Journal: Frontiers in Immunology

Article Title: PD0325901 alleviates thrombin-inhibited osteogenic differentiation through an IL-1β-activated feedback loop between MEK-Erk1/2 and NF-κB signal pathways: insights from bioinformatics and experimental verification

doi: 10.3389/fimmu.2026.1730337

Figure Lengend Snippet: The mutual regulatory effects of MEK-Erk1/2 and Stat3 following treatment with PD03 and C188-9, and the role of Stat3 pathways in osteogenic differentiation. (A, B) The phosphorylation levels of Erk1/2, Stat3 (Y705), and Stat3 (S727) in osteoblasts were examined by western blot following treatment with thrombin and PD03 at different time points, and the relative protein levels of p-Erk1/2 to Erk1/2, p-Stat3(Y705) to Stat3, and p-Stat3(S727) to Stat3 were measured using Image J software. (C, D) The protein levels of p-Erk1/2, p-Stat3 (Y705), and p-Stat3 (S727) in osteoblasts were analyzed by western blot after being treated with thrombin and C188–9 at different time points, and the relative protein levels of p-Erk1/2 to Erk1/2, p-Stat3(Y705) to Stat3, and p-Stat3(S727) to Stat3 were evaluated using Image J software. (E) Osteoblasts were stained for ALP and Fluo-4 AM after being induced with the osteogenic differentiation medium for 7 days. (F) An enzymatic assay quantified ALP activities in cell lysates. (G) Western blot analysis was used to examine the expression levels of Col1α1, Runx2 and OCN. (H) The relative protein levels of Col1α1/β-actin, Runx2/β-actin and OCN/β-actin were measured using ImageJ software. (I) The expression levels of Col1α1, Runx2, Osterix, OPG, and OCN were assessed by qPCR following thrombin and C188–9 treatment for 7 days. Data are presented as mean ± SD (n = 3). P-values were determined by one-way ANOVA (multi-group comparisons) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, P >0.05). Scale bar: 100 μm.

Article Snippet: The following antibodies were used in this study: β-actin (AF5001, Beyotime, China), Col1α1 (ab270993, Abcam, UK), Runx2 (AF2593, Beyotime, China), OCN (AF6297, Beyotime, China), Igf1 (AF7179, Beyotime, China), Wnt5a (29793-1-AP, Proteintech, USA), Tgfb3 (AF8142, Beyotime, China), Spp1 (A5427, Bimake, USA), COX-2 (F0327, Bimake, USA), Matrix Metalloproteinase-9 (MMP-9, AF5234, Beyotime, China), Minichromosome Maintenance Complex Component 2 (MCM2, A5172, Bimake, USA), Proliferating Cell Nuclear Antigen (PCNA, SC-25280, Santa, USA), PAR-1 (AF6837, Beyotime, China), IL-1RA (AF7218, Beyotime, China), p65 (8242S, CST, USA), p-p65 (3033T, CST, USA), Erk1/2 (4695T, CST, USA), p-Erk1/2 (4370S, CST, USA), p-Stat3 (Y705) (9145S, CST, USA), p-Stat3 (S727) (9134S, CST, USA), and Stat3 (4904T, CST, USA).

Techniques: Phospho-proteomics, Western Blot, Software, Staining, Enzymatic Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Danggui Shaoyao San ameliorates neuroinflammation in a D-galactose-induced Alzheimer’s disease rat model by suppressing the JAK2/STAT3 pathway and modulating Th17/Treg -related immune dysregulation

doi: 10.3389/fcell.2026.1763180

Figure Lengend Snippet: DSS alleviates neuroinflammation in AD rats via inhibition of the JAK2/STAT3 signaling pathway. (A) Representative Western blot bands of JAK2, p-JAK2, STAT3, and p-STAT3 proteins in hippocampal tissue. (B) Quantitative analysis of p-JAK2 protein expression levels. (C) Quantitative analysis of p-STAT3 protein expression levels. (D) Statistical analysis of p-JAK2-positive cells by immunofluorescence (IF). (E) Statistical analysis of p-STAT3-positive cells by immunohistochemistry (IHC). (F) Representative immunofluorescence images showing p-JAK2 (red) and DAPI (blue) in the hippocampal CA2 region. (G) Representative immunohistochemistry (IHC) images of p-STAT3 expression in the hippocampus and the CA2 region (×100 and ×400 magnification). Data are presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. model group.

Article Snippet: Other reagents included sodium pentobarbital (Merck KGaA, Germany, #P3761), donepezil hydrochloride (MCE, United States, #HY-B0034), D-galactose (Sigma-Aldrich, Shanghai, China, #V900922), primary antibodies targeting STAT3 and Foxp3 (Proteintech, Wuhan, China, #102532-AP, #22228-1-AP), ROR-γt and p-STAT3-Y705 antibodies (HUABIO, Hangzhou, China, #HA722121, #ET1603-40), JAK2 antibody (Aifang, Hunan, China, #AFRM99330), p-JAK2-Y1007/1008 antibody (ABclonal, Wuhan, China, #AP0531), β-actin antibody (Affinity Biosciences, China, #AF7018), postsynaptic density protein 95 (PSD95) and synaptophysin I antibody (abcam, Cambridge, United Kingdom, #ab18258,#ab254349), secondary antibodies (Elabscience, Wuhan, China, #E-AB-1003), Iba1 antibody (Wako Pure Chemical Industries, Ltd., Japan, #011-27991), primers for β-actin, IL-1β, IL-6, etc., were designed using Primer 6.0 software based on sequences from the NCBI database and synthesized by Sangon Biotech (Shanghai), China, reverse transcription kit (Novoprotein, Suzhou, China, #E047-01B), SYBR Mixture premix and RIPA protein lysis buffer (Comwin Biotech, Jiangsu, China, #CW3008M, #CW2333S), ELISA kits (Jianglai Biotechnology, Shanghai, China, #JL13427, #JL20880), BCA protein concentration assay kit (Elabscience Biotechnology, Wuhan, China, #E-BC-K318-M), TRIzol, SlowFadeTM Gold Antifade Mountant with DAPI, and donkey anti-goat Alexa Fluor 647 fluorescent secondary antibody (red) (Thermo Fisher Scientific, United States, #15596-026, # S36942 , #A32849), universal two-step detection kit (Zhongshan Golden Bridge, Beijing, China, #PV-9000), Toluidine blue, Triton X-100, and antifade mounting medium with DAPI (Solarbio Science & Technology Co., Ltd., Beijing, China, #G3663, #T8200, #S2110), hematoxylin staining solution and eosin staining solution (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China, #ZLI-9610, #ZLI-9613), Fixation/permeabilization kits, dyes, and antibodies against CD4, Foxp3, and IL-17A for phenotypic analysis of Th17 and Treg cells were purchased from BD Pharmingen (United States), detailed information is provided in .

Techniques: Inhibition, Western Blot, Expressing, Immunofluorescence, Immunohistochemistry, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Danggui Shaoyao San ameliorates neuroinflammation in a D-galactose-induced Alzheimer’s disease rat model by suppressing the JAK2/STAT3 pathway and modulating Th17/Treg -related immune dysregulation

doi: 10.3389/fcell.2026.1763180

Figure Lengend Snippet: DSS rescue Th17/Treg -related immune dysregulation both in the brain and periphery in a D-galactose-induced Alzheimer’s disease rat model. DSS exerts its anti-neuroinflammatory effects by inhibiting JAK2 and STAT3 phosphorylation, reducing their nuclear translocation, and consequently suppressing Th17 differentiation and pro-inflammatory cytokine production.

Article Snippet: Other reagents included sodium pentobarbital (Merck KGaA, Germany, #P3761), donepezil hydrochloride (MCE, United States, #HY-B0034), D-galactose (Sigma-Aldrich, Shanghai, China, #V900922), primary antibodies targeting STAT3 and Foxp3 (Proteintech, Wuhan, China, #102532-AP, #22228-1-AP), ROR-γt and p-STAT3-Y705 antibodies (HUABIO, Hangzhou, China, #HA722121, #ET1603-40), JAK2 antibody (Aifang, Hunan, China, #AFRM99330), p-JAK2-Y1007/1008 antibody (ABclonal, Wuhan, China, #AP0531), β-actin antibody (Affinity Biosciences, China, #AF7018), postsynaptic density protein 95 (PSD95) and synaptophysin I antibody (abcam, Cambridge, United Kingdom, #ab18258,#ab254349), secondary antibodies (Elabscience, Wuhan, China, #E-AB-1003), Iba1 antibody (Wako Pure Chemical Industries, Ltd., Japan, #011-27991), primers for β-actin, IL-1β, IL-6, etc., were designed using Primer 6.0 software based on sequences from the NCBI database and synthesized by Sangon Biotech (Shanghai), China, reverse transcription kit (Novoprotein, Suzhou, China, #E047-01B), SYBR Mixture premix and RIPA protein lysis buffer (Comwin Biotech, Jiangsu, China, #CW3008M, #CW2333S), ELISA kits (Jianglai Biotechnology, Shanghai, China, #JL13427, #JL20880), BCA protein concentration assay kit (Elabscience Biotechnology, Wuhan, China, #E-BC-K318-M), TRIzol, SlowFadeTM Gold Antifade Mountant with DAPI, and donkey anti-goat Alexa Fluor 647 fluorescent secondary antibody (red) (Thermo Fisher Scientific, United States, #15596-026, # S36942 , #A32849), universal two-step detection kit (Zhongshan Golden Bridge, Beijing, China, #PV-9000), Toluidine blue, Triton X-100, and antifade mounting medium with DAPI (Solarbio Science & Technology Co., Ltd., Beijing, China, #G3663, #T8200, #S2110), hematoxylin staining solution and eosin staining solution (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China, #ZLI-9610, #ZLI-9613), Fixation/permeabilization kits, dyes, and antibodies against CD4, Foxp3, and IL-17A for phenotypic analysis of Th17 and Treg cells were purchased from BD Pharmingen (United States), detailed information is provided in .

Techniques: Phospho-proteomics, Translocation Assay

Journal: iScience

Article Title: NF-κB and STAT3 signaling uniquely stratify survival in female glioblastoma patients

doi: 10.1016/j.isci.2026.114761

Figure Lengend Snippet: High expression of NF-κB and IL-6/JAK/STAT3 genes is a biomarker of shorter survival in female patients Survival analysis performed from TCGA data downloaded from the GlioVis portal ( N = 368; 225 males and 143 females). (A) Forest plots of hazard ratios from univariate Cox regression survival analysis in all patients, male patients, or female patients for individual NF-κB mesenchymal genes (Bhat et al., Cancer Cell , 2013). (B) Kaplan-Meier survival curves stratified by high and low expression of IL-6 in all patients, male patients, or female patients. (C) Hazard ratios from univariate Cox regression survival analysis of NF-κB mesenchymal genes in male, female, or all patients. (D) Hazard ratios from univariate Cox regression survival analysis of IL-6/JAK/STAT3 genes (Human Molecular Signatures Database). In (A), significant genes ( p < 0.05) are indicated in red; in (B), significance was determined by the log rank test; hazard ratios in (C) and (D) are presented as median ± interquartile range. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗; p < 0.0001 as determined by Kruskal-Wallis test followed by a Dunn’s post-hoc pairwise comparison with multiplicity corrections. See also and and .

Article Snippet: Primary antibodies purchased from Cell Signaling Technologies include p-STAT3 Y705 (1:1000, 9145, RRID: AB_2491009 ), STAT3 (1:2500, 9139, RRID: AB_331757 ), p-p65 S536 NF-κB (1:1000, 3033, RRID: AB_331284 ), p65 NF-κB (1:2500, 6956, RRID: AB_10828935 ), β-actin (1:5000, 8457, RRID: AB_10950489 ), p-IκBα S32 (1:1000, 2859, RRID: AB_561111 ), IκBα (1:2500, 4814, RRID: AB_390781 ), p-PKA C T197 (1:1000, 5661, RRID: AB_10707163 ), and PKA C (1:2000, 5842, RRID: AB_10706172 ).

Techniques: Expressing, Biomarker Discovery, Comparison

Journal: iScience

Article Title: NF-κB and STAT3 signaling uniquely stratify survival in female glioblastoma patients

doi: 10.1016/j.isci.2026.114761

Figure Lengend Snippet: Female murine transformed astrocytes are more sensitive to regulation and inhibition of STAT3 signaling than male cells (A) Correlation between IL-6 expression and p-STAT3 (Y705) expression in full serum conditions ( n = 5 lots). Male: r = 0.51, R 2 = 0.26, ns; female: r = 0.96, R 2 = 0.93, ∗∗, p < 0.01 (Pearson’s correlation). (B and C) Western blot (B) and quantification (C) of p-STAT3 (Y705) levels in serum-starved conditions following TNF-α treatment (10 ng/mL) with blocking using 10 μg/mL of either α-IgG control or α-IL-6 for 24 h in female cells ( n = 3 lots). Western blot representative of n = 3 independent experiments. (D) Western blot of p-STAT3 (Y705) levels in serum-starved conditions with or without supplemental 10 ng/mL epidermal growth factor (EGF) in male and female cells ( n = 5 lots). Replicate lot 6 results serve for normalization between blots. (E and F) Quantification of p-STAT3 Y705 levels from (D), segregated by sex ( n = 5 lots; E) or strength of NF-κB gene signature ( n = 6 weak, n = 4 strong; F); 6F is the abbreviation for lot 6 females. (G) Correlation between p-STAT3 (Y705) levels and IC 50 of AZD1480 in serum-starved conditions in murine transformed astrocytes ( n = 5 lots per sex with or without supplemental EGF). Male: r = −0.26, R 2 = 0.25, ns; female: r = −0.79, R 2 = 0.53, ∗∗, p < 0.01 (Spearman’s correlation). (H and I) Western blot (H) and quantification (I) of p-STAT3 (Y705) and p-p65 (S536) levels in serum-starved conditions following treatment with vehicle-control (DMSO) or H-89 (10 μM) for 18 h in lot 6 male and female cells. Western blot representative of n = 3 independent experiments. Expression levels normalized to vehicle-treated (DMSO) female cells. (J) Hazard ratios from univariate survival analysis of wild-type (WT) or altered (alt) EGFR tumors in all ( N = 164 WT, N = 198 alt), male-only ( N = 94 WT, N = 125 alt), or female-only ( N = 70 WT, N = 73 alt) patients of IL-6/JAK/STAT3 genes from the Human Molecular Signatures Database. Data are presented as the median ± interquartile range. In (C), (E), (F), and (I), data are presented as the mean ± SD. In (C), (E), (F), (I), and (J), ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗; p < 0.0001; ns, not significant as determined by Tukey’s post hoc pairwise comparison following two-way ANOVA test (C, E, F, and I) or Kruskal-Wallis test followed by a Dunn’s test for post-hoc pairwise comparisons with multiplicity corrections (J). (C) Lot 6: α-IL-6 (∗, p < 0.05); TNF-α (∗∗∗, p < 0.001); interaction (ns, not significant). Lot 8: α-IL-6 (∗, p < 0.05); TNF-α (∗, p < 0.05); and interaction (ns, not significant). Lot 12: α-IL-6 (∗∗∗, p < 0.001); TNF-α (ns, not significant); and interaction (∗∗, p < 0.01) by two-way ANOVA. (E) Sex (ns, not significant); EGF (∗, p < 0.05); and interaction (ns, not significant) by two-way ANOVA. (F) NF-κB (∗, p < 0.05); EGF (∗∗, p < 0.01); and interaction (ns, not significant) by two-way ANOVA. (I) p-p65 (S536): sex (ns, not significant); H-89 (ns, not significant); and interaction (ns, not significant). P-STAT3 (Y705): sex (∗∗∗∗; p < 0.0001); H-89 (∗∗, p < 0.01), and interaction (∗∗, p < 0.01) by two-way ANOVA. Blue and red boxes in (D) and (H) indicate male and female murine transformed astrocytes, respectively. All data in (A)–(I) are compiled from n = 3 independent experiments (technical replication). In (A), (D), (E), (F), and (G), all data are compiled from each of the 5 lots (biological replication). See also and .

Article Snippet: Primary antibodies purchased from Cell Signaling Technologies include p-STAT3 Y705 (1:1000, 9145, RRID: AB_2491009 ), STAT3 (1:2500, 9139, RRID: AB_331757 ), p-p65 S536 NF-κB (1:1000, 3033, RRID: AB_331284 ), p65 NF-κB (1:2500, 6956, RRID: AB_10828935 ), β-actin (1:5000, 8457, RRID: AB_10950489 ), p-IκBα S32 (1:1000, 2859, RRID: AB_561111 ), IκBα (1:2500, 4814, RRID: AB_390781 ), p-PKA C T197 (1:1000, 5661, RRID: AB_10707163 ), and PKA C (1:2000, 5842, RRID: AB_10706172 ).

Techniques: Transformation Assay, Inhibition, Expressing, Western Blot, Blocking Assay, Control, Comparison

Journal: bioRxiv

Article Title: Pneumococcus drives STAT3 activation of lower airway epithelium in a strain and burden-dependent manner

doi: 10.64898/2026.02.13.705726

Figure Lengend Snippet: Differential canonical and non-canonical STAT3 Activation in A549 Cells Upon Spn Challenge Is Dependent on Strain and Infection Dose A . Representative immunofluorescence images showing nuclear localization of phosphorylated STAT3 at tyrosine 705 (p-STAT3 [Y705]) in A549 cells 2 h after challenge with Streptococcus pneumoniae (Spn) strains TIGR4 or 6B at multiplicities of infection (MOIs) of 5, 20, or 100, compared with uninfected controls (un.). Cells were stained with an anti-p-STAT3 (Y705) antibody (green; GFP-labeled secondary antibody) and DAPI for nuclear staining (magenta; pseudocolored). B. Quantification of nuclear p-STAT3 (Y705) immunofluorescence (IF) intensity in A549 cells 2 h after infection with Spn TIGR4 or Spn 6B at the indicated MOIs. Nuclear segmentation was based on DAPI staining. IF intensity values were normalized to the mean of the uninfected (un.) control within each experiment and log₂-transformed. Each point represents an individual nucleus (un., grey; TIGR4, purple; 6B, green). Black circles indicate the mean of each of the n = 3 independent experiments, and red horizontal lines denote replicate means. Statistical significance was assessed using two-way ANOVA followed by Tukey’s post-hoc test; ns, not significant (p > 0.05); **p < 0.01; ****p < 0.0001. C. Representative immunofluorescence images showing nuclear localization of phosphorylated STAT3 at serine 727 (p-STAT3 [S727]) in A549 cells 2 h after challenge with Spn strains TIGR4 or 6B at MOIs of 5, 20, or 100, compared with uninfected controls (un.). Cells were stained with an anti-p-STAT3 (S727) antibody (green; GFP-labeled secondary antibody) and DAPI for nuclear staining (magenta; pseudocolored). D. Quantification of nuclear p-STAT3 (S727) immunofluorescence (IF) intensity in A549 cells 2 h after infection with Spn TIGR4 or Spn 6B at the indicated MOIs. Nuclear segmentation was based on DAPI staining. IF intensity values were normalized to the mean of the uninfected (un.) control within each experiment and log₂-transformed. Each point represents an individual nucleus (un., grey; TIGR4, purple; 6B, green). Black circles indicate the mean of each of n = 3 independent experiments, and red horizontal lines denote replicate means. Statistical significance was assessed using two-way ANOVA followed by Tukey’s post-hoc test; ns, not significant (p≥0.05); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Primary antibody staining was carried out overnight at 4 °C in 5% BSA with the following antibodies (all at 1:200 dilution): Anti-STAT3 (124H6) Mouse mAb (Cell Signaling #9139), Anti-p-STAT3 (Y705) (D3A7) XP Rabbit mAb (Cell Signaling #9145), Anti-p-STAT3 (S727) Rabbit mAb (Abcam #ab32143).

Techniques: Activation Assay, Infection, Immunofluorescence, Staining, Labeling, Control, Transformation Assay

Journal: bioRxiv

Article Title: Pneumococcus drives STAT3 activation of lower airway epithelium in a strain and burden-dependent manner

doi: 10.64898/2026.02.13.705726

Figure Lengend Snippet: S. pneumoniae Infection Drives Expression of STAT3-Regulated Genes in A549 cells A Semi-quantitative real-time PCR analysis of SOCS3, a negative feedback regulator of STAT3, in A549 cells following infection with Spn TIGR4 or Spn 6B at MOI 20 or 100 for 2 h. Data are shown as log₂ fold changes relative to the uninfected (Un.) control from n = 4 biological replicates (MOI 20) and n = 3 biological replicates (MOI 100). Gene expression was normalized to the housekeeping gene GAPDH and subsequently to the uninfected control. Statistical comparisons between Spn TIGR4 and Spn 6B at each MOI were performed on −ΔΔCt values using Student’s t -test. *p < 0.05; *p < 0.01. B Heatmap of real-time PCR results showing expression of selected STAT3-associated genes of interest (GOIs) in A549 cells after 2h-challenge with Spn TIGR4 or 6B at MOI 20 and 100. Plotted values represent log₂ fold changes relative to uninfected. Statistical comparisons were made between Spn strains TIGR4 and 6B for MOI 20 and 100 using Student’s t-test on ΔCt-values. p-values (pV) are stated in the right most column,* any p < 0.05. Color scale reflects relative expression levels (see scale bar).

Article Snippet: Primary antibody staining was carried out overnight at 4 °C in 5% BSA with the following antibodies (all at 1:200 dilution): Anti-STAT3 (124H6) Mouse mAb (Cell Signaling #9139), Anti-p-STAT3 (Y705) (D3A7) XP Rabbit mAb (Cell Signaling #9145), Anti-p-STAT3 (S727) Rabbit mAb (Abcam #ab32143).

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction, Control, Gene Expression

Journal: bioRxiv

Article Title: Pneumococcus drives STAT3 activation of lower airway epithelium in a strain and burden-dependent manner

doi: 10.64898/2026.02.13.705726

Figure Lengend Snippet: A. Semi-quantitative real-time PCR analysis of SOCS3 expression in A549 cells following exposure to IL-6 (50 ng/mL) for 30 min or 2 h. Data are shown as log₂ fold changes relative to the uninfected control from n = 5 (30 min) and n = 2 (2 h) biological replicates. Gene expression was normalized to the housekeeping gene GAPDH and subsequently to the uninfected control (ΔΔCt-method). B. Heatmap of real-time PCR results showing expression of selected STAT3-associated genes of interest (GOIs) after 30 min and 2 h exposure to IL-6 in A549 cells. Values represent log₂ fold changes relative to uninfected. The color scale indicates relative expression levels (see scale bar ).

Article Snippet: Primary antibody staining was carried out overnight at 4 °C in 5% BSA with the following antibodies (all at 1:200 dilution): Anti-STAT3 (124H6) Mouse mAb (Cell Signaling #9139), Anti-p-STAT3 (Y705) (D3A7) XP Rabbit mAb (Cell Signaling #9145), Anti-p-STAT3 (S727) Rabbit mAb (Abcam #ab32143).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Gene Expression

Journal: bioRxiv

Article Title: Pneumococcus drives STAT3 activation of lower airway epithelium in a strain and burden-dependent manner

doi: 10.64898/2026.02.13.705726

Figure Lengend Snippet: Validation of STAT3 Knockdown Efficiency by RNAi in A549 Cells A. Representative immunofluorescence images showing total STAT3 localization in A549 cells treated for 48 h with STAT3-targeting RNAi (STAT3 RNAi) or scrambled control RNAi (scr RNAi). B. Quantification of mean total STAT3 fluorescence intensity within the nucleus (segmented by DAPI) in RNAi-treated A549 cells (n = 3 biological replicates) after 48 h of RNAi treatment, followed by either a 2 h challenge with Spn TIGR4 at MOIs 20 or 100, or stimulation with IL-6 (50 ng/mL) for 15 min. Percent knockdown between scr RNAi and STAT3 RNAi is indicated in the figure. C. Representative brightfield images (10× magnification) of trypan blue membrane exclusion assay in RNAi-treated A549 cells following 2-hour challenge with Spn TIGR4 and 6B at MOI 20. Cells with compromised membrane integrity show dark blue nuclear staining due to trypan blue uptake. D. Lactate dehydrogenase (LDH) release assay in A549 cells after 2-hour challenge with Spn TIGR4 or Spn 6B at MOIs 20 and 100, stimulation with IL-6 (50 ng/mL, 2h), and uninfected controls. LDH release is expressed as a percentage of total cell lysis induced by chemical detergent. Bars represent mean ± SD of n = 4 biological replicates.

Article Snippet: Primary antibody staining was carried out overnight at 4 °C in 5% BSA with the following antibodies (all at 1:200 dilution): Anti-STAT3 (124H6) Mouse mAb (Cell Signaling #9139), Anti-p-STAT3 (Y705) (D3A7) XP Rabbit mAb (Cell Signaling #9145), Anti-p-STAT3 (S727) Rabbit mAb (Abcam #ab32143).

Techniques: Biomarker Discovery, Knockdown, Immunofluorescence, Control, Fluorescence, Membrane, Exclusion Assay, Staining, Lactate Dehydrogenase Assay, Lysis

Journal: bioRxiv

Article Title: Pneumococcus drives STAT3 activation of lower airway epithelium in a strain and burden-dependent manner

doi: 10.64898/2026.02.13.705726

Figure Lengend Snippet: STAT3 knockdown does not affect A549 cell integrity during S. pneumoniae challenge A . Adhesion of Spn to A549 cells after 48 h of transfection with STAT3-targeting RNAi (STAT3 RNAi) or scrambled control RNAi (scr RNAi). Cells were challenged for 2 h with Spn TIGR4 or Spn 6B at MOIs of 20 or 100, after which adherent bacteria were quantified as CFUs from n = 3 independent biological replicates. B . Quantification of trypan blue–positive nuclei in RNAi-treated A549 cells following a 2 h challenge with Spn TIGR4 or Spn 6B at MOI 20. Four to six microscopy fields per condition and experiment were manually analyzed, and the mean percentage of trypan blue–positive cells was determined. Percentages from individual biological replicates (n = 3) are shown. Statistical comparisons between STAT3 RNAi and scr RNAi were performed for each condition using a two-sided Student’s t-test. C . Quantification of mean nuclear phosphorylated STAT3 (p-STAT3 [Y705]) fluorescence intensity in RNAi-treated A549 cells (n = 3 biological replicates) after 48 h of RNAi treatment, followed by either a 2 h challenge with Spn TIGR4 at MOI 100 or stimulation with IL-6 (50 ng/mL) for 15 min. Nuclear segmentation was based on DAPI staining. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post-hoc test. Statistical significance: ns, not significant (p > 0.05); *p < 0.05; **p < 0.01.

Article Snippet: Primary antibody staining was carried out overnight at 4 °C in 5% BSA with the following antibodies (all at 1:200 dilution): Anti-STAT3 (124H6) Mouse mAb (Cell Signaling #9139), Anti-p-STAT3 (Y705) (D3A7) XP Rabbit mAb (Cell Signaling #9145), Anti-p-STAT3 (S727) Rabbit mAb (Abcam #ab32143).

Techniques: Knockdown, Transfection, Control, Bacteria, Microscopy, Fluorescence, Staining

Journal: bioRxiv

Article Title: Pneumococcus drives STAT3 activation of lower airway epithelium in a strain and burden-dependent manner

doi: 10.64898/2026.02.13.705726

Figure Lengend Snippet: Live S. pneumoniae and Pneumolysin Production Are Required to Trigger Canonical STAT3 Activation Quantification of phosphorylated STAT3 immunofluorescence intensity (IF) over the nucleus in A549 cells, segmented based on DAPI nuclear staining. Fluorescence intensities were normalized to the mean of the uninfected control (un.) for each experiment and log₂-transformed. Each colored point represents an individual nucleus; black circles indicate the mean of biological replicates, and horizontal lines show the overall mean. Statistical significance was assessed using two-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05; **p < 0.01; ***p < 0.001. A. Quantification of p-STAT3 (Y705) IF intensity over the nucleus of A549 cells challenged with live Spn TIGR4 and PFA-inactivated TIGR4 (PFA TIGR4) at MOIs of 20 and 100. Color coding: un. (grey), live TIGR4 (purple), inactivated TIGR4 (mauve). (n = 4). B. Quantification of p-STAT3 (Y705) IF intensity over the nucleus of A549 cells challenged withSpn TIGR4 and Spn TIGR4Δ ply (pneumolysin-deficient) at MOIs 20 and 100. Color coding: un. (grey), TIGR4 (purple), TIGR4Δ ply (yellow). (n = 3). C. Quantification of p-STAT3 (Y705) IF intensity over the nucleus of A549 cells challenged withSpn TIGR4 and Spn TIGR4Δ spxB (pyruvate oxidase-deficient) at MOIs 20 and 100. Color coding: un. (grey), TIGR4 (purple), TIGR4Δ spxB (salmon). (n = 3).

Article Snippet: Primary antibody staining was carried out overnight at 4 °C in 5% BSA with the following antibodies (all at 1:200 dilution): Anti-STAT3 (124H6) Mouse mAb (Cell Signaling #9139), Anti-p-STAT3 (Y705) (D3A7) XP Rabbit mAb (Cell Signaling #9145), Anti-p-STAT3 (S727) Rabbit mAb (Abcam #ab32143).

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Control, Transformation Assay

Journal: bioRxiv

Article Title: Pneumococcus drives STAT3 activation of lower airway epithelium in a strain and burden-dependent manner

doi: 10.64898/2026.02.13.705726

Figure Lengend Snippet: Live S. pneumoniae and Pneumolysin Production Are Required to suppress non-canonical nuclear STAT3 localization and recombinant Pneumolysin Alone Is Not Sufficient to Induce Canonical STAT3 Activation in A549 Cells Quantification of phosphorylated STAT3 immunofluorescence intensity (IF) over the nucleus in A549 cells, segmented based on DAPI nuclear staining. Fluorescence intensities were normalized to the mean of the uninfected control(un.) for each experiment and log₂-transformed. Each colored point represents an individual nucleus; black circles indicate the mean of biological replicates, and horizontal lines show the overall mean. A. Quantification of p-STAT3 (S727) IF intensity over the nucleus of A549 cells challenged with live Spn TIGR4 and PFA-inactivated TIGR4 (PFA TIGR4) at MOIs of 20 and 100. Color coding: un. (grey), live TIGR4 (purple), inactivated TIGR4 (mauve). (n = 4). Statistical significance was assessed using two-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05; ns, not significant. B. Quantification of p-STAT3 (S727) IF intensity over the nucleus of A549 cells challenged with Spn TIGR4 and Spn TIGR4Δ ply (pneumolysin-deficient) at MOIs 20 and 100. Color coding: un. (grey), TIGR4 (purple), TIGR4Δ ply (yellow). (n = 3). Statistical significance was assessed using two-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05; ns, not significant. C. Quantification of p-STAT3 (S727) IF intensity over the nucleus of A549 cells challenged with Spn TIGR4 and Spn TIGR4Δ spxB (pyruvate oxidase-deficient) at MOIs 20 and 100. Color coding: un. (grey), TIGR4 (purple), TIGR4Δ spxB (salmon). (n = 3). Statistical significance was assessed using two-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05; ns, not significant. D Quantification of nuclear p-STAT3 (Y705) IF intensity over the nucleus of A549 cells following a 2 h challenge with Spn TIGR4 (MOI 100) or recombinant pneumolysin (PLY) at 0.1, 2, or 10 nM. Color coding: un., grey; TIGR4, purple; PLY, lilac. (n=2). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple-comparisons test versus uninfected; *p ≤ 0.05; ns, not significant.

Article Snippet: Primary antibody staining was carried out overnight at 4 °C in 5% BSA with the following antibodies (all at 1:200 dilution): Anti-STAT3 (124H6) Mouse mAb (Cell Signaling #9139), Anti-p-STAT3 (Y705) (D3A7) XP Rabbit mAb (Cell Signaling #9145), Anti-p-STAT3 (S727) Rabbit mAb (Abcam #ab32143).

Techniques: Recombinant, Activation Assay, Immunofluorescence, Staining, Fluorescence, Control, Transformation Assay