Fig 1: Binding of HIV-1 gp41 HR2 Antibodies to MAPK14(A) Representative ELISA graphs show the reactivity of blood anti-gp41 antibodies against overlapping peptides covering the extracellular portion of gp41 (gp41EC). The sequences of the peptides recognized are indicated on the top.(B) Representative ELISA graphs show the binding of biotinylated mucosal anti-gp41 to YU-2 gp140 in the presence of potential competitor antibodies (blood-derived anti-gp41 IgG). Error bars indicate the SEM of duplicate values.(C) Protein microarray plots show the reactivity profile of blood anti-gp140 antibodies against human proteins. For each protein spot, the mean fluorescence intensity (MFI) given by the reference (mGO53) and test antibody are depicted on the y and x axes, respectively. Each dot represents the average of duplicate array proteins. The diagonal lines indicate equal binding for reference and test antibodies.(D) Bar graphs comparing the MFI values (Left) and Z scores (Right) for the binding of the selected anti-gp41 antibodies to MAPK14 immobilized on the protein array.(E) Representative ELISA graph comparing the reactivity of blood anti-gp140 antibodies to purified MAPK14 proteins. The 6-195 and mGO53 (green) were used as positive and negative control, respectively. Error bars indicate the SEM of duplicate values.(F) Affinity of anti-gp41 antibodies to MAPK14. (Left) Representative SPR curves comparing the binding overtime of selected anti-gp41 IgGs (at 250 nM) to purified MAPK14 immobilized on the sensor chip (250 RUs). (Right) Dot plots comparing the relative affinity (KD) of anti-gp41 antibodies to MAPK14. No binding could be detected (ND) for the following antibodies: 5-25, 10-137, 10-437, and 10-1304.See also Figure S6.
Fig 2: Cross-Reactivity of Mucosal HIV-1 Antibodies against Human Self-Antigens(A) Bar graph shows the polyreactivity levels of mucosal HIV-1 gp140 antibodies as measured in duplicate by ELISA against KLH, dsDNA, insulin, and LPS in Figure S5A. The x axis indicates cumulative AUC (CAUC) values for polyreactivity (polyR).(B) Antibody binding to HEp-2 cells was assayed by immunofluorescence assay. Ctr+, positive control of the kit. mGO53 and ED38 are negative and positive control antibodies, respectively. The scale bars represent 15 µM.(C) Frequency histograms show the log10 protein displacement (s) of the mean fluorescent intensity (MFI) signals for the selected antibodies (n = 9) compared with non-reactive antibody mGO53. The polyreactivity index (PI) corresponds to the Gaussian mean of all array protein displacements.(D) Representative protein microarray plots show the reactivity profile of mucosal anti-gp140 antibodies against human proteins. For each protein spot, the MFI given by the reference (mGO53) and test are depicted on the y and x axes, respectively. Each dot represents the average of duplicate array proteins. The diagonal lines indicate equal binding for reference and test antibodies. Blue dots indicate immunoreactive proteins with a Z score > 5 that were identified in two independent experiments as shown in Figure S5B.(E) Representative ELISA graph comparing the reactivity of mucosal anti-gp140 antibodies to purified MAPK14. mGO53 is the negative control (green). Error bars indicate the SEM of duplicate values.(F) SPR sensorgram showing the binding overtime of 6-195 to purified MAPK14 proteins immobilized on the sensor chip (50 RUs).See also Figure S5.
Fig 3: Interactions of Anti-HR2 Cluster II Antibodies with MAPK14(A) Representative ELISA graph shows the binding of biotinylated anti-cluster II D50 antibody to MN gp41 in the presence of potential competitor antibodies (anti-gp41HR2 IgGs). mGO53 is the negative control (green).(B) Representative ELISA graph shows the binding of murine (D50) and human (167-D IV, 98-6, and 5F3) anti-cluster II antibodies to purified MAPK14. mGO53 is the negative control (green).(C) Surface representations show the electrostatic potentials of the gp41 cluster II (residues 642–664) (deposited in the Protein Data Base under PDB: 1ENV) calculated with PyMOL, colored blue (positive electrostatic potential) to red (negative electrostatic potential).(D) Representative ELISA graph shows the area under the curves of OD405 nm values (AUC) for the binding of the selected IgG antibodies to purified MAPK14 in presence of increasing salt concentration.(E) (Top) Amino acid sequence alignment of the gp41 region spanning residues 642–665. Amino acid variations are shown in red. (Bottom) Heat map comparing the mean AUC values for the ELISA binding of anti-gp41 and control antibodies to the selected HIV-1 Env strains (n = 8) as measured in Figure S7. Color intensity is proportional to the reactivity level, with darker-blue colors indicating high binding, whereas light colors show moderate binding (white, no binding).(F) Diagram shows the sequence alignment (Top) based on the structural superposition (Bottom) between a gp41HR2 cluster II peptide (red, from PDB: 1ENV) and MAPK14 (blue, PDB: 5ETI).(G) ELISA graph comparing the binding of mutated anti-gp41 antibodies (black straight lines) and their germline counterparts (red dotted lines) to trimeric YU-2 gp140, gp41, and MAPK14.Error bars in (A), (B), (D), and (G) indicate the SEM of duplicate values. See also Figure S7.
Fig 4: Combined pharmacological blockade of EZH2 and p38 enzymatic activities reduces neoplastic functions(A) Immunoblots of MDA-MB-231 cells treated with GSK-343 (3 µM for 48 h), SB202190 (p38i, 20 µM for 48 h), or the combination.(B) Cells in A were subjected to growth assays.(C) Synergistic effect of EZH2 inhibitor and p38 inhibitor in MDA-MB-231 cells incubated with various doses of GSK-343 and SB202190 for 4 d. A matrix for synergy score was calculated (Ianevski et al., 2017).(D and E) Wound healing assay to quantify cell migration (D) and reconstituted Boyden basement membrane-invasive chamber assay of MDA-MB-231 cells treated as in (A). Representative chambers after crystal violet staining are shown above bars. Data for B-E are from at least three independent experiments carried out in at least triplicate. Data for B, (D and E) are presented as mean ± SEM. *p = 0.05; **p = 0.01; ***p = 0.005; ****p = 0.0001.
Fig 5: pEZH2-T367 and p-p38 are significantly upregulated in human breast cancer metastasis compared to matched primary tumors from the same patient(A) Representative images of matched primary human breast carcinomas and metastasis (n = 16 patients) immunostained for pEZH2-T367 and p-p38 (600x). pEZH2-T367 is upregulated in the cytoplasm of metastatic cells compared to the primary tumor. Bars 50 µm.(B) Distribution of pEZH2-T367 protein expression in the 16 primary breast carcinomas and matched metastasis; the number of primary and metastatic breast carcinomas with low and high pEZH2-T367.(C) Association between pEZH2-T367 and p-p38 expression in primary and metastatic carcinomas of the breast; 56.75% of metastasis exhibit high expression of both pEZH2-T367 and p-p38.
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