Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: misPLA analyses of phosphorylation states and protein-protein interactions in SK-BR cells with or without EGF stimulation. A) Phosphorylation targets: SK-BR cells were analyzed for phosphorylation of STAT5a (pSTAT5a), STAT3 (pSTAT3), AKT (pAKT), ERK (pERK), and EGFR (pEGFR), under unstimulated and EGF-stimulated conditions. All targets were visualized three at a time in sequential detection cycles and are shown simultaneously (upper panels, “All targets”) and subsequently as individual channels. PLA signals (red, cyan, green, purple) reflect activated protein states detected via dual-recognition proximity ligation. DAPI (blue) labels nuclei. Scale bars = 50 µm. B) Protein-protein interactions: Visualization of the following protein-protein interactions investigated by misPLA in unstimulated and EGF-stimulated SK-BR cells: MEK1–ERK2, GRB2–MEK1, EGFR–GRB2, STAT3–STAT5a, JAK1–JAK3, JAK1–PI3K, JAK2–STAT5a, JAK1–STAT3, and JAK2–JAK3. Upper panels (“All targets”) represent simultaneous visualization of all targets, imaged three at a time in sequential detection cycles, followed by separated signals per interaction. Scale bars = 50 µm.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Phospho-proteomics, Protein-Protein interactions, Ligation

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: misPLA mapping of signaling interactions in a lymph-node Hodgkin lymphoma, mixed cellularity (right neck); Hodgkin lymphoma, lymphocyte-depleted (neck); Hodgkin lymphoma, lymphocyte-predominant (left neck); Hodgkin lymphoma, mixed cellularity (left neck); and thymoma type B3 (mediastinum). Top row (visualization cycle 1) displays MEK1–ERK2 (FITC), EGFR–GRB2 (Cy5) and GRB2–MEK1 (Cy3N) together with DAPI. Middle row (cycle 2) shows STAT3–STAT5a (FITC), JAK1–JAK3 (Cy5) and JAK1–PI3Kp85 (Cy3N). Bottom row (cycle 3) presents JAK2– STAT5a (FITC), JAK2–JAK3 (Cy5) and JAK1–STAT3 (Cy3N). All nine pairs of antibody-oligonucleotide conjugates were applied and then amplified in a single incubation. The RCA products were revealed using detection oligonucleotides conjugated with three fluorophores in three visualization cycles. A standard three-channel fluorescence microscope was used with identical settings for all three fluorophores. Scale bars, 50 µm.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Amplification, Incubation, Fluorescence, Microscopy

Journal: Discover oncology

Article Title: Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway.

doi: 10.1007/s12672-024-01700-y

Figure Lengend Snippet: Fig. 3 SNORD52 mediates M2 macrophage polariza- tion and activates JAK2/ STAT6 pathway. A Following transfection of OE-SNORD52/ OE-NC in THP-1 and U937 for 48 h, the mRNA expression of SNORD52 was detected by qRT-PCR. ****P < 0.0001 vs. OE-NC. B Following trans- fection of OE-SNORD52/ OE-NC in THP-1 and U937 for 48 h, the protein levels of Arginase-1 and CD163 were determined through Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. OE-NC. C The ratio of CD206-positive or CD163-positive cells following transfection of OE-SNORD52/ OE-NC was analyzed by flow cytometry. ***P < 0.001 vs. OE-NC. D Following transfec- tion of OE-SNORD52/OE-NC in THP-1 and U937 for 48 h, the levels of JAK2/STAT6 pathway-related proteins were determined through Western blotting. ***P < 0.001 vs. OE-NC. E The IL-4 and IL-13 treated THP-1/U937 mac- rophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the mRNA expression of SNORD52 was detected by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001 vs. ASO-NC. F The IL-4 and IL-13 treated THP-1/U937 mac- rophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the protein levels of Arginase-1 and CD163 were determined through Western blotting. ***P < 0.001 vs. ASO-NC. G The ratio of CD206-positive or CD163-positive cells following transfection of SNORD52- ASO-1/-2 or ASO-NC was analyzed by flow cytometry. ***P < 0.001 vs. ASO-NC. (H) The IL-4 and IL-13 treated THP-1/ U937 macrophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the levels of JAK2/STAT6 pathway-related proteins were determined through Western blotting. ***P < 0.001 vs. ASO-NC. ns: no significance

Article Snippet: Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Incubation

Journal: Discover oncology

Article Title: Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway.

doi: 10.1007/s12672-024-01700-y

Figure Lengend Snippet: Fig. 5 Huh7 cells-derived exosomal SNORD52 mediates M2 macrophage polarization through activating JAK2/STAT6 pathway. A Following the establishment of co-culture model, AG-490 (a specific JAK2 inhibitor) was added and the levels of JAK2/STAT6 pathway-related pro- teins in receptor THP-1 macrophages were measured by Western blotting. B Following the establishment of co-culture model, AG-490 (a specific JAK2 inhibitor) was added and the ratio of CD206-positive or CD163-positive THP-1 macrophages was analyzed by flow cytometry. **P < 0.01, ***P < 0.001. ns: no significance

Article Snippet: Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

Techniques: Derivative Assay, Co-Culture Assay, Western Blot, Flow Cytometry

Journal: Molecules (Basel, Switzerland)

Article Title: Extract from Black Soybean Cultivar A63 Extract Ameliorates Atopic Dermatitis-like Skin Inflammation in an Oxazolone-Induced Murine Model.

doi: 10.3390/molecules27092751

Figure Lengend Snippet: Figure 6. A63 extract prevents eosinophil infiltration toward the inflammatory site via CCL26 expression downregulation and the inactivation of JAK1/STAT6 signaling. qPCR analysis of CCL26 expression in IL-4/IL-13-induced HS68 cells (A). Western blot results of IL-4/IL-13-induced HS68 cells (B). Results are expressed as mean ± standard deviation (### p < 0.001 versus control group, *** p < 0.001 versus IL-4/13 treated group).

Article Snippet: The membranes were blocked with a 5% BSA solution and treated with primary antibodies for anti-phospho-p44/42 MAPK (ERK1/2), phospho-p38 MAPK, phospho-SAPK/JNK, phospho-STAT3, phospho-STAT6, phospho-JAK1, p44/42 MAPK (ERK1/2), p38 MAPK, JNK, STAT3, STAT6, JAK1, and GAPDH (Cell Signaling Technology, MA, USA, 1:1000).

Techniques: Expressing, Western Blot, Standard Deviation, Control

Journal: Viruses

Article Title: ZIKV Strains Elicit Different Inflammatory and Anti-Viral Responses in Microglia Cells

doi: 10.3390/v15061250

Figure Lengend Snippet: Infection of BV2 microglia cells with ZIKV PE243 and ZIKV MR766 strains differentially modulate the pro-inflammatory miRNA-155 and anti-inflammatory and anti-viral factors. BV2 cells were infected with both ZIKV PE243 or ZIKV MR766 , with an MOI of 1, at 12, 24, 48, and 72 hpi using mock-treated (Mock) as a negative control and LPS + ATP (LPS) as a positive control. ( a ) miRNA-155, ( b ) SOCS1, ( d ) SOCS3 and ( e ) SHIP1 mRNA expression levels were measured by RT-qPCR. ∆∆Ct plotted results were normalized to hprt1 and mock values for mRNAs, and U6 and mock for miRNAs. ( c ) The protein SOCS1 expressions were analyzed by Western analysis. The membrane was stained with anti-SOCS1 and anti-β-actin as a loading control. Statistical analysis was performed by One-Way ANOVA, considering * p < 0.05, ** p < 0.01, and *** p < 0.001 as significant differences.

Article Snippet: Primary antibodies against PPAR-γ (Thermo Fisher Scientific Inc., PA3-821A) and SOCS1 (Boster Biological Technology Co. Ltd, Pleasanton, CA, USA, PA1074) were added to the membranes, and then secondary antibodies against mouse (Cell Signaling Technology, 7076S, Danvers, MA, USA) and rabbit (KPL, 04-15-06) were used.

Techniques: Infection, Negative Control, Positive Control, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Staining, Control

Journal: Viruses

Article Title: ZIKV Strains Elicit Different Inflammatory and Anti-Viral Responses in Microglia Cells

doi: 10.3390/v15061250

Figure Lengend Snippet: Comparison of inflammatory, anti-inflammatory, and anti-viral marker expressions by BV2 microglial cell line after infection with Brazilian and African ZIKV strains (ZIKV PE243 and ZIKV MR766 ).

Article Snippet: Primary antibodies against PPAR-γ (Thermo Fisher Scientific Inc., PA3-821A) and SOCS1 (Boster Biological Technology Co. Ltd, Pleasanton, CA, USA, PA1074) were added to the membranes, and then secondary antibodies against mouse (Cell Signaling Technology, 7076S, Danvers, MA, USA) and rabbit (KPL, 04-15-06) were used.

Techniques: Comparison, Marker, Infection