin in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24h.

(H) Confocal microscopy images of β-catenin in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) for 24h. Scale bar: 10 μm.

(I) Co-immunoprecipitation and immunoblot analyses of β-catenin ubiquitination in CRIP1 knockdown MHCC-97H cells treated with mildronate (5 mM) and EX527 (20 μM) for 24 h.

Data information: Data represent the mean ± SD of at least three independent experiments. **P <0.01 and ***P < 0.001. Differences were tested using an unpaired 2-tailed Student's t-test (A, C and D).