Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

doi: 10.1186/s13046-024-03106-8

Figure Lengend Snippet: Fig. 1 The JAK/HDACi and regorafenib combination reduces cell proliferation and colony formation of CRC cells. A Western blotting demonstrating the key targets of JAK/HDACi and regorafenib in tissue lysates of CRCs (T) along with their adjacent, uninvolved normal tissues (N). B Figure represents relative protein expression (normalized to their respective β-actin) of drug targets illustrated in Fig A. C MTT assay analyzing the effect on cell proliferation of JAK/HDACi (0.25 – 5.0 µM) and regorafenib (0.25 – 5.0 µM) in single and their combination drug treatments in normal primary colonic epithelium cells. Results are presented as percents of vehicle control (DMSO). D MTT assay evaluating the efficacy of JAK/HDACi (500nM) and regorafenib (500nM) alone and in combination drug treatments on proliferation of CRC cell lines. Results are presented as percents of vehicle control (DMSO) in each set (i) SW480, (ii) HT29, (iii) HCT116, and (iv) RKO. E Colony assay for four CRC cell lines (SW480, HT29, HCT116, and RKO) showing the efficacy on colony formation of JAK/HDACi (500nM) and regorafenib (500nM) alone and in combination drug treatments. F Quantification of colonies was performed by a 10% acetic acid extraction method. Results are presented as averages of triplicates from each treatment group and as percents of vehicle control (DMSO) (i) SW480, (ii) HT29, (iii) HCT116, and (iv) RKO. P-values were labeled as follows: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001

Article Snippet: We tested JAK/HDACi (Cat. # HY-126141) and regorafenib (Cat. # HY-10331) obtained from MedChem Express (Monmouth Junction, NJ).

Techniques: Western Blot, Expressing, MTT Assay, Control, Colony Assay, Extraction, Labeling

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

doi: 10.1186/s13046-024-03106-8

Figure Lengend Snippet: Fig. 2 The JAK/HDACi and regorafenib combination induces cell arrest at the G0-G1 phase, delays the S phase, and induces apoptosis. A Cell cycle arrest was analyzed by flow cytometry of SW480 cells exposed to DMSO (control), JAK/HDACi, regorafenib, or the combination. B The average of three experiments was quantified for each treatment group and plotted as percent cell arrest. C-F Apoptosis was analyzed by flow cytometry quantifying FITC-conjugated annexin and propidium iodide (PI) positive cells. Average of three experiments (sum of % early and late apoptosis) were quantified for each treatment group and plotted as percent apoptotic cells. C SW480 cells, D HT29 cells, E HCT116 cells, F RKO cells. P-values were labeled as follows: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001

Article Snippet: We tested JAK/HDACi (Cat. # HY-126141) and regorafenib (Cat. # HY-10331) obtained from MedChem Express (Monmouth Junction, NJ).

Techniques: Flow Cytometry, Control, Labeling

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

doi: 10.1186/s13046-024-03106-8

Figure Lengend Snippet: Fig. 3 Combination treatment of JAK/HDACi and regorafenib decreases kinase activity in CRC cells. A-B Network models of altered kinase activity is displayed. A (i) JAK/HDACi, (ii) Regorafenib, and (iii) Combination altered kinases in SW480 cells. B (i) JAK/HDACi, (ii) Regorafenib, and (iii) Combination, altered kinases in RKO cells. Networks were generated using MetaCore (portal.genego.com) with an auto expand network set with 75 maximum nodes, with metabolites, and orphan nodes were excluded. Input nodes (kinases) have large blue circles around them. The smaller blue circles represent that kinase activity decreased, while smaller red circles represent that kinase activity increased/unaltered. Arrowheads denote the direction of interactions, and color of the lines indicates type of interaction (green; positive, red; inhibitory, grey; complex). The key for symbols is provided in Table S5. C Table summarizes Mean Kinase Statistics (MKS) of kinases modulated by treatment of JAK/HDACi and regorafenib and their combination in SW480 and RKO cells. Mean Kinase Statistic for each treatment groups values are represented as change from DMSO control. D Western blotting demonstrating inactivation of kinases. In lysates of four CRC cell lines, JAK/HDACi targets pJAK1, pJAK2, pJAK3, and pSTAT3Y705, and regorafenib targets pEGFR and pERK 1/2. β-Actin was used as a loading control. E Western blots demonstrating that JAK/HDACi and the combination treatment inhibit deacetylation of histone H3K9, H4K8 and alpha tubulin, as illustrated by increased proteins of acetylated-histone H3K9, H4K8, and α-tubulin. β-Actin was used as a loading control. P-values were labeled as follows: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001

Article Snippet: We tested JAK/HDACi (Cat. # HY-126141) and regorafenib (Cat. # HY-10331) obtained from MedChem Express (Monmouth Junction, NJ).

Techniques: Activity Assay, Generated, Control, Western Blot, Labeling

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

doi: 10.1186/s13046-024-03106-8

Figure Lengend Snippet: Fig. 4 Whole transcriptomic analysis shows that combination treatment of JAK/HDACi and regorafenib modulates important pathways. Results are presented from of duplicates (n = 2) from each treatment group. A, B Venn diagram illustrating number of genes up-regulated/down-regulated by JAK/HDACi (500 nM), regorafenib (500 nM), and in their combination treatment. C Volcano plots illustrating statistically significant genes in three comparisons (i) JAK/HDACi (JH) vs. DMSO, (ii) regorafenib (Reg) vs. DMSO, and (iii) combination (combo) vs. DMSO, for SW480 cells. D significantly enriched KEGG pathways in genes uniquely altered by combination treatment. E Heatmap of genes uniquely modulated in combination treatment. Genes that are differentially expressed with log2 fold change ≥ 1 or ≤ -1 and p-value < 0.05 in only combination treatment are referred as uniquely modulated genes. F, G Quantitative PCR showing expression of gene targets (normalized to GAPDH). F The upregulated gene targets are shown (i) PCDHA10, (ii) ITIH5, (iii) PCDHB10, (iv) MYO18B, and (iv) CYP4F12, and G the down-regulated gene targets are shown (i) HSPH1 (ii) HSPD1 (iii) LYAR and (iv) EPHA2. H, I Western blots showing HSPH1 and cyclin D1 protein expression in cell lysates of H SW480, and I RKO cells. β-Actin was used as a loading control. The values above western blots represent protein expression (normalized to their respective β-actin) compared to their DMSO controls. P-values were labeled as follows: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001

Article Snippet: We tested JAK/HDACi (Cat. # HY-126141) and regorafenib (Cat. # HY-10331) obtained from MedChem Express (Monmouth Junction, NJ).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Labeling

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

doi: 10.1186/s13046-024-03106-8

Figure Lengend Snippet: Fig. 5 Combination treatment leads to reductions in tumor size, volume, and weight in CRC-PDXs. A Image illustrating harvested CRC-PDXs were smaller in the combination treated groups compared to the vehicle control, JAK/HDACi and regorafenib treated groups. B Tumor progression kinetics illustrating the efficacy of treatment. The statistical significance is calculated at 28th day of treatment. P-values were labeled as follows: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. D H&E staining, illustrating necrotic regions as shown in dotted line. E IHC analysis showing decreased numbers of Ki67-positive cells in tissues treated with the combination, indicating fewer proliferating cells (upper panel). Increased TUNEL-positive cells present in the combination-treated group, indicating induced apoptosis (bottom panel)

Article Snippet: We tested JAK/HDACi (Cat. # HY-126141) and regorafenib (Cat. # HY-10331) obtained from MedChem Express (Monmouth Junction, NJ).

Techniques: Control, Labeling, Staining, TUNEL Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

doi: 10.1186/s13046-024-03106-8

Figure Lengend Snippet: Fig. 6 Combination treatment decreases metastasis of luciferase-tagged HT-29 cells injected into NSG mice. A Bioluminescence imaging of vehicle- and drug-treated groups at 0, 10, and 30-day intervals, highlighting efficacy of the treatment for the combination group, as evidenced by reduced bioluminescence intensity of luciferase-tagged HT29 cells. B Quantification of bioluminescence intensity on day 30th of vehicle and drug-treated groups, showing reduced intensities in the regorafenib- and combination-treated groups. P-values were labeled as follows: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. C H&E staining of FFPE sections from lung at 10X magnification (top panel) and kidney at 2X magnification (bottom panel) showing reduced tumor burdens in these organs in combination treatment. D Toxicity analysis in serum samples of mice with the vehicle- and drug-treated HT29 metastatic model. Values were within the reference range, illustrating no evident toxicity was observed by either by JAK/HDACi and regorafenib alone or their combination treatment

Article Snippet: We tested JAK/HDACi (Cat. # HY-126141) and regorafenib (Cat. # HY-10331) obtained from MedChem Express (Monmouth Junction, NJ).

Techniques: Luciferase, Injection, Imaging, Labeling, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Combination of dual JAK/HDAC inhibitor with regorafenib synergistically reduces tumor growth, metastasis, and regorafenib-induced toxicity in colorectal cancer.

doi: 10.1186/s13046-024-03106-8

Figure Lengend Snippet: Fig. 7 Combination treatment reduces tumor growth in an MC38 syngeneic mouse model, and increased infiltration of CD45 and cytotoxic CD8 T cells. A Image illustrating that harvested syngeneic tumors were smaller in treated groups compared to vehicle control B Tumor progression curve illustrating efficacy of the treatment; in the combination-treated group the tumors regressed, and statistical significance is calculated at 28th day of treatment. P-values were labeled as follows: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. C Tumor weights (in grams) were reduced in the combination-treated group. D Table summarizing the functional aspects of modulated genes in the combination-treated group. E Higher Log2 abundance scores of Cd45 and cytotoxic cells were evident for the combination-treated group. F Gene expression of targets (i) Cx3cr1, (ii) Gzmb, and (iii) Gzme were altered in JAK/HDACi, regorafenib and the combination-treated group. G H&E and IHC staining for CD8, CD45, pSTAT3Y705 and pERK1/2 are shown. H Western blots illustrating expressions of pJAK2, pERK1/2, and granzyme B. I Plasma analysis for cytokines released after drug treatment (i)TNF-Alpha and (ii) KC/GRO. J Pharmacokinetics in plasma samples of C57BL/6 mice after JAK/HDACi and regorafenib alone and in their combination treatment at 1, 7, 21, and 48 h

Article Snippet: We tested JAK/HDACi (Cat. # HY-126141) and regorafenib (Cat. # HY-10331) obtained from MedChem Express (Monmouth Junction, NJ).

Techniques: Control, Labeling, Functional Assay, Gene Expression, Immunohistochemistry, Western Blot, Clinical Proteomics, Drug discovery

Journal: Biochemistry and biophysics reports

Article Title: NanoLuciferase technology-based detection of TMPRSS2 as attempt to develop anti-coronavirus agents.

doi: 10.1016/j.bbrep.2024.101783

Figure Lengend Snippet: Fig. 1. Normalized TMPRSS2-HiBiT signal under treatment of compounds from (A) diversified building block library, (B) bioactive compound library, or (C) human endogenous metabolite library, at concentration of 10 μM and adjusted to corresponding ATP-Glo signal.

Article Snippet: Diversified building block compound library, bioactive compound library and endogenous metabolite compound library were from TargetMol.

Techniques: Blocking Assay, Drug discovery, Concentration Assay

Journal: Archives of Medical Science : AMS

Article Title: Inhibitory effect of PDGF-BB and serum-stimulated responses in vascular smooth muscle cell proliferation by hinokitiol via up-regulation of p21 and p53

doi: 10.5114/aoms.2018.75085

Figure Lengend Snippet: Effect of hinokitiol on PDGF-BB signaling pathway in VSMCs. A – Confluent cells were pre-treated in the presence or absence of hinokitiol (1–100 μM) in a serum-free medium, and then stimulated with 10 ng/ml PDGF-BB for 10 min. The cells were lysed, and the levels of ( A ) p-ERK1/2, ( B ) p-Akt, ( C ) p-PI3K and ( D ) p-JAK2 were analyzed by SDS-PAGE and immunoblotting. The respective total proteins and a-tubulin were used for the normalization of phosphorylated ones. Data are expressed as mean ± SEM ( n = 3). * p < 0.05 compared to the PDGF-BB stimulated cells

Article Snippet: Anti-phospho-ERK1/2 (Thr 202 /Tyr 204 ), anti-phospho-AKT (Ser 473 ), antiphospho-PI3K, and antiphospho-JAK2 monoclonal antibodies (mAbs) were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: SDS Page, Western Blot

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: Oncogene

Article Title: SUMOylation and SENP3 regulate STAT3 activation in head and neck cancer

doi: 10.1038/onc.2016.124

Figure Lengend Snippet: NNK induces STAT3 phosphorylation and increase of SENP3 protein levels in HNC cells in an ROS-dependent manner. ( a ) The levels of STAT3 phosphorylation at tyrosine 705 (in brief, pY705-STAT3) were determined by IB in Hep-2 cells exposed to 1 μ m NNK and 20 ng/ml IL-6 respectively for the indicated time (left), and in KB or HN30 cells exposed to 1 μ m NNK for indicated time (right). ( b ) The pY705-STAT3 levels were determined by IB in Hep-2 cells exposed to 1 μ m NNK in the presence or absence of 1 μg/ml neutralized antibody against IL-6 (left) and 10 μ m JAK inhibitor (right) for the indicated time. Anti-IL-6 antibody and JAK inhibitor were pre-administered for 1 h. ( c ) The pY705-STAT3 levels were determined by IB in Hep-2 cells exposed to lower doses of NNK (0.01 μ m ) or IL-6 (2 ng/ml) alone or in combination for the indicated time. ( d ) ROS level was determined by DCFH-DA staining and flow-cytometric analysis in Hep2 cells exposed to 1 μ m NNK for the indicated time. Error bars represent±s.d. for triplicate experiments. *** P <0.001. ( e ) The SENP3 and pY705-STAT3 levels were determined by IB in Hep-2, KB and HN30 cells exposed to 1 μ m NNK in the presence or absence of 5 m m NAC for the indicated time.

Article Snippet: When needed, the anti-oxidant NAC (Sigma, St Louis, MO, USA) was pre-administered for 4 h, and the JAK inhibitor (Santa Cruz Biotechnology, Dallas, TX, USA) and anti-IL-6 (InvivoGen, San Diego, CA, USA) were pre-administered for 1 h before other treatments.

Techniques: Phospho-proteomics, Staining