EMBO/soda-nel huggingface dataset:
A selective ER-phagy exerts procollagen quality control via a CALNEXIN-FAM134B complex (2018)
Figure 7

Figure 7. CANX and FAM134B interact and deliver PC to autophagosomes.

(A) Schematic representation of FAM134B WT, lir mutant and delta reticulon constructs.

(B) U2OS cells were transfected with empty vector control (pcDNA3), FAM134B-HA WT or mutant constructs as indicated. (C) U2OS cells were transfected with HALO-PC2, FAM134B-HA or FAM134Blir-HA constructs, treated with 100 nM BafA1 for 6h and with CST where indicated. (B, C) Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against HALO, CANX, FAM134B, LC3 and β-ACTIN. 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.

(D) Proposed model of collagen recognition by autophagy. After synthesis, PC chains are subjected to quality control operated by the CANX/CRT chaperone system. Properly folded PC associates with HSP47 and is then secreted, whereas the misfolded fraction is sequestered by the CANX-FAM143B complex and delivered to lysosomes through ER-phagy.

Expanded View Figures
Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphomamphoma. (2018)
Figure 1

Figure 1 - MYC controls mTORC1 signaling through regulation of the TSC1.

A Immunoblot of expression levels of MYC, TSC1, TSC2, and β-actin loading control in high MYC Burkitt's lymphoma (BL) cells compared to low MYC Hodgkin lymphoma (HL) cells.

B Immunoblots showing expression levels of MYC, TSC1, TSC2 or β-actin loading control in P493-6 cells treated with tetracycline for 72 hours (+Tet) or in untreated cells (-Tet).

C Relative TSC1 and TSC2 mRNA expression levels determined by qRT-PCR for high MYC (-Tet) versus low MYC (+Tet) P493-6 cells treated for 24 h with tetracycline (mean ± st.dev., n=3 technical replicates) *p< 0.05; **p< 0.01; statistical relevance was determined by unpaired t-test (two-tailed).

D qRT-PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h-72h (+Tet). Immunoblots for 24h and 48h (+Tet) show S6K and phosphorylation (P-) of S6K as downstream mTORC1 target, and β-actin loading control. For 72 h (+Tet) the immunoblots show expression of MYC and phosphorylation (P-) of downstream mTORC1 targets S6K and S6, and α-tubulin as loading control.

E Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1-specific sh-RNAs compared to scrambled control sh-RNA in P493-6 cells. Other blots show the expression levels of TSC2, S6K/P-S6K, S6/P-S6 and α-tubulin for loading control.

F Immunoblots of indicated proteins in P493-6 cells with high MYC (-Tet, 72 hours) or low MYC (+Tet, 72 hours) levels either treated with rapamycin or solvent.
Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphomamphoma. (2018)
Figure 2

Figure 2 - Levels of TSC1 and MYC correlate in Burkitt's lymphoma.

A Elevated TSC1 and MYC expression in BL (cohort 1). Example of immune staining of TSC1, MYC, the B-cell marker CD20, and DAPI nuclear DNA-staining in germinal centers of control lymph nodes (upper rows) and samples from BL patients (lower rows). Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples (see materials and methods) (the horizontal line shows the median, whiskers show maximum and minimum data points and the box represents the first to the third quartiles, n=56 fields for tumor samples and n=21 fields for control germinal centers; scale bar = 100 μm).

B Quantification of immunoblot analysis of TSC expression in BL (cohort 2) (n=13) compared to healthy tonsils (n=3) or reactive lymph nodes (n=3) (mean ± st. dev.) shown in Fig EV2B.

Data information: In all graphs *p< 0.05; **p< 0.01; ***p<0.001, statistical relevance was determined by unpaired t-test (two-tailed).
Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphomamphoma. (2018)
Figure 3

Figure 3 - TSC1 is crucial for survival of Burkitt's lymphoma (BL) cells.

A Left graph shows the multiplication rate of P493-6 (-Tet) cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Right graph shows percentage of apoptotic P493-6 (-Tet) cells expressing scrambled control shRNA or a TSC1-specific sh-RNA determined by FACS analysis of AnnexinV/7AAD stained cells (mean ± st.dev., n=3 biological replicates).

B Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).

C TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control sh-RNA or TSC1-specific sh-RNA were treated with hydroxytamoxifen to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± st.dev., n=3 biological replicates).

D Survival rate of different BL cell lines upon TSC1 knockdown. Graphs show numbers of viable cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA 3 days after seeding of equal number of viable cells (determined by trypan blue exclusion; mean ± st.dev., n=3 biological replicates).

E Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity (10nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± st.dev., n=3 biological replicates); (Bl2 cells were selected for stable knockdown with Puromycin, DG75 cells without selection).

F Ramos cells expressing either a TSC1-specific or a control shRNA were inoculated into NOD/SCID mice and tumor volume was measured regularly. The immunoblot shows the level of knockdown of TSC1 (sh-TSC1b) compared to control (sh-contr), and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation. Tumor growth curves show mean ± SEM (n=8/group). Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation. The top panels show tumors derived from Ramos cells stably expressing control sh-RNA (sh-contr); the lower panels show tumors derived from Ramos cells stably expressing TSC1-specific sh-RNA (sh-TSC1b).

Data information: In all graphs * p< 0.05; ** p< 0.01; ***p<0.001, ****p<0.0001statistical relevance was determined by unpaired t-test (two-tailed).
Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphomamphoma. (2018)
Figure 4

Figure 4 - TSC1 function restricts oxidative phosphorylation and ROS production.

A and B TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in P493-6 (-Tet) cells expressing scrambled control sh-RNA or TSC-specific sh-RNA, treated with 20 nM rapamycin for 12 h, or tetracycline to repress MYC for 48 h where indicated (A) or in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated (B) (mean ± st. dev., n=6 biological replicates).

C and D Ratio of mitochondrial to genomic DNA (C) and expression of cytochrome C (CYCS) or ATP5G1 mRNAs (D) determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).

E TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner. FACS analysis of DCF-DA stained P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated (mean ± st.dev., n=3 biological replicates).

F TSC1 knockdown results in increased phosphorylation of SAPK/JNK. Immunoblot of SAPK/JNK Thr183/Tyr185-phosphorylation in P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA. α-tubulin expression serves as loading control.

G Antioxidant treatment rescues cells from death caused by TSC1 knockdown. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 3 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 10 μM of the antioxidant BHA where indicated (mean ± st.dev., n=3 biological replicates).

Data information: In all graphs *p< 0.05; **p< 0.01; ***p<0.001, statistical relevance was determined by unpaired t-test (two-tailed).
Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphomamphoma. (2018)
Figure 5

Figure 5 - MYC controls TSC1 expression through transcriptional and miR-15a-mediated regulation.

A Accumulation of EU-labeled (Click-It, Invitrogen) TSC1-mRNA over 3 h in high MYC (-Tet) versus low MYC (+Tet) P493-6 cells (mean ± st. dev., n=3 technical replicates).

B Drawing of the TSC1 promoter and luciferase-reporter constructs with the predicted E-box (CACGTG, pos. -317/-322) indicated. Relative luciferase units (RLU) of co-transfection experiments using the indicated TSC1-promoter-reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells (mean ± st. dev., n=3). Immunoblot shows MYC overexpression and α-tubulin as loading control.

C TSC1 mRNA turnover determined by pulse and chase (Click-It, Invitrogen) over 8 h in P493-6 cells, either treated with tetracycline to repress MYC or left untreated (mean ± st.dev., n=3 technical replicates).

D miR-15a expression determined by qRT-PCR in P493-6 cells treated with tetracycline for 3 days (+Tet) to suppress MYC expression or in untreated (-Tet) cells (mean ± st. dev., n=3 technical replicates).

E Immunoblots showing expression levels of TSC1, S6K/P-S6K and α-tubulin in P493-6 and HEK293T cells overexpressing miR-15a or a control miRNA. At the right miR-15a overexpression was determined by qRT-PCR in P493-6 and HEK293T cells (mean ± st. dev., n=3 technical replicates).

F Immunoblot showing TSC1 expression, S6K/P-S6K and α‑tubulin in MCF-7 cells transfected with LNA modified anti-sense miR-15a oligos or control LNAs. At the right, successful miR-15a knockdown determined by qRT-PCR in MCF-7 cells is shown (mean ± st. dev., n=3 technical replicates).

G Relative luciferase activities (RLU) derived from reporter constructs containing TSC1-3´UTR wild type sequences or with mutated miR-15a seed sequence binding site on co-transfection with miR-15a in MCF-7 cells (mean ± st. dev., n=3 biological replicates). The drawing at the left shows the TSC1-3'UTR with the position of the miR-15a/16-1 seed sequence and used mutation indicated.

H Rate of oxygen consumption in P493-6 (-Tet) cells ectopically expressing miR-15a or control-miRNA vector, under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated (mean ± st. dev., n=8 biological replicates).

Data information: In all graphs *p< 0.05; **p< 0.01; ***p<0.001, statistical relevance was determined by unpaired t-test (two-tailed).
Blood molecular markers associated with COVID-19 immunopathology and multi-organ damage (2020)
Figure 3

Figure 3. Protein variation associated with COVID-19 pathophysiology

Different expression of proteins in samples collected at the first timepoint. The heatmap (left panel) indicated expression patterns of proteins overrepresented (fold change > 2) in healthy control (1,656 proteins), mild (1,547 proteins) and severe (2,362 proteins) patient groups. The top categories enriched for clusters are shown. Values for each protein in each sample (columns) are color-coded based on expression level, low (blue) and high (red) z-scored FOT. The line plots (middle panel) indicated selected gene sets with up-up (upregulation in both mild and severe patients compared with healthy controls) or up-down or down-down regulation trend during disease progression. The bar plot (right panel) indicated Gene Ontology annotations with up-up or up-down or down-downregulation trend during disease progression (Fisher-exact test, p <0.05).

The Weighted Gene Co-Expression Network Analysis (WGCNA) of 31 COVID-19 samples shows modules that are highly correlated with clinical features (left heatmap). Among the 33 modules (ME0-ME32), six of them showed significant correlation with clinical parameters (asterisk in the heatmap). Gene Ontology (GO) enrichment analysis were performed on these six modules and the enriched pathways were presented on the right panel (Fisher-exact test, p <0.05).

Boxplots showing differences of the APTT time (left) and ME1-enriched protein expressions (right) between mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. * p <0.05, ** p <0.01, *** p <0.001. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.

Boxplots showing differences of IL-6 level, IL-10 level (left) and ME15-enriched protein expressions (right) between mild (n=13) and severe (n=18) COVID-19 patients. Differences between groups were estimated using Mann-Whitney-Wilcoxon test. * p <0.05, ** p <0.01, *** p <0.001. The horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Blood molecular markers associated with COVID-19 immunopathology and multi-organ damage (2020)
Figure 6

Figure 6. Differences in immune responses among COVID-19 patients.

Transcriptional profiles reflect the dynamic immune response in COVID-19. GSEA (FDR < 0.25; 1,000 permutations) was used to identify positive (red), negative (blue) or no (white) enrichment of BTMs (gene sets). The graph shows the normalized enrichment score (NES) of each selected BTM in the different time points (T1, T2 and T3) for patients with mild or severe COVID-19 illness, in comparison to healthy controls. The timepoints 1, 2, 4 or 1, 3, 5 were used as T1, T2 and T3 for patients with four or five sampling timepoints, respectively.

Expression levels of NETs' markers for individual transcripts in severe (n=37) versus mild (n=179) comparisons. Data were represented as means ± SEM and differences between groups were estimated using a Student's t test. *p < 0.05; ** p < 0.01; ***p < 0.001.

Heatmap of IFNG, GZMB and PRF1 gene expression in COVID-19 patients. Average expression values were centered and scaled. Red indicates a higher expression and blue indicates a lower expression.

Heatmap of the expression of genes enriched in IL-17 signaling pathway between healthy control and COVID-19 patients. Average expression values were centered and scaled. Red indicates a higher expression and blue indicates a lower expression.

T cell and innate immune response elucidate immunopathology of COVID-19.
Blood molecular markers associated with COVID-19 immunopathology and multi-organ damage (2020)
Figure 7

Figure 7. COVID-19-associated metabolomic changes in blood plasma.

Plasma metabolomic changes revealed a trajectory in COVID-19 severity, from healthy control (n=12), mild (n=29), to severe (n=17).

Changes in the concentration of plasma metabolites are associated with COVID-19 severity. The discharge group consist of all patients (mild and severe) that were recovered and discharged. L1, low-density lipoprotein subclass-1 (LDL1); VL, very low- density lipoprotein (VLDL); L5, LDL5; L6, LDL6; H2: high-density lipoprotein subclass-2 (HDL2); H3: HDL3; H4: HDL4.

COVID-19 severity is associated with significant changes in lipoprotein subclasses including high-density lipoprotein subclass-1 (HDL1), HDL4, low-density lipoprotein subclasses (LDL1, LDL4, LDL5), very low-density lipoprotein subclass-5 (VLDL5) and their compositional components (ApoA1, triglycerides, cholesterol). TG: triglycerides; FC: free cholesterol; CE: cholesteryl esters; CH: total cholesterol (i.e., FC + CE); PL: total phospholipids; A1: ApoA1; A2: ApoA2; L1TG: TG in LDL1; L1TG%: percentages of L1TG in total lipids of LDL1; L1%: percentage of LDL1 in all LDL; L-TG%: percentages of L-TG(TG in LDL) in total lipids of LDL; V5FC%, V5CE%: percentages of V5FC(FC in VLDL5) and V5CE(CE in VLDL5) in total lipids of VLDL5; L5CE%, L5CH%: percentages of L5CE(CE in LDL5) and L5CH(CH in LDL5) in total lipids of LDL5; H1FC%: percentages of H1FC (FC in HDL1) in total lipids of HDL1; H-A2: ApoA2 in both HDL and nascent HDL; H4A1, H4A2, H4CE, H4CH, H4FC, H4PL: ApoA1, ApoA2, CE, CH, FC and PL in HDL4;

Plasma levels of key enzymes and proteins directly involving lipoprotein metabolism are indicators for COVID-19 severity. Data were represented as means ± SD and differences between groups were estimated using a Student's t test. * p < 0.05; ** p < 0.01; *** p < 0.001. Control, n=12; mild, n=29; severe, n=17; discharge, n=16; sLDLR: soluble low-density lipoprotein receptor; LCAT: lecithin-cholesterol acyltransferase; CEPT: cholesteryl-ester transfer protein.

COVID-19 caused dysregulation in lipoprotein metabolism, glycolysis and TCA cycle. The three boxes from left to right are control, mild and severe, in which gray means normal, blue means decrease, red means increase. CS: Citrate synthase; IDH: Isocitrate dehydrogenase; ACO2: Aconitase; OGDH: α-ketoglutarate dehydrogenase; DLD: Dihydrolipoyl dehydrogenase; SDH: Succinic dehydrogenase; MDH: Malate dehydrogenase; PDH: Pyruvate dehydrogenase; PDK: Pyruvate dehydrogenase kinase; ACLY: ATP citrate lyase; ACAC: Acetyl coenzyme A carboxylase; FASN: Fatty acid synthetase; LDH: Lactate dehydrogenase.
Potent virus neutralization by human IgM against SARS-CoV-2 is impaired by class switch (2022)
Figure 1

Figure 1. Spike-protein specific antibodies of classes M, G, and A in human serum.

A. Spike-binding antibodies in serum. Binding by antibodies of classes M, G, and A is detected with secondary antibodies conjugated to different fluorochromes, and the results are shown on the left, middle, and right plots, respectively. Binding (geometric mean fluorescence intensity - GMFI- of corresponding secondary antibody) to TE cells expressing SARS-CoV-2 spike protein is plotted on the vertical axis, and binding to untransfected cells on the horizontal axis. Each point represents a value from one donor. Samples from donors with no known exposure to SARS-CoV-2 antigens (n = 86) are plotted with blue circles, recently infected donors (n = 34) with red circles, and vaccinated donors (n = 14) with green diamonds. Red circles with black triangles correspond to donors from whose B cells monoclonal antibodies were isolated (n = 5, Appendix Table S2). P values are derived from a two-way analysis of variance followed by Tukey's test to compare the specific binding, i.e., (binding to spike-expressing cells)/(binding to untransfected cells) between conditions (convalescent or vaccinated against unexposed) within each antibody class. The experiment was independently repeated three times, and results shown come from the third replicate.

B. Change in antibody binding following vaccination against SARS-CoV-2. Binding of antibodies from sera assessed in the experiment shown in (A), is shown for two samples of serum from each of 7 donors. Data are plotted as in (A) with binding to spike-expressing cells on the vertical axis, and to untransfected cells on the horizontal, using blue symbols for samples from before vaccination, and red symbols for samples from 15-29 days after the first vaccination (but before any second vaccination). Black lines connect points corresponding to pre/post pairs of samples from each donor. Left plot shows results for IgM and right plot for IgG.

C. Comparison of post-vaccination increase in binding to untransfected cells between IgG and IgM. The increase in binding to untransfected TE 0 cells between pre- and post-vaccination samples is shown on the left with blue symbols for IgG and on the right with black symbols for IgM. The horizontal line at y=0 is the expected result when no increased binding is observed. P value is derived from a two-tailed, Wilcoxon matched pairs signed rank test.

D. Comparison of spike-specific IgM, as measured by flow cytometry or ELISA. The left two "FC" are the results of flow cytometry, and the left verical axis shows the ratio of serum IgM binding to spike-expressing cells, vs binding to non-expressing control cells, as plottted in A. The right two columns show spike-RBD-specific IgM as measured by an IgM-capture ELISA, and the right vertical axis shows the optical densities (OD). P values are calculated by two-way analysis of variance, followed by Sidak's multiple comparisons test. The entire experiment was repeated three times, and the data shown are derived from the third replicate.