EMBO/soda-nel huggingface dataset:
Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy (2021)
Figure 4

Figure 4. Pdia4 is involved in the regulation of cell death, ROS accumulation, and the activity of ETC and NOX.

(A) The islets of WT, Pdia4-/- (KO) and Pdia4tg/tg (TG) BKS mice were isolated and grown in complete DMEM medium containing 3.3 mM (LG) and 30 mM glucose (HG) for 12 h. The islets stained with propidium iodide (PI), photographed, and quantified. Scale bar = 50 μm.

(B) The islets from the mice (A) were treated with 3.3 mM (LG) or 30 mM glucose (HG). After lysis, the lysates underwent immunoblotting analysis using the antibody against Beclin 1, LC3 , p62 and actin.

(C-D) The islets from the mice (A) were incubated with MitoGreen plus MitoSOX (C) or Hoechst 33342 (Ho) plus CM-H2DCFDA (D) in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). Mitochondrial (C) and cytosolic ROS (D) in the islets were visualized and quantified. Scale bar = 100 μm.

(E) Mitochondria isolated from the mouse islets (A) were measured for the activity of ETC CI (left), CII/III (middle) and CIV (right) using MitoCheck assays.

(F) The membrane fraction of the pancreas from the mice (A) was measured for Nox activity using luminometric assays.

Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy (2021)
Figure 5

Figure 5. Pdia4 affects mitochondria and insulin granules in β-cells.

(A) Ultrastructure of mitochondria and insulin granules in typical β-cells of 3 mouse lines on BKS and Leprdb/db backgrounds. Arrows indicate the mitochondria whereas arrowheads indicate insulin granules. Scale bar = 1 μm.

(B) Average number (left), average area (middle), and swelling percentage (right) of the β-cell mitochondria from the mouse pancreatic islets (A) in microscopic sections.

(C) The average number of insulin granules in β-cells from the mouse pancreatic islets (A) in microscopic sections.

Data information: A total of 15-23 images per group were analyzed. Data from each group are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy (2021)
Figure 6

Figure 6. Binding domain mapping analysis of Pdia4 and Ndufs3 or p22phox.

(A) Mitochondrial (Mito, top left), ER (bottom left) and membrane (Mem, right) fractions of Min6 cells were precipitated with isotype (Iso) or anti-Pdia4 (Pdia4) antibody. The fractions and immunoprecipitates (IP) underwent immunoblotting analysis.

(B) Confocal analysis of the indicated proteins in Min6 cells. Min6 cells were intracellularly stained with anti-Pdia4 and anti-Ndufs3 antibodies plus MitoRed (top). Min6 cells were surface stained with anti-Pdia4 antibody, followed by intracellular staining with anti-p22phox antibody (bottom). Scale bar = 5 μm.

(C) The construct encoding Flag-Pdia4 and that expressing Myc/Flag-tagged Ndufs3 (top) or p22phox (bottom) were co-transfected into 293T cells. Total lysates of the cells were incubated with anti-Myc (left) or anti-Pdia4 (right) antibodies plus protein G beads. Their total lysates (TL) and immunoprecipitates (IP) underwent immunoblotting analysis anti-Flag antibody.

(D) His-tagged Pdia4 was incubated with the recombinant proteins, Gst-TecSH3, Gst-Ndufs3, and Gst-p22phox. The immunoprecipitates (IP) and recombinant proteins underwent immunoblotting analysis with anti-Gst and anti-His antibodies.

(E) The construct encoding Flag-Pdia4 or its mutants and that expressing Myc/Flag-tagged Ndufs3 (left) or p22phox (right) were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis. N-terminal Flag tag, catalytic domains (a, a' and a''), non-catalytic domains (b and b'), and C-terminal KEEL are indicated. Pdia4* is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs (a*, a'* and a''*).

(F) The construct encoding Myc/Flag-tagged p22phox, Ndufs3 or their mutants and that expressing Flag-tagged Pdia4 were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis using anti-Flag antibody.
Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy (2021)
Figure 7

Figure 7. Pdia4 regulates ROS generation involving Ndufs3 and p22phox in Min6 cells.

(A) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK), a Pdia4 RNAi (KD) and a Pdia4 cDNA (OVE) were sorted and tested for the Pdia4 protein level (left). The cells were incubated with NAC (1 mM) and then stained with MitoSOX or CellROX in response to 0.5 mM (LG) and 25 mM (HG) glucose for an additional 30 min. Signal from MitoSOX (middle) and CellROX (right) was re-plotted into histograms.

(B) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK) and an RNAi of Ndufs3 (Ndufs3 KD) or p22phox (p22phox KD) were selected and tested for levels of Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX), p22phox (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG) glucose.

(C) The same experiments as (B) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 (tNdufs3) and p22phox (tp22phox).

(D) Min6 GK or KD cells infected with a lentivirus expressing a cDNA of full-length Ndufs3 or p22phox were selected and tested for the level of Pdia4 and Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX), Pdia4 and p22phox (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG) glucose.

Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.
Pdia4 regulates β-cell pathogenesis in diabetes: molecular mechanism and targeted therapy (2021)
Figure 8

Figure 8. Effect of GHTT on diabetes development and ROS production in Leprdb/db mice.

(A) Molecular docking indicating the interaction between GHTT and active motifs (AMs) of Pdia4. AM 1, 2 and 3 represent the first, second and third CGHC domains. The sulfur, nitrogen and oxygen atoms are shown in yellow, blue, and red, respectively. The hydrogen bond and hydrophobic interaction between GHTT (light pink) and the amino acid residues (gray) of Pdia4 model are shown by the green and yellow dashed lines, respectively.

(B) Insulin-based turbidity assays were used. Insulin was incubated with recombinant Pdia4 in the presence of PBS and inhibitors at 25°C. After 30 min, the reaction was stopped and measured the absorbance at 595 nm. The relative activity (%) of Pdia4 was obtained by the formula, 100% × (OD595 of PBS − OD595 of GHTT)/(OD595 of PBS).

(C) Min6 cells were incubated with medium containing low glucose (LG, 0.5 mM) and high glucose (HG, 25 mM) in the absence or presence of 28 μM GHTT (HG+GHTT) and then stained with MitoSOX or CellROX for an additional 30 min. Signal from CellROX (right) and MitoSOX (left) was quantified and re-plotted into histograms.

(D) 293T cells, which were transfected with the construct encoding Flag-Pdia4 and that expressing Myc/Flag-tagged p22phox or Ndufs3, were treated with GHTT (28 μM) for 30 min. The cells were lysed and incubated with anti-Pdia4 antibodies plus protein G beads. Their total lysates (TL) and immunoprecipitates (IP) underwent immunoblotting analysis with anti-Flag antibody.

(E) Eight-week-old diabetic Leprdb/db mice were orally fed with a daily dose of PBS (CTR), sitagliptin (STG, 90 mg/kg) and GHTT (1, 5 and 25 mg/kg) from 8 to 24 weeks. Their FBG and PBG were measured.

(F) The sections of pancreata of the mice (E) were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions.

(G) A schema illustrating the regulation of Ndufs3 and p22phox by Pdia4 via a CGHC-dependent intermolecular interaction. Pdia4 interacts with Ndufs3 and p22phox, leading to an increased activity of ETC CI and Nox. Consequently, the exuberant ROS induces β-cell pathology and diabetes. Conversely, Pdia4 deficiency and inactivation of Pdia4 by GHTT can abolish the interaction of Pdia4 and p22phox or Ndufs3, leading to reduction of ROS production and β-cell failure and mitigation of diabetes.

Data information: Data from 3 experiments (B, C and F) and more (E) are presented as the mean ± SD. Log rank (E) and one-way ANOVA test (B, C and F) were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. The number of mice (n) is indicated in parentheses.
Deep mutational scanning of the Neisseria meningitis major pilin reveals the importance of pilus tip-mediated adhesion (2019)
Figure 2

Figure 2: Implication of the amino-terminal region in aggregation

(A) The level of aggregation relative to piliation level is represented as the Log2 of the ratio between the frequency of each single point mutation in the aggregation library relative to the frequency of the same mutation in the piliation library. Each round dot represents a point mutant. Mutation type is color-coded as indicated in the legend. Squares on the x-axis correspond to the original amino acids in the SB sequence with the protein secondary structure indicated below. Dotted lines represent a two-fold threshold value. Bottom insets illustrate the mutations generated de novo and further analyzed in Figure 2 and Figure 3.

(B) Scanning electron microscopy of single bacteria on a cellulose filter. Scale bars: 500 nm.

(C) Two representative immunofluorescence labelling of TFP. Scale bar: 1 µm.

(D) Piliation per bacterium as a function of the proportion of piliated bacteria normalized to pilESB values as measured by flow cytometry using the 20D9 monoclonal antibody. Black dots indicate all the individual de novo generated PilE mutants that were characterized. Blue dots indicate mutants of the piliation machinery and green dots mutants in the α1N region. Solid lines indicate 95% confidence interval.

(E) Quantification of aggregation normalized to pilESB for a subset of outliers and corresponding pilT double mutants. Mean values ± SEM are indicated for each strain. N≥3 independent experiments. Paired t-test. p<0.05 (*), p<0.01 (**).
Deep mutational scanning of the Neisseria meningitis major pilin reveals the importance of pilus tip-mediated adhesion (2019)
Figure 3

Figure 3: Carboxy-terminal region of the pilin involved in aggregation

(A) Pilus structure (PDB: 5KUA) showing a cluster of charged amino acids surrounding the protruding K140 involved in aggregation. N138 is in light blue, Q122 and K140 in orange and E99, K103, K144 and H149 in dark blue.

(B) Piliation per bacterium as measured by flow cytometry using the 20D9 monoclonal antibody and the F10 nanobody.

(C) Visualization of type IV pili by immunofluorescence either using the 20D9 antibody or F10 nanobody as indicated (magenta). In the case of the K140Q mutant labeled with the 20D9 antibody the background was made visible to convincingly show absence of pilus labeling. Scale bar: 2 µm.

(D) Aggregation expressed as a function of piliation per bacterium measured with 20D9 antibody except for pilEQ122E and pilEK140Q for which the F10 nanobody value is reported. Each dot represents values for a PilE point mutant relative to the SB strain. Hypo-aggregative mutants are highlighted in orange while hyper-aggregative mutants are in light blue. solid lines indicate the 95% confidence interval.

(E) Quantification of PilX present in purified pili preparations sheared from the bacterial surface using western blot. PilX levels were normalized using PilE as a reference and normalized to pilESB values. Mean ratio ± SEM is indicated. N=3 independent experiments. Paired t-test.
Deep mutational scanning of the Neisseria meningitis major pilin reveals the importance of pilus tip-mediated adhesion (2019)
Figure 4

Figure 4: Residues of the pilin localized towards the distal end of pili are involved in early adhesion

(A) The ratio between the adhesion and aggregation mutation scores for each amino acid in the PilE sequence is represented as dots. Mutation type is color-coded as indicated in the legend. The superimposed magenta line represents the average ratio averaged over a moving window of 5 amino acids (right axis). Shaded areas highlight the two regions identified as important for adhesion. The yellow to red heat map along the pilin sequence indicates the axial position of the amino acid in PilE as shown on the structure of PilE (distal tip in red, proximal tip in yellow).

(B) Early adhesion of pilE mutants as a function of aggregation. Values were normalized to that of pilESB. Each dot represents a pilE mutant and the pilV, pilD, pilX and pilC1 reference mutants. Mutants in the region surrounding A131 are in green and those surrounding Y50 in purple. A blown-up view of the zone of interest (lower left quarter) is presented on the right panel. The shaded area highlights the absence of significant adhesion.

(C) Early adhesion of PilE mutants as a function of piliation per bacterium normalized to that of pilESB.

(D) Piliation length of a selection of mutants determined by immunofluorescence. Average pilus length ± SEM is indicated. N=6 experiments with at least 50 bacteria analyzed in each experiment. Paired t-test.

(E) Number of pili per bacteria ± SEM determined as in (E). Paired t-test. p<0.05 (*).

(F) Amino acids from the two regions identified in panel A are highlighted in purple and green. The SM1 epitope is colored in light purple.
Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/β-catenin modulator Annexin A1 (2019)
Figure 1

Figure 1. F. nucleatum preferentially binds, invades and stimulates the growth of cancerous colorectal cells via Annexin A1.

(A) Lung cancer cells PC-9, prostate cancer cells 22RV1, bladder cancer cells UMUC3, breast cancer cells MCF-7, colonic adenoma-derived non-cancerous cells AA/C1 (aka C1) and AA/C1/SB (aka SB), or cancerous cells AA/C1/SB/10C (aka 10C) were incubated with wild-type F. nucleatum 12230 (Fn), the fadA-deletion mutant US1 (US1), or E. coli DH5α (E. coli) at multiplicity of infection (MOI) of 1,000:1. Cells numbers are mean values ± SEM. The experiment was performed in triplicates and repeated three times. **p<0.01, ***p<0.001, compared to untreated controls; #p<0.05, ### p<0.001, compared to US1-treated cells (two-way ANOVA).

(B) Attachment (left panel) and invasion (right panel) of wild-type F. nucleatum 12230 (Fn) to the non-cancerous SB cells, either untreated or transfected with antisense or sense ANXA1, and to the cancerous 10C cells, either untreated or transfected with control or ANXA1- or CDH1-specific siRNA or both (MOI 50:1). F. nucleatum attachment and invasion to the untreated SB cells were designated as 100%, respectively; all other values were expressed as relative to those obtained with untreated SB. Data are mean values ± SEM. The experiment was performed in triplicates and repeated four times. *p<0.05, **p<0.01 and ***p<0.001 (one-way ANOVA).

(C) Left panel: qPCR analysis of Villin1 (VIL1) mRNA levels in 10C cells treated with control siRNA or VIL1-specific siRNA, demonstrating knocking down of Villin1. Right panel: attachment of F. nucleatum 12230 to 10C cells treated with control siRNA or VIL1-specific siRNA. Data are mean values ± SD. The experiment was performed in triplicates and repeated twice. *p<0.05 (student's t-test).
Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/β-catenin modulator Annexin A1 (2019)
Figure 2

Figure 2. Annexin A1 is a novel colon cancer prognosis marker.

Relationship between ANXA1 mRNA expression levels and disease-free survival (DFS) in colon cancer patients. The relationship between ANXA1 mRNA expression levels and DFS was investigated in a database of 466 primary colon carcinomas, assembled by pooling four independent gene-expression array datasets from the NCBI-GEO online repository (GSE14333, GSE17538, GSE31595, GSE37892), as previously described [34]. The association between ANXA1 expression levels and DFS was tested using Kaplan-Meier survival curves, after patient stratification in groups with high, medium and low ANXA1 expression, using three different methods: (A) based on the median of ANXA1 mRNA expression levels (high 50% vs. low 50%); (B) based on the quartile distribution of ANXA1 mRNA expression levels (high 25% vs. middle 50% vs. low 25%); and (C) based on ANXA1 mRNA expression thresholds calculated using the StepMiner algorithm (low vs. high), as previously described [35, 36]. Overall, high ANXA1 mRNA expression levels were associated with a statistically significant reduction in DFS (p<0.001, log-rank test), irrespective of the method used for the stratification. Differences in ANXA1 mRNA expression levels did not appear to correlate with differences in each tumor's relative content of epithelial cells in the analyzed biospecimens (i.e. tumor cell density) as revealed by the lack of visual correlations with the epithelial cell marker Desmoplakin (DSP).
Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/β-catenin modulator Annexin A1 (2019)
Figure 3

Figure 3. Annexin A1 is selectively expressed in proliferating cancerous colorectal cells and a novel CRC growth factor.

(A) Real-time qPCR analysis of ANXA1 expression using mRNA extracted from the non-cancerous SB, cancerous 10C, and human CRC cell lines HCT116, DLD1 and RKO, each grown to 50% or 100% confluency. All results were normalized to the ANXA1 mRNA levels in SB cells of 100% confluency, which was designated as 1. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. **p<0.01 and ***p<0.001 (student's t-test).

(B) Confocal microscopy analysis of SB and 10C cells grown to 20% (top panels) or 100% (bottom panels) confluency followed by immuno-fluorescence staining of Annexin A1 (green). The nuclei were stained with DAPI (blue). A series of 20-50 consecutive images in the z-axis were stacked together to generate the 3-D figure at 400x magnification. Annexin A1 is most abundantly expressed on the outer layer of 20% confluent 10C (pointed by arrows; also see movie EV1), compared to 100% confluent 10C, or SB of either confluency. Scale bars, x = 1 μm, y = 1 μm, z = 1.6 μm. The experiment was repeated at least twice.

(C) Cell proliferation assay of adenoma-derived non-cancerous SB and cancerous 10C and human CRC cell lines HCT116, DLD1, SW480, HT29 and RKO either untreated (black lines) or following treatment with control siRNA (gray lines) or ANXA1-specific siRNA (green lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *p<0.05, ** p<0.01 and *** p<0.001, compared to the untreated cells (two-way ANOVA).

(D) Cell proliferation assay of SB (left panel) and RKO (right panel) cells transfected with ANXA1 (red line), as compared to the control cells (black lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA).

(E) Xenografted tumor growth in nude mice following subcutaneous and bilateral inoculation of HCT116 cells transfected with control siRNA or ANXA1-specific siRNA. The tumor volumes were measured after 7 days post injection (left panel, n=4). For each mouse, the tumor resulting from ANXA1-specific siRNA treated cells was normalized to that from control siRNA-treated cells, which was designated 100%. The line represents the average. *p<0.05 (paired t-test). The individual tumor pairs are shown on the right panel: top, tumors arising from control siRNA treated cells; bottom, tumors arising from ANXA1-specific siRNA treated cells.
Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/β-catenin modulator Annexin A1 (2019)
Figure 4

Figure 4. FadA induces Annexin A1 expression through E-cadherin.

(A) Real-time qPCR analysis of ANXA1 mRNA levels in SB, 10C, HCT116 and DLD1 cells incubated with wild-type F. nucleatum 12230 (Fn) or fadA-deletion mutant US1 (US1) at MOI of 50:1 for the indicated time periods. The results were normalized to those obtained with untreated cells and were the mean of 3 independent experiments each performed in triplicates. *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA).

(B) Flow cytometry analysis of Annexin A1 expression in 10C cells either untreated, or incubated with BSA (1000 µg/ml), or mFadA (1000 µg/ml), or FadAc (100, 300, or 1000 µg/ml) for 1 hr. Data are mean values ± SD. The experiment was performed in triplicates and repeated more than three times. **p<0.001 (one-way ANOVA).

(C) Flow cytometry analysis of Annexin A1 in 10C cells transfected with control siRNA (dotted black line) or CDH1-specific siRNA (dotted red line) followed by no treatment, or incubation with BSA (1000 µg/ml) or FadAc (1000 µg/ml) for 1 hr. C, untreated control. Data are mean values ± SD. The experiment was performed in triplicates and repeated twice. ***p<0.001 (two-way ANOVA).

(D) Confocal microscopy analysis of SB and 10C cells either untreated or following incubation with CFSE-labeled F. nucleatum 12230 (Fn) for 1 hr at MOI of 5:1. Annexin A1 was stained green and E-cadherin blue. Imagines are 800x magnification. Note the enhanced expression of Annexin A1 in 10C compared to SB and its location on the outer layer of the cell mass. The experiment was repeated three times. Scale bar, 250 nm.