EMBO/soda-nel huggingface dataset:
S1P Defects Cause a New Entity of Cataract, Alopecia, Oral Mucosal Disorder, and Psoriasis-like Syndrome (2022)
Figure 4

Figure 4. S1P dysfunction impairs ETF flavination and stability.

A Representative immunohistochemical images of S1P, ETFA and ETFB in normal controls and patient 1. Scale bars: 100 μm.

B Unchanged mRNA levels and decreased protein levels of ETFA and ETFB were observed in the skin biopsy of patient 1. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates of normal controls; n = 6 technical replicates of patient 1) and immunohistochemistry (lower panel, n = 6 biological replicates; n = 3 technical replicates) of the S1P, ETFA and ETFB levels.

C Representative immunohistochemistry images of S1P, ETFA and ETFB in both Mbtps1-conditional knockout (cKO) and Mbtps1-loxP mice. Scale bars: 100 μm.

D MBTPS1 gene knockout led to decreases in the ETFA and ETFB protein levels in vivo. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates) and immunohistochemistry (lower panel, n = 6 biological replicates) of the S1P, ETFA and ETFB levels in Mbtps1-cKO mice and Mbtps1-loxP mice.

E Cycloheximide (CHX) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells. Left panel: Representative western blotting images of the ETFA and ETFB protein levels during CHX chase. Right panel: Quantification of the immunoblotting results corresponding to the left panel (n = 3 biological replicates).

F Mutant S1P (p.Val355Gly and p.Ter1053Arg) only weakly interacted with ETFA and ETFB. We constructed Flag-tagged wild-type and mutant S1P HaCaT cell lines and then performed a Co-IP assay with Flag antibody, and the interaction between S1P and ETFA/ETFB was detected by immunoblotting.

G Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), decreased the association between ETFA and ETFB. We performed a Co-IP assay with an ETFA antibody, and the interaction between ETFA and ETFB was detected by immunoblotting.

H Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), enhanced the incorporation of FAD into the ETF complex. The visible spectra of flavin show two shoulder peaks at 420 nm and 460 nm, indicating the incorporation of FAD in the ETF complex. In the presence of the wild-type S1P protein, the two peaks were shifted by 0.02 OD units, whereas the additional mutant S1P (p.Val355Gly and p.Ter1053Arg) weakly shifted the two peaks by 0.005-0.01 OD units.

Data information: The data are presented as the means ± SDs. Statistical significance was assessed by unpaired two-tailed Student's t test (B: upper panel, B: lower panel for S1P and ETFB, D: upper panel, D: lower panel for S1P and ETFB, E: right panel) or the mean-Whitney two-tailed U test (B: lower panel for ETFA, D: lower panel for ETFA). ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Three biological replicates were included in the experiment (F, G).
S1P Defects Cause a New Entity of Cataract, Alopecia, Oral Mucosal Disorder, and Psoriasis-like Syndrome (2022)
Figure 5

Figure 5. S1P dysfunction impairs cellular OXPHOS and increases glycolysis.

Mitochondrial respiration (OCR) in control (Ctrl) and MBTPS1-knockout (KO) HaCaT cells was quantified in real time using a Seahorse extracellular flux analyser. Right subpanels: Quantiﬁcation of basal respiration, maximal respiration, and OCR-coupled ATP production in mitochondrial respiration (n = 5 biological replicates).

B Quantiﬁcation of relevant metabolites in mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis in Ctrl and MBTPS1-KO HaCaT cells (n = 4 biological replicates).

C Nonmitochondrial respiration (ECAR) was quantified in real time using a Seahorse extracellular flux analyser. Quantification of non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve in Ctrl and MBTPS1-KO HaCaT cells (n = 4 biological replicates).

D MBTPS1 knockout led to a decrease in global ATP production in HaCaT cells. Control and MBTPS1-KO HaCaT cells were cultured in 6-well dishes. A standard curve was generated to calculate the sample ATP concentrations using an ATP Lite Luminescence Assay Kit (n = 4 biological replicates).

E MBTPS1 knockout significantly increased the generation of mitochondrial reactive oxygen species (Mito SOX) in HaCaT cells. Left panel: Representative immunofluorescence images of Mito SOX in HaCaT cells. Scale bars: 100 µm. Right panel: The immunofluorescence intensity was quantified to calculate the relative Mito ROS level in HaCaT cells (n = 4 biological replicates).

F Schematic representation of OXPHOS and glycolysis upon S1P dysfunction.

Data information: The data are presented as the means ± SDs. Statistical significance was assessed by unpaired two-tailed Student's t test (A-E). ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
S1P Defects Cause a New Entity of Cataract, Alopecia, Oral Mucosal Disorder, and Psoriasis-like Syndrome (2022)
Figure 6

Figure 6. Riboflavin therapy rescues the OXPHOS defect by restoring ETF stability.

A The S1P deficiency-induced decrease in ETFA and ETFB was significantly reversed by riboflavin (Rib) in a concentration-dependent manner in MBTPS1-KO HaCaT cells. GAPDH was used as a loading control. MBTPS1-KO cells were initially cultured in DMEM for 24 h and supplemented with 0, 2.5, 5, or 10 µM riboflavin for 3 days. The expression of ETFA and ETFB was then detected by immunoblotting.

B The S1P deficiency-induced abnormalities in mitochondrial respiration can be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells. Left panel: Mitochondrial respiration (OCR) was quantified in real time using a Seahorse extracellular flux analyser. Right panel: Quantification of non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve in HaCaT cells (n = 3 biological replicates).

C The decreased global ATP production caused by S1P deficiency could be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells (n = 4 biological replicates).

D Inflammatory lesions in CAOP syndrome were significantly improved by riboflavin supplementation. The representative head image of patient 1 before therapy is shown in Figure 1A (panel 1).

Data information: The data are presented as the means ± SDs. Statistical significance was assessed by unpaired two-tailed Student's t test (B, C). *P < 0.05; **P < 0.01; ***P < 0.001. Three biological replicates were included in the experiment (A).
Dysregulation of the miR-30c/DLL4 axis by circHIPK3 is essential for KSHV lytic replication (2022)
Figure 1

Figure 1: KSHV dysregulates inhibitory miRNA

(A) Heat map of differentially expressed miRNAs during KSHV replication at 0, 16 and 24 hour identified through miR-Seq. miR-29b and miR-30c are highlighted in red, with T test analysis.

(B) qPCR analysis of miR-29b and miR-30c levels in cells at 0 and 24 hours post-lytic induction. SNORD68 was used as a housekeeper (n=3).

(C, D) qPCR analysis of TREx 0 and 24 post-induction (n=3) using GAPDH as a housekeeper, analysis is of levels of pri-miR-29b/pri-miR-30c (C) and pri+pre-miR-29b/pri+pre-miR-30c (D).

(E) Representative western blot of scr or miR-30c transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3.

(F) Representative western blot of scr or miR-29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3.

(G) qPCR analysis of ORF57 DNA levels for viral load at 72 hours post induction, with scr and miR-30c transfected TREx cells with uninduced cells as a control and GAPDH as a housekeeper (n=3).

(H) qPCR analysis of ORF57 in HEK-293T cells for reinfection assay.

Data information: In B-H data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
Dysregulation of the miR-30c/DLL4 axis by circHIPK3 is essential for KSHV lytic replication (2022)
Figure 3

Figure 3: circHIPK3 is essential for KSHV replication

(A) qPCR analysis of circHIPK3 levels in scr and circHIPK3 KD stably expressing TREx cells. GAPDH was used as a housekeeper (n=3).

(B) qPCR analysis of HIPK3 levels in scr and circHIPK3 KD stably expressing TREx cells. GAPDH was used as a housekeeper (n=3).

(C) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control. Densitometry analysis of n=3 western blots.

(D) qPCR analysis of ORF57 DNA levels for viral load at 72 hours post induction, with scr and circHIPK3 KD TREx cells with uninduced cells as a control and GAPDH as a housekeeper (n=3).

(E) qPCR analysis of ORF57 in HEK-293T cells for reinfection assay (n=3).

(F) qPCR analysis of levels of miR-29b, miR-30c and miR-27a in scr and circHIPK3 KD cells 0 and 24 hours post-induction. SNORD68 was used as a housekeeper (n=3).

Data information: In A-F data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
Dysregulation of the miR-30c/DLL4 axis by circHIPK3 is essential for KSHV lytic replication (2022)
Figure 5

Figure 5: DLL4 is targeted by circHIPK3 and miR-30c

(A) qPCR analysis of DLL4 levels in TREx cells 0 and 24 hours post induction. GAPDH was used a housekeeper (n=3).

(B) Representative western blot of DLL4 expression in TREx cells 0 and 24 hours post induction. GAPDH was used a loading control (n=3).

(C) qPCR of DLL4 levels in scr and circHIPK3 KD stable TREx cells at 0 and 24 hours post induction with GAPDH as a housekeeper (n=3).

(D) Representative western blot of DLL4 levels in scr and circHIPK3 stable TREx cells at 0 and 24 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3.

(E) qPCR analysis of DLL4 levels in TREx cells that have been transfected with either scr or miR-30c 24 hours pre-induction, analysis performed 0 and 24 hours post induction. GAPDH was used a housekeeper (n=3).

(F) Representative western blot of DLL4 levels in scr or miR-30c transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3

(G) Fold enrichment of GAPDH and DLL4 in biotinylated miR-30c RIPs over Scr transfected into HEK-293Ts. (n=3)

(H) Luciferase Reporter Assay from HEK-293Ts co-transfected with DLL4 3'UTR reporter plasmid and either scr or miR-30c. Data presented is relative to an internal firefly control (n=3).

(I) qPCR analysis of DLL4 levels in TREx cells that have transfected with either scr or a miR-30c antagomiR 24 hours pre-induction, with analysis performed at 0 and 24 hours post induction. GAPDH was used as a housekeeper (n=3).

(J) Representative western blot of DLL4 levels in scr or miR-30c antagomiR transfected TREx cells at 0, 24 and 48 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3.

(K) qPCR analysis of DLL4 expression in Ago2 RIPs as a percentage of input. TREx Cells were transfected with either a scrambled control or a miR-30c antagomiR. (n=3)

(L) Luciferase Reporter Assay from HEK-293Ts co-transfected with DLL4 3'UTR reporter plasmid and either scr or miR-30c antagomiR. Data presented is relative to an internal firefly control (n=3).

Data information: In A-L data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
Dysregulation of the miR-30c/DLL4 axis by circHIPK3 is essential for KSHV lytic replication (2022)
Figure 6

Figure 6: circHIPK3:miR-30c:DLL4 dysregulation affects cell cycle

(A) qPCR of DLL4 expression in stable expression scr and 2 DLL4 KD TREx cell lines at 0 and 24 hours post-induction. GAPDH was used as a housekeeper (n=3).

(B) Representative western blot of DLL4 levels in scr and DLL4 KD TREx cells with GAPDH as a loading control, densitometry analysis is n=3.

(C) Representative western blot of ORF65 levels at 0, 24 and 48 hours post induction in scr and DLL4 KD TREx cells, GAPDH was used as a loading control and densitometry analysis performed on n=3.

(D) qPCR analysis of ORF57 DNA levels for viral load at 72 hours post induction, with scr and DLL4 KD cells with uninduced cells as a control and GAPDH as a housekeeper (n=3).

(E) qPCR analysis of ORF57 in HEK-293T cells for reinfection assay (n=3).

Data information: In A-E data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
Non-productive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immune responses (2022)
Figure 2

Figure 2. Exposure of PBMCs to SARS-CoV-2, and to a much lower extent SARS-CoV, triggers a JAK/STAT-dependent cell-intrinsic innate immune response

(A-C) RNA extracted from Ruxolitinib-treated or mock-treated, and SARS-CoV-, SARS-CoV-2- or mock-exposed PBMCs was analyzed for (A) IFIT1, (B) IFNA1 and IFNB1 (C) mRNA expression by Q-RT-PCR at indicated time points. Suspension and adherent cell fractions were analyzed separately, except at the four hours time point. Values were normalized to cellular RNASEP expression and are shown as fold change over mock-inoculated conditions. The dotted line indicates the expression level of mock-inoculated cell cultures and is set to 1.

(C) Supernatants from Ruxolitinib- or mock-treated and SARS-CoV-, SARS-CoV-2 or mock-inoculated PBMCs were collected 48 hours post exposure and cytokine expression of IFN-α2, IP-10/CXCL10, MCP-1/CCL2 and MCP-3/CCL7 were quantified using a Luminex-based immunoassay. PHA- or LPS-treated PBMCs were used as a positive control. Bars represent the results of a pool of four individual samples per condition.

(D) Supernatants collected from SARS-CoV-, SARS-CoV-2- or mock-exposed PBMCs at indicated time points were analyzed for the release of bioactive IFN using the HL116 reporter cell assay.

Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M (A-B, D) Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor and time points. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown). n.d. = not detectable.
Non-productive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immune responses (2022)
Figure 4

Figure 4. Exposure to SARS-CoV-2 induces a global innate immunity-related gene profile in PBMCs with cell type-specific signatures

(A) Pseudotime cell trajectory analysis and GSEA analysis using genes differentially regulated between mock-, SARS-CoV- and SARS-CoV-2-challenged conditions for indicated cell types.

(B) Representative UMAPs showing IFIT1 and ISG15 mRNA expression in the indicated conditions.

(C) Volcano plot of all DEGs in SARS-CoV-2-exposed cells compared to mock-exposed cells in the indicated cell types. Known ISGs were colored in green based on their presence in the interferome database (http://www.interferome.org/; v2.01).

(D) Cell trajectory maps of indicated cell types with cells colored by expression of the genes in an IFN-module gene set.

(E) Dot plot depicting expression of selected ISGs and cytokines. Expression levels are color-coded, the percentage of cells expressing the respective gene is coded by symbol size.

Data information: Data shown in this figure are based on the analysis of two donors.
Targeting of GPCR and EGF receptor overcomes PI3K inhibitor resistance in PTEN-null breast cancer (2020)
Figure 2

Figure 2. Anti-tumour effects of combinatorial inhibition of PI3Kβ and EGFR on triple-negative breast cancers in vivo

(A) Tumor volume of MDA-MB-468 mammary fat-pad xenografts treated with vehicle, AZD8186 (50 mg/kg, og twice/day), erlotinib (50 mg/kg IP once/day) alone or in combination (5-6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test **P=0.0028 and ***P<0.0001.

(B) Tumor volume of HCC-70 mammary fat-pad xenografts treated with vehicle, AZD8186 (150 mg/kg, og once/day), erlotinib (50 mg/kg IP once/day) alone or in combination (6-7 mice per group, mean±SEM). Growth curves were compared using two-way ANOVA statistical test. Statistical significance of two-way ANOVA statistical test ***P=0.0006 and ****P<0.0001.

(C) Waterfall representation of changes in the volume of individual HCC-70 tumors during the treatment. Statistical comparison between different treatment groups by Mann-Whitney test **P=0.0023 and ***P=0.0006.

(D) Change in the body weight of mice harbouring HCC70 xenograft tumors (6-7 mice per group, mean±SEM) during 21 days of treatment.

(E) A cell line derived from a mammary tumor spontaneously developed in a Wap-cre:Ptenfl/fl:Tp53fl/fl mouse was cloned and injected in the mammary fat pad of syngeneic C57BL6/J recipient mice. Tumours grew in 12 out of 35 transplanted mice and only these tumours were selected for the treatments described in the figure. When tumours reached an average volume of 100mm3, they were treated with vehicle or a combination of AZD8186 (150mg/kg, og once/day) and erlotinib (50mg/kg IP once/day). Tumours were then measured during the treatment (6 mice per group, mean±SEM). Statistical significance of two-way ANOVA statistical test ****P<0.0001.
Targeting of GPCR and EGF receptor overcomes PI3K inhibitor resistance in PTEN-null breast cancer (2020)
Figure 3

Figure 3. Biochemical effects of EGFR and PI3K pathway combined inhibition.

(A) MDA-MB-468 cells were treated for 24h with vehicle, PI3Kβi (AZD8186 250 nM), EGFRi (gefitinib 3 μM) or cetuximab (100 μg/ml), alone or in combination. The cell lysates were probed with the indicated antibodies.

(B) MDA-MB-468 cells were treated for 24h with vehicle, AKTi (MK2206 450 nM), gefitinib (3 μM) or cetuximab (100 μg/ml), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.

(C, D) HCC70 (C) and ZR-75-1 (D) parental cells or PI3Kβi-Res (derivative cells with acquired resistance to AZD8186), or AKTi-Res (acquired resistance models to MK2206) were treated with vehicle, PI3Kβi (AZD8186 250 nM) or AKTi (MK2206 1 μM) for 24 hours. The whole cell lysates were then probed with the indicated antibodies. Spliced images of parental and resistant paired samples were taken from the same original blot and blots showing P-S6 and S6 tot in (D) have been cut and reassembled for figure purposes.

(E) HCC70 parental, PI3Kβi-Res or AKTi-Res cells were treated with vehicle, AKTi (MK2206 250 nM) or gefitinib (3 μM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.

(F) HCC70 MK res (acquired resistance models to MK2206) were treated with serial dilutions of gefitinib, alone or in combination with MK2206 (810 nM), as indicated, and viability measured after 4 days of treatment. Average ± SD of triplicates and representative of two independent experiments.

(G) HCC70 AZD res (acquired resistance models to AZD8186) were treated with serial dilutions of gefitinib, alone or in combination with AZD8186 (270 nM), as indicated, and viability measured after 4 days. Average ± SD of triplicates and representative of two independent experiments.

(H,I) p110β co-immunoprecipitates with EGFR in MDA-MB-468 (H) and in HCC70 (I). Cells were pre-treated with different inhibitors, including vehicle, pan-PI3Ki GDC0941 (1 μM for MDA-MB-468 or 0.5 μM for HCC70), EGFRi (gefitinib 3 μM), PI3Kβi (AZD8186 250 nM) or a combination of EGFRi and PI3Kβi. Cell lysates were incubated with IgG control or anti-EGFR antibody and the immuno-complexes or the total lysates were immune-blotted with the indicated antibodies.

(J) EGF-induced increase in phospho-AKT is dependent on p110β kinase activity. MDA-MB-468 were starved in 0% FBS and pre-treated with vehicle or PI3Kβi (AZD8186 250 nM) for 1h. Cell lysates were probed with the indicated antibodies. Phospho-AKT and pan-AKT bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT normalised to the control (left hand lane) is shown.
Targeting of GPCR and EGF receptor overcomes PI3K inhibitor resistance in PTEN-null breast cancer (2020)
Figure 4

Figure 4. A CRISPR-Cas9 screening identified GNB2 as a target to potentiate the inhibition mediated by pan-PI3K inhibitor.

(A, B) Results of CRISPR-Cas9 screening in combination with AZD8186 100nM (A) or GDC0941 400nM (B). The dot-plots show for each gene knocked-out by sgRNAs the fold change (log2) between treated conditions (AZD8186 or GDC0941, respectively) and vehicle in the fluorescence signal (anti-phosphoS6 immunofluorescence) versus the p-value of the difference calculated by two sided t-test. The plots represent means of biological triplicates. Genes for which it was calculated a p<0.0001 and Log2 (fold change)>0.25 are reported and highlighted in green; genes having a 0.0001<p<0.05 and a Log2 (fold change)>0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A).

(C) Box & Whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941-treated conditions for MDA-MB-468 cells transduced with non-target Control, GNB2 or GNG5 sgRNAs (N=2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values within 1.5 times the inter-quartile range. Statistical significance of unpaired t-test ****P<0.0001.

(D) Biochemical analysis of GNB2 KO cells. Cell lysates of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software.
Targeting of GPCR and EGF receptor overcomes PI3K inhibitor resistance in PTEN-null breast cancer (2020)
Figure 5

Figure 5. GNB2 KO modifies the sensitivity to different inhibitors of EGFR-PI3K pathway.

(A) MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones were treated with vehicle, GDC0941 1 μM, gefitinib 3 μM or lapatinib 1 μM for 24 hours. The cell lysates were probed with the indicated antibodies.

(B-D) Viability assays of MDA-MB-468 parental cells and three MDA-MB-468 GNB2 KO clones treated with serial dilutions of the indicated drugs for six days. Mean ± SD of triplicates and representative of two or three independent experiments.

(E) Patient samples from METABRIC dataset classified as TNBCs (N=299) were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of GNB2 between the PTEN low or mut and the PTEN-WT groups. Box & Whisker plot (Median/IQR/1.5*IQR Whiskers). P value calculated by unpaired t-test.