Journal: Bioorganic chemistry

Article Title: Bromo-substituted indirubins for inhibition of protein kinase-mediated signalling involved in inflammatory mediator release in human monocytes.

doi: 10.1016/j.bioorg.2024.107470

Figure Lengend Snippet: Fig. 3. Impact of indirubins 1 and 2 on the activation status of protein kinase pathways. A-F) Human monocytes were pre-treated with indirubins 1 or 2 at the indicated concentrations, or with inhibitors of GSK-3β (SB216763, 5 µM), CDK8 (CCT-251921, 40 nM), CDK9 (JSH-150, 20 nM) or CDK1 (Ro-3306, 10 µM) for 15 min prior to stimulation with LPS 100 ng/mL for 18 h. (A) Protein expression of β-catenin compared to GAPDH as housekeeping protein. (B) Levels of phosphorylated GSK-3β compared to total expression of GSK-3β protein. (C,D) Levels of phosphorylated STAT1 (C, Ser727 and D, Tyr701) vs. β-actin. (E) Levels of phosphorylated NF-κB (Ser276) compared to GAPDH. (F) Levels of phosphorylated Rb (Ser807/Ser811) vs. β-actin. Protein amounts were evaluated by Western Blot and densito metric analysis thereof. n = 3–4 biological replicates. Statistics: Data are shown as A-E) individual values and means ± SEM. Statistics were calculated by one-way ANOVA for multiple comparisons with Dunnett’s correction, testing treatments against vehicle (0.1 % (v/v) DMSO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit monoclonal anti-COX-2, 1:1000 (12282, Cell Signaling, Danvers, MA), mouse monoclonal anti-β-catenin, 1:1000 (610153, BD Biosciences, San Jose, CA) mouse monoclonal anti-GSK-3β, 1:1000 (9832, Cell Signaling), rabbit monoclonal anti-phospho-GSK-3β (Ser9), 1:1000 (9323S, Cell Signaling), rabbit monoclonal anti-phosphoSTAT1 (Ser727), 1:1000 (ab109461, Abcam, Cambridge, UK), rabbit monoclonal anti-phospho-STAT1 (Tyr701), 1:1000 (ab109457, Abcam), rabbit monoclonal anti-phospho-NF-κB p65 (Ser276), 1:1000 (ab183559, Abcam), rabbit monoclonal anti-phospho-Rb (Ser807/811), 1:1000 (9308S, Cell Signaling), monoclonal anti-β-actin, 1:1000 (4970, Cell Signaling), mouse monoclonal anti-GAPDH, 1:1000 (97166S, Cell Signaling).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

doi: 10.1186/1742-2094-9-113

Figure Lengend Snippet: Effects of SAHA on IFN-γ-induced phosphorylation of STAT1 and STAT3 in human astrocytes. Human astrocytes were incubated with or without SAHA at the concentrations indicated for 1 h. The cells were subsequently stimulated with IFN-γ for 30 minutes. Astrocytes in the control group were incubated with medium only. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr 701 -STAT1 (pSTAT1) and total STAT1 (A) or phospho-Tyr 705 -STAT3 (pSTAT3) and total STAT3 (C) . The density ratios of phosphorylated to total protein are shown as mean ± S.E.M. of three independent experiments (B, D) . *Significantly different from IFN-γ stimulation only.

Article Snippet: The protein was transferred to a PVDF membrane at 70 V for 2 h. The membrane was blocked with 5% skim milk plus 3% bovine serum albumin (BSA) in PBS at room temperature (RT) for 1 h. Subsequently, the membrane was incubated with specific rabbit antibodies against phospho-Tyr 701 -STAT1 (1:2,000), total STAT1 (1:1,000), phospho-Tyr 705 -STAT3 (1:2,000) or total STAT3 (1:1,000) at 4°C overnight and then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000) at RT for 1 h. All antibodies used for immunoblotting were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Incubation, Control, SDS Page

Journal: Advanced Science

Article Title: Calhm6 Governs Macrophage Polarization Through Chp1‐Camk4‐Creb1 Axis and Ectosomal Delivery in Inflammatory Responses

doi: 10.1002/advs.202502395

Figure Lengend Snippet: Loss of Calhm6 enhances M1‐like macrophages through Creb1 inactivation. A ) RT‐qPCR analysis of CHIL3, IL10, ARG1, TNFA, NOS2, and IL6 in shCtrl or shCalhm6 cell line treated with LPS, IFNγ combined or IL‐4 for 24 h. B,C) Morphology of wild‐type or Calhm6 knockout BMDMs B) polarized to M1‐like or M2‐like phenotypes by treatment with LPS and IFNγ combined or IL‐4 for 24 h, and cell length C) was measured by ImageJ ( n =55). Scale bars(C), 100 µm. D) Transcriptome analysis of M1‐of M2‐like markers expressed by LPS, IFNγ combined or IL‐4 for 24 h in wild‐type or Calhm6 knockout BMDMs. E,F) Seahorse analysis of OCR (E) and ECAR (F) in BMDMs form wild‐type or Calhm6 knockout mice. G,H) Flow cytometric analysis of CD86 + /CD206 + macrophage populations in the spleen (G) and lymph node (H) of wild‐type and Calhm6 knockout mice, as determined with anti‐F4/80, anti‐CD11b, anti‐CD86, and anti‐CD206 antibodies. I,J) Immunoblot analysis of (p‐) Stat1, Stat3, Creb1, Stat5, Stat6, Calhm6, and β‐actin in wild‐type or Calhm6 knockout BMDMs stimulated for 0, 30, 60 min with LPS, IFNγ combined (I) or IL‐4 (J). K) Fluorescence microscopy of wild‐type or Calhm6 knockout BMDMs treated with CaCl 2 (2 m m ) or IL‐4 for 3 h, then washed, fixed, and immunostained with anti‐Creb1 (red) and counterstained with DAPI (blue). Scale bars, 50 µm. L) Immunoblot analysis of (p‐) Creb1 and β‐actin in EV or OE‐Calhm6 cells treated with or without KG‐501 or 666‐15. M) RT‐PCR analysis of CHIL3, IL10, ARG1, TNFA, NOS2, and IL6 in EV or OE‐Calhm6 cells treated with or without KG‐501 or 666‐15. N) Calcium influx over time in wild‐type or Calhm6 knockout BMDMs treated with LPS (200 ng mL −1 ) and stained by the Rhod2‐AM. O)Immunoblot analysis of (p‐) Creb1 and β‐actin in EV or OE‐Calhm6 cells treated with or without BAPTA‐AM (20 µ m ) or Ca 2+ ‐free DMEM for 0.5, 1, 3, 6 h. The data represent the mean ± S.D. ( n = 3). NS, not significant ( p > 0.05); * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.00001 compared with control, Student's t‐test.

Article Snippet: Antibody to phosphorylated (p‐) NFκB (AP0475), (p‐) IκBα (AP0731), (p‐) Stat1 (AP0054), (p‐) Stat3 (AP0070), (p‐) Stat6 (AP0456), (p‐) Creb1 (AP1421), (p‐) Stat5 (AP0758), anti‐CaMK4 (A5304), anti‐Chp1 (A15791), anti‐Mmp2 (A19080), anti‐CD63 (A5271), anti‐Arg1 (A1847) and anti‐Nos2 (A0312) were from Abclonal.

Techniques: Quantitative RT-PCR, Knock-Out, Western Blot, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Staining, Control

Journal: Advanced Science

Article Title: Calhm6 Governs Macrophage Polarization Through Chp1‐Camk4‐Creb1 Axis and Ectosomal Delivery in Inflammatory Responses

doi: 10.1002/advs.202502395

Figure Lengend Snippet: Calhm6 promotes Creb1 activation and M2‐like polarization of macrophages via the Chp1‐CaMK4 axis. A) Identification of Calhm6, Chp1, and CaMK4 by mass spectrometry in a Flag‐tagged Calhm6‐precipitation assay in cell lysates of OE‐Calhm6 cells, untreated or treated with IL‐4 (20 ng mL −1 ) or CaCl 2 (2 m m ). B) RT‐PCR of TNFA, NOS2, IL6, CHIL3, IL10, and ARG1 in Sh‐Scr or Sh‐Chp1 cells treated with LPS, IFNγ combined, or IL‐4 (dose as above). C) Immunoblot analysis of phosphorylated (p‐) p38, NFκB, IκBα, and β‐actin in total lysates of Raw264.7 cells treated with Scramble‐shRNA (Sh‐Scr) or Chp1‐specific shRNA (Sh‐Chp1), left treated for 0, 30, 60 min with LPS (200 ng mL −1 ) or LTA (100 ng mL −1 ). D,E) Seahorse analysis of OCR (D) and ECAR (E) in BMDMs from Sh‐Scr or Sh‐Chp1 cells. F) Immunoblot analysis of (p‐) Stat1, IκBα, Creb1, Stat3, Stat6, and β‐actin in Sh‐Scr or Sh‐Chp1 cells stimulated for 0, 30 min with LPS (200 ng mL −1 ) and IFNγ (10 ng mL −1 ) combined or IL‐4 (20 ng mL −1 ). G) Immunoblot analysis of p‐Creb1 and Chp1, CaMK4, β‐actin in Raw264.7 cells expressing Flag‐tagged CaMK4 and Myc‐tagged Chp1. H) HeLa cells were transfected with plasmids expressing Myc‐tagged CaMK4 and HA‐tagged Chp1. Before fixing the cells, they were treated with CaCl 2 , BAPTA‐AM, or Ca 2+ ‐free DMEM for 3 h. Confocal microscopy shows the co‐localization of Chp1 (red) with CaMK4 (green) at the cell periphery. Images shown are representative of approximately 20 cells. Scale bar, 20 µm. I) Immunoassay of 293T cells expressing various combinations (above lanes) of Flag‐tagged CaMK4 and Myc‐tagged Chp1, and after being treated with PBS or the CaCl 2 , BAPTA‐AM, or Ca 2+ ‐free DMEM for 3 h, assessed in anti‐Flag immunoprecipitates or total cell lysates, probed with various tag antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. J) Immunoassays of 293T cells expressing various combinations (above lanes) of Flag‐tagged Chp1 and the fragment of GFP‐tagged CaMK4 as indicated; immunoprecipitation with anti‐Flag and analysis by immunoblot with the tag antibodies; below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. K) Immunoassays (as in J) of 293T cells expressing various combinations (above lanes) of Flag‐tagged CaMK4 and the fragment of GFP‐tagged Chp1 as indicated. L) Phos‐tag analysis of 293T cells expressing Flag‐tagged wild‐type Chp1 or Chp1 (S99A) and HA‐tagged CaMK4 (above lanes). M) Immunoassay of lysates of 293T cells expressing various combinations (above lanes) of Flag‐tagged wild‐type Chp1 or Chp1 (S99A) and HA‐tagged CaMK4, immunoprecipitated with anti‐Flag and analyzed by immunoblot with anti‐HA (α‐HA) or anti‐Flag (α‐Flag); below, immunoblot analysis of total cell lysates (TCL) without immunoprecipitation. N) Immunoblot analysis of (p‐) and total CaMK4 and β‐actin in BMDMs treated with LPS, IFNγ combined, or IL‐4 for 30 min. O) Immunoblot analysis of (p‐) and total CaMK4 and β‐actin in BMDMs treated with IL‐4 for 30 min after pretreatment with BAPTA‐AM (20 µ m ) or Ca 2+ ‐free DMEM for 3 h. NS, not significant ( p > 0.05); * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control, Student's t‐test.

Article Snippet: Antibody to phosphorylated (p‐) NFκB (AP0475), (p‐) IκBα (AP0731), (p‐) Stat1 (AP0054), (p‐) Stat3 (AP0070), (p‐) Stat6 (AP0456), (p‐) Creb1 (AP1421), (p‐) Stat5 (AP0758), anti‐CaMK4 (A5304), anti‐Chp1 (A15791), anti‐Mmp2 (A19080), anti‐CD63 (A5271), anti‐Arg1 (A1847) and anti‐Nos2 (A0312) were from Abclonal.

Techniques: Activation Assay, Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, shRNA, Expressing, Transfection, Confocal Microscopy, Immunoprecipitation, Control

Journal: iScience

Article Title: C9ORF72 suppresses JAK-STAT mediated inflammation

doi: 10.1016/j.isci.2023.106579

Figure Lengend Snippet:

Article Snippet: rabbit anti-pSTAT1 , R and D Systems , Cat# AF2894, RRID: AB_2198137.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Bradford Protein Assay, CRISPR, Plasmid Preparation, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software