EMBO/soda-nel huggingface dataset:
Biogenesis of secretory immunoglobulin M requires intermediate non-native disulfide bonds and engagement of the protein disulfide isomerase ERp44 (2021)
Figure 5

Figure 5. Formation of intra-subunit C575 bonds is needed for efficient polymerization.

A. Schematic view of the mutants generated to evaluate the functional role of the C575 intra-subunit bonds. HEK293T cells expressing λ chains were transfected with µHaloC575A-C414A (HC575A, depicted in red in the figure), µC414A (µ, in blue) or both mutants together.

B. To selectively label intracellular HC575A, cells were treated with 1 nM Halo-tag TMR Direct Ligand for 24 hours. The left panel shows aliquots of the lysates of the indicated transfectants, resolved under non-reducing conditions and decorated with anti-λ antibodies. In the two right panels, samples were precipitated with NP-Sepharose and electrophoresed under non reducing conditions. The signal for the Halo-tag TMR was checked directly in the gel. The arrows point at the various assemblies between µ, HC575A and λ chains.

C. The experiment shown aims to determine whether ERp44 can bind also to a single C575, or rather to non-native bonds. Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP-Sepharose, resolved under non reducing conditions and the blot decorated with anti-λ antibodies or anti-ERp44 as indicated. Black asterisks point to covalent complexes of Halo-ERp44 with µs2L2.
Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic disorder (2020)
Figure 1

Figure 1 - A detailed spatial map of the nucleolar human proteome.

A. Schematic overview of the nucleolus and its substructures: fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC).

B. UTP6 in U-2 OS cells exemplify proteins localized to whole nucleoli (HPA025936).

C. Fibrillar center localization shown by a UBTF IF staining in U-2 OS cells (HPA006385).

D. UMAP visualization of the IF images generated in the HPA Cell Atlas (also shown in Fig 3A). The images from singularly localizing nuclear proteins are highlighted in purple (nucleoli), blue (fibrillar center), brown (nuclear speckles) and orange (nuclear bodies). Multilocalizing nucleolar proteins are highlighted in yellow.

E. Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP-tagged HeLa cells (magenta), also supported by IF antibody staining using HPA040741 (green).

F. The multilocalizing ribosomal protein RPL13 detected in the nucleoli, cytosol and ER in MCF-7 cells (HPA051702).

Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.
Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic disorder (2020)
Figure 2

Figure 2 - Previously unknown nucleolar proteins.

A. KRI1 localized to the nucleoli in MCF-7 cells (HPA043574).

B. GPATCH4 localized to the nucleoli in U-2 OS cells (HPA054319).

C. FOXL2NB localized to the fibrillar center in SiHa cells (HPA061017).

D. FAM71E1 localized to the fibrillar center in U-2 OS cells (HPA048111).

E. LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533).

F. ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690).

G. METTL5 localized to the nucleoli in U-2 OS cells (HPA038223).

Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.
Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic disorder (2020)
Figure 3

Figure 3 - The nucleoli rim localization.

A. UMAP visualization of the IF images generated in the HPA Cell Atlas (also shown in Fig 1D), specifically highlighting the nucleolar protein clusters. The images from singularly localizing nucleolar proteins are highlighted in purple, fibrillar center proteins in blue and nucleoli rim proteins in green. Multilocalizing nucleolar proteins are highlighted in yellow.

B. IF staining of MKI67 shows localization to nucleoli rim in U-251 cells (CAB000058).

C. IF staining of GNL3 shows localization to nucleoli rim in U-2 OS cells (HPA036743).

D. HEK 293T cells expressing endogenous levels of N-terminus mNG-MKI67 show a faint nucleoli rim localization, although still visible. White arrows indicate cells where the rim could be seen.

E. Fixed HEK 293T cells expressing mNG-tagged MKI67 show a clear nucleoli rim localization.

F. HEK 293T cells expressing endogenous levels of C-terminus tagged GNL3 show nucleoli rim localization. White arrows indicate cells where the rim could be seen.

Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.
Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic disorder (2020)
Figure 4

Figure 4 - Nucleolar proteins recruited to mitotic chromosomes.

A. MKI67 (HPA000451).

B. NOC2L (HPA044258).

C. EMG1 (HPA039304).

D. BMS1 (HPA043081).

E. BRIX1 (HPA039614).

F. RSL1D1 (HPA043483).

Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.
Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic disorder (2020)
Figure 5

Figure 5 - Intrinsic disorder level for mitotic chromosome and nucleolar proteins as predicted by IUPRED2A.

A. The fraction of disordered residues per protein for the nucleolar proteins is significantly higher (*, adjusted P < 1x10-4, Dunn's test with Bonferroni correction. n = 848) compared to the cytosolic proteome as defined in the HPA Cell Atlas (n = 2,054). Additionally, the disorder levels for the nucleoli rim proteins (**, adjusted P = 4x10-4, n = 153) and the mitotic chromosome proteins (***, adjusted P < 1x10-4, n = 65) are significantly higher compared to the other nucleolar proteins. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median. Outliers are shown as dots.

B. Fraction of proteins (in percent) having at least one functional disordered domain (>30 consecutive amino acids) for the mitotic chromosome proteins compared to the cytosolic proteome. More than 81% of the mitotic chromosome proteins have at least one long disordered domain.

C-E. IF stainings of previously uncharacterized mitotic chromosome proteins predicted to be highly disordered. Protein of interest shown in green and DAPI in blue. Scale bar 10 µm. C. CCDC86 (HPA041117), 100% predicted disorder level. D. RRP15 (HPA024639), 87% predicted disorder level. E. PRR19 (HPA070350), 67% predicted disorder level.
Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic disorder (2020)
Figure 6

Figure 6 - Two recruitment phenotypes among mitotic chromosome proteins revealed.

A. MKI67 is detected on the chromosomes throughout all mitotic phases and exemplifies proteins in the early recruitment category.

B-F. IF stainings of proteins where localization to mitotic chromosomes is seen after metaphase, showing a typical late recruitment expression pattern. B. NOL12 (HPA003547). C. TIGD1 (HPA041717). D. BYSL (HPA031219). E. ACTL6B (HPA045126). F. ZNF202 (HPA059229).

Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.
Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic disorder (2020)
Figure 7

Figure 7 - Mitotic chromosome proteins in MKI67 KO HeLa cells.

A-B. GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells. A. GNL3 (HPA036743). Median overlapKO = 0.539, median overlapWT = 0.795 (P = 9.34x10-9) and median Pearson correlationKO = -0.335, median Pearson correlationWT = 0.134 (P = 3.58x10-6). B. PES1 (HPA040210). Median overlapKO = 0.516, median overlapWT = 0.755 (P = 1.77x10-13) and median Pearson correlationKO = -0.293, median Pearson correlationWT = 0.129 (P = 4.06x10-13).

C. No protein aggregates were observed in MKI67 KO cells for PWP1 (HPA038708), despite being localized to mitotic chromosomes in WT cells. Median overlapKO = 0.523, median overlapWT = 0.861 (P = 1.05x10-13) and median Pearson correlationKO = -0.190, median Pearson correlationWT = 0.515 (P = 8.71x10-15).

D-E. Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells. White arrows indicate where protein aggregates can be seen. D. DENND4A (HPA065343). Median overlapKO = 0.671, median overlapWT = 0.803 (P = 1.02x10-4) and median Pearson correlationKO = -0.224, median Pearson correlationWT = 0.236 (P = 1.91x10-5). E. NOL12 (HPA003547). Median overlapKO = 0.468, median overlapWT = 0.820 (P = 1.01x10-17) and median Pearson correlationKO = -0.318, median Pearson correlationWT = 0.381 (P = 1.81x10-10). White arrow indicates metaphase cell.

Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.
LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy (2021)
Figure 3

Figure 3. veal2 lncRNA interacts and negatively regulates Prkcbb protein in Zebrafish

A. Schematic of the methodology adopted for the identification of protein interacting partners of veal2 using a RAP-MS based approach.

B. RAP- MS followed by western blotting validated Prkcbb and Ephb3 as interacting protein partners with veal2.

C. RNA immunoprecipitation of veal2 was performed by pull-down of Prkcbb and Epbhb3, followed by qRT-PCR. IgG pull-down was performed as control. Gel image shows the amplification of veal2 in different RNA immunoprecipitation samples, along with 10% input control, as marked by arrowheads.

D. qRT-PCR based quantification of veal2 transcript across Prkcbb, Ephb3 and IgG immunoprecipitations. actb was used as normalization control. Data from three different biological replicates represented as mean fold change values ± standard deviation.

E. Relative kinase activity of human PRKCB2 under standard conditions and in the presence of various concentrations of the WT veal2 RNA, veal2-Δ8 RNA, veal2-AS RNA. 52nM of the PRKCB2 protein was used per reaction. Data from three different experiments plotted as mean fold change values ± standard deviation.

F-K. Enzastaurin treatment rescues hemorrhage phenotype in veal2gib005Δ8/+ zebrafish embryos indicated by rescue of the vascular integrity defects in veal2gib005Δ8/+. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. Experiment was repeated in biological replicates and total no of embryos scored are mentioned on figure. (F-I) Magnification-5X, Scale bar-100μm, (J-K) Magnification-20X, Scale bars-50μm.

L. Relative number of animals that displayed the hemorrhage phenotype across the progeny of the outcross of veal2gib005Δ8/+ zebrafish. Number of phenotypic animals upon treatment with Enzastaurin was normalized with the number of phenotypic animals when treated with DMSO. Data from three different experiments plotted as mean fold change values ± standard deviation.

Data information: All the experiments N≥3, *** p-value<1E-3, **** p-value<1E-4. Statistics: (D,L) unpaired two-tailed T-tests. (E) Two-way ANOVA with Bonferroni's multiple data comparison.
LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy (2021)
Figure 4

Figure 4. veal2 interacts with DAG binding C1 domain of Prkcbb protein and regulates kinase activation

A-B. In-silico docking of veal2 and Prkcbb identified putative 4 interaction motifs in Prkcbb. Motif1-(549, 551-553, 559, 560) in Purple, motif2-(584-587) in Orange, motif3-(678-680) in Red, motif4-(844-846) in Green. The 8bp deletion in veal2-Δ8 RNA led to change in folding and altered interaction with Prkcbb.

C. Scatter plot showing the number of animals which displayed the hemorrhage phenotype across the progeny derived from an outcross of veal2gib005Δ8/+ zebrafish injected with IVTs of different veal2 variants. Control indicates veal2gib005Δ8/+ zebrafish without complementation. Data from three different experiments plotted as individual values; the middle bar represents mean percentage and the error bar represents ± standard deviation.

D. The site of interaction of the motif3 of veal2 (CLM) with the Prkcbb protein indicates that it lies in a previously known DAG binding site. Both RNA (pink) and protein (purple) structures are shown as ribbon models. The distances between nucleotides of motif-3 of veal2 and the 2 amino acids of Prkcbb are given in Å units.

E. Relative kinase activity of human PRKCB2 with various concentrations of DAG without or with different concentrations of veal2 WT RNA (0, 0.025nM, 0.25nM, 2.5nM, 25nM). PRKCB2 activity without DAG was used for normalization. Data from three different experiments plotted as mean fold change values ± standard deviation.

F. Abundance of Prkcbb protein in total cell (T), cytoplasmic (C) and membrane (M) fractions of cells from 2 dpf gib004Tg(fli1a:EGFP;gata1a:DsRed) and veal2gib005Δ8/+ zebrafish embryos. Arrowhead indicates Prkcbb enrichment in the membrane fraction of veal2gib005Δ8/+ zebrafish embryos.

G. Relative quantification of Prkcbb localization in cytoplasmic and membrane fractions of control and veal2gib005Δ8/+ embryos. Data from three different experiments plotted as mean percentage values ± standard deviation.

Data information: All the experiments N≥3.
LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy (2021)
Figure 5

Figure 5. A conserved human VEAL2 (VEAL2) interacts and regulates human protein kinase c beta protein.

A. Schematic of the methodology adopted for the identification of RNA interacting partners of PRKCB in HUVECs using a RIP-seq based approach.

B. RIP-Seq of PRKCB in HUVECs identified a candidate lncRNA with significant q-value (0.002) and high expression.

C. Representation of genomic location of AC008440.2- human vascular endothelial associated lncRNA 2 (VEAL2) transcript. It is an antisense to 3' UTR of known protein coding myeloid-associated differentiation marker (MYADM) gene. 3'RACE analysis confirmed VEAL2 as an independent transcript. Reads from PRKCB-RIPseq and IgG-RIPseq mapping to VEAL2 loci are also given. Blue highlights reads mapping to +ve strand and Red highlights reads mapping to -ve strand.

D. Absolute quantification of VEAL2 in HUVECs revealed 0.024 copies per cell. 102 to 108 copies of VEAL2 RNA were used to make the standard curve. Data from three different experiments plotted as mean values ± standard deviation.

E-H e-GFP fusion assay confirms lack of peptide formation from veal2 sequence. (E-F) mitfa-eGFP fused transcript. (G-H) VEAL2-eGFP fused transcript. Arrowheads indicating e-GFP expression in mitfa-eGFP injected embryos. Scale bar-100μm.

I-N. Complementation of VEAL2 in veal2gib005Δ8/+ embryos significantly rescued hemorrhage phenotype. Arrowheads show the presence of hemorrhage due to the vascular integrity defects. (I-L) Magnification-5X, Scale bar-100μm (M-N) Magnification-20X, Scale bars-50μm.

O-Q. Single molecule-FISH (smFISH) of VEAL2 in HUVECs shows its cytoplasmic localization. (O) VEAL2 in CAL-fluor red(610nM). (P) DAPI. (Q) Merged image for VEAL2 and DAPI. Magnification-100X, scale bar-5μm.

R-T. Co IF for PRKCB and smFISH of VEAL2 highlight their colocalization. (R) PRKCB in the GFP channel. (S) VEAL2 in CAL-fluor red (610nM). (T) Merged image for PRKCB and VEAL2. Magnification-100X, scale bar-20μm.

U. Bar graph represents the colocalization rate (%) of VEAL2 with PRKCB and CAMKIID proteins in HUVECs. Data from three different experiments plotted as individual values with mean percentage values ± standard deviation.

V. Relative kinase activity of human PRKCB2 under standard conditions and in the presence of various concentrations of the WT VEAL2 RNA and VEAL2-AS RNA. 52nM of the PRKCB2 protein was used per reaction. Data from three different experiments plotted as mean fold change values ± standard deviation.

Data information: All the experiments N≥3. *** p-value<1E-3. Statistics: (U) Unpaired two-tailed T-test. (V) Two-way ANOVA with Bonferroni's multiple data comparison.
LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy (2021)
Figure 7

Figure 7. VEAL2 is involved in diabetic retinopathy and can recover associated microvascular complications.

A. Bar graph representing relative expression of VEAL2 (in fold change) in choroid tissue isolated from control and diabetic retinopathy (DR) patients. Data obtained from 8 biological replicates (patients) and represented as individual values with mean fold change ± standard deviation.

B. Bar graph representing relative fold change of VEAL2 expression in blood samples of patients with different diabetic stages with aggravating vascular dysfunctions from diabetic melitus (DM) to non proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR) compared to control patients. Data was collected from 50 different patients in each condition and represented as individual values with mean fold change ± standard deviation.

C. ROC curve shows sensitivity and specificity of VEAL2 as a diagnostic biomarker for proliferative diabetic retinopathy with endothelial dysfunction.

D. Complementation of VEAL2 and veal2 reverted increased permeability levels in the HUVEC monolayer model for hyperglycemia. Bar graph representing the effect of overexpression of VEAL2 and veal2 on permeability levels in hyperglycemia disease model, measured as efflux of dextran conjugated FITC. Data obtained from 3 different biological replicates and plotted as mean percentage fold change values ± standard deviation.

E-S. Modeling hyperglycemia in HUVEC resulted in dysregulation of junctional assembly of CDH5 and CTNNB1 proteins and increased membrane localization of PRKCB protein. Complementation of VEAL2 and veal2 in hyperglycemic conditions reverted junctional disassembly of CDH5 and CTNNB1 and also kept PRKCB in cytoplasm to mitigate pathological conditions associated with hyperglycemia. (E-H,Q) CDH5 protein, (I-L,R) CTNNB1 protein, (M-P,S) PRKCB protein. (E-P) Magnification-60X, Scale bar-15μm. Arrowheads indicate representation of signals of proteins in HUVECs. (Q-S) Data from cells of different fields of 3 technical replicates of 1 biological replicate is presented as representation. Data is shown as individual values; the middle bar represents the mean and the error bar represents ± standard deviation.

Data information: All the experiments N≥3. **p-value<1E-2, ***p-value<1E-3, ****p-value<1E-4. Statistics: (A) Unpaired two-tailed T-test, (B,D,Q-S) One-way ANOVA with Bonferroni's multiple data comparison, (C) Wilson/Brown method
Cytoskeletal tension actively sustains the migratory T cell synaptic contact (2019)
Figure 1

Figure 1. Synapse polarization involves actin remodeling in mouse naïve CD4+ T cells ('T cells' henceforth) and proceeds despite of integrin augmentation.

(A) General schematic of the assay system used in this study for examining the process of synapse sustenance vs. polarization, post initial T cell adhesion and spreading on the antigen presenting surface (APS). Cell-APS interface of T cells was imaged for synapse establishment and maturation (5') and eventual symmetry breaking and polarization (20').

(B-D) T cell interface actin (phalloidin) re-organization during synapse symmetry breaking, imaged using SIM. Quantification of the shape elongation (AR; B, n= 44 for 5', 39 for 20'), or relative fluorescence signal of the indicated proteins in the SIM images (C, D) normalized to the mean of values at 5'; points represent values for individual cells. Arrowheads in (C) indicate foci. p values are: ***P< 0.0001 for AR, **P =0.003 for foci, **P= 0.008 for talin, and p =0.36 for total F-actin using Mann-Whitney two-tailed test between populations of cells within the same experiment.

(E) Relationship between AR and foci at individual cell level, each point in the scatter plot represents value obtained from a single cell.

(F-H) Integrin augmentation does not rescue synapse symmetry breaking. Cells were allowed to adhere to the APS for 5' or 10', and were then treated with vehicle control, 0.5mM MnCl2 or 100nM A286982 for subsequent 10'. Cells were fixed, stained with phalloidin and anti-Talin antibody, and imaged using SIM (F) and analyzed for AR (G) as well as talin, total F-actin and actin foci at the synapse (H). In (E-H), n for 5'= 40, for 20'= 62, for MnCl2= 59, for A286982= 53. The values in the plots represent the intensity values normalized to the mean of 5' in each set. p values in the graph, n.s. > 0.05; ***P <0.001; *P= 0.01; **P<0.009 using Mann-Whitney two-tailed test between populations of cells within the same experiment.

Data information: Central values in the graphs represent Mean and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5µm.
Cytoskeletal tension actively sustains the migratory T cell synaptic contact (2019)
Figure 2

Figure 2. Synapse symmetry involves temporal regulation of WASP and WASP-dependent actin kinetics.

(A) Time-lapse imaging of LifeAct-GFP expressing T cell using Lattice lightsheet microscopy (LLSM) during synapse polarization. Shown are snapshots at selected timepoints (left) and a kymograph of actin foci (denoted by arrows in the first image panel) over time (right) for a representative T cell.

(B) Quantification of changes in actin foci and cellular aspect ratio in LLSM images during symmetry breaking. The values in the plot are the raw values normalized to their mean in each case.

(C-E) T cells incubated with APS for the indicated time periods were processed for phalloidin- or immune-staining (C, D) or for western blotting (E). Graph in (D) shows relative levels of total and active WASP (WASP-phosphorylated at Y293, pWASP), normalized to the mean levels at 5'; for pWASP n in 5'= 49, in 20'= 44; for WASP, n in 5'= 56, 20'= 56. P values, P**= 0.0027 for pWASP and P**= 0.0035 for WASP using Mann-Whitney two-tailed test. The scatterplot in (D) shows the relationship between active WASP and foci on a per cell basis. Numbers in the graph in (E) indicate WASP band intensity divided by the actin band intensity, and resulting ratios normalized to the values at the 0' time point (non-activated cells), from three independent experiments (n=3). P value *= 0.025, using paired two tailed t-test.

(F-H) Actin dynamics in mature synapse of LifeAct-GFP expressing T cell using TIRFM. The images show the foci (pseudocolored red) extracted from the total synaptic F-actin (F-G), and kymograph shows the time course of foci and lamellar activity over a period of 210 sec. Histograms of foci and lamellar protrusion and retraction (fluctuations) dynamics in WT T cell (H); Lifetime of individual foci and lamellar events (graph on the bottom right, each dot represents a single foci/lamellar protrusion-retraction event n= 126 for foci and n= 77 for lamellar protrusions). ***P<0.0001 using Mann-Whitney two-tailed test between populations of cells within the same experiment.

(I) Kymograph of the lamellar activity of WASP-deficient cells that lack actin foci.

Data information: Central values in the graphs represent Mean and error bars represent ± SEM. These experiments were repeated at least thrice with similar results. Scale bar, 5µm.