Journal: bioRxiv

Article Title: Middle-aged mice treated with GHK-Cu peptide administered intraperitoneally or intranasally show behavioral rescue but divergent hippocampal aging programs

doi: 10.64898/2026.04.09.717524

Figure Lengend Snippet: Positive area-staining by antibody in intranasally-treated GHK-Cu mice of both sexes for (A) Synaptophysin, (B) GFAP, (C) MCP-1, (D) PSD-95, and (E) TGF-β. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Following blocking, sections were incubated overnight at 4°C with primary antibodies against synaptophysin (1:250, Invitrogen MA5-16402), PSD95 (1:250, Abcam ab18258), phospho-SMAD2 (1:50, Invitrogen 44-244G), MCP-1 (1:800, Novus NBP1-07035), p21 (1:200, Abcam ab188224), TGF-β (1:50, Abcam ab215715), and GFAP (1:1500, Invitrogen: PA1-10019).

Techniques: Staining

Journal: bioRxiv

Article Title: Middle-aged mice treated with GHK-Cu peptide administered intraperitoneally or intranasally show behavioral rescue but divergent hippocampal aging programs

doi: 10.64898/2026.04.09.717524

Figure Lengend Snippet: Positive area-staining by antibody in intraperitoneally-treated GHK-Cu mice of both sexes for (A) TGF-β, (B) GFAP), (C) MCP-1, (D) p21, (E) Synaptophysin, (F) pSMAD-2, and (G) PSD-95. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Following blocking, sections were incubated overnight at 4°C with primary antibodies against synaptophysin (1:250, Invitrogen MA5-16402), PSD95 (1:250, Abcam ab18258), phospho-SMAD2 (1:50, Invitrogen 44-244G), MCP-1 (1:800, Novus NBP1-07035), p21 (1:200, Abcam ab188224), TGF-β (1:50, Abcam ab215715), and GFAP (1:1500, Invitrogen: PA1-10019).

Techniques: Staining

Journal: Redox Biology

Article Title: The electrophilic metabolite of kynurenine, kynurenine-CKA, requires C151 in Keap1 to derepress Nrf2

doi: 10.1016/j.redox.2026.104009

Figure Lengend Snippet: AhR inhibition has no effect on the anti-inflammatory actions of Kyn-CKA in BMDMs. (A) Mouse AhR reporter cells expressing luciferase under the control of the xenobiotic response element (XRE) were incubated with Kyn-CKA or kynurenine for 24 h prior to luminescence measurement (n = 3). Data were fitted to a sigmoidal four-parameter logistic curve (L-Kyn: r 2 = 0.824, IC 50 = 28 μM, span = 3218 RFU. Kyn-CKA: r 2 = 0.970, IC 50 = 13 μM, span = 47,901 RFU). (B) Inhibition of AhR-dependent luciferase expression by CH223191 (AhRinh). (C–F) Kyn-CKA inhibits the expression of the NF-κB-regulated genes IL6, MCP1, Nos2 and IL1β in BMDM following 5 h incubation with LPS (0.5 μg/mL) and the indicated concentrations of Kyn-CKA both in the presence or absence of CH223191 (10 μM). Data are n = 3–4, ∗∗∗∗p < 0.0001 by one-way ANOVA and Tukey's post-test. (G – H) Extracellular cytokine levels from BMDM treated as in C–F for 5 h. Data are n = 3–4 (all replicates shown), ∗∗∗∗p < 0.0001 by one-way ANOVA and Tukey's post-test.

Article Snippet: ELISA kits for MCP1 (DY479 and MJE00B) and IL6 (DY406) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Expressing, Luciferase, Control, Incubation

Journal: Redox Biology

Article Title: The electrophilic metabolite of kynurenine, kynurenine-CKA, requires C151 in Keap1 to derepress Nrf2

doi: 10.1016/j.redox.2026.104009

Figure Lengend Snippet: The low-dose anti-inflammatory effects of Kyn-CKA in BMDMs are dependent on Nrf2. (A – B) Treatment with Kyn-CKA (5 h) fails to induce the expression of Nqo1 and Gclm in BMDMs obtained from Nrf2-knockout (Nrf2 −/− ) mice. Data are n = 3, ∗∗∗∗p < 0.0001 by two-way ANOVA and Tukey's post-test. (C – G) Effects of Kyn-CKA on the expression of NF-κB−regulated genes in WT and Nrf2-KO BMDMs following 5 h incubation with LPS (0.5 μg/mL) and the indicated concentrations of Kyn-CKA. Data are n = 6, ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA and Tukey's post-test. Nrf2 was identified as a significant source of variation for MCP1, IL6, TNFα, Nos2, (p < 0.0001) and IL1β (p < 0.005).

Article Snippet: ELISA kits for MCP1 (DY479 and MJE00B) and IL6 (DY406) were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Knock-Out, Incubation

Journal: Frontiers in Oncology

Article Title: The STAT3–ZEB1 axis contributes to CCL2-mediated resistance to osimertinib in lung cancer

doi: 10.3389/fonc.2026.1699471

Figure Lengend Snippet: Gain- and loss-of-function analyses reveal that CCL2 regulates osimertinib resistance by suppressing apoptosis. (A) CCL2 mRNA levels in HCC827-mock and HCC827-CCL2 cells were quantified by RT-qPCR (***p < 0.001). (B) Cell viability after 48 h of osimertinib treatment was assessed by MTT assay. DMSO-treated cells were normalized to 100%. Data represent the mean ± SD from four independent experiments, each performed in triplicate (***p < 0.001, Student’s t-test). (C) CCL2 mRNA levels in HCC827/gef cells transfected with CCL2 siRNA were quantified by RT-qPCR. Data represent the mean ± SD from three independent biological replicates (***p < 0.001, Student’s t-test). (D) Cell viability after 72 h of osimertinib treatment was measured by MTT assay. (E) CCL2 mRNA levels in H1975/AZD-18 cells transfected with CCL2 siRNA were quantified by RT-qPCR. Data represent the mean ± SD from three independent biological replicates (***p < 0.001, Student’s t-test). (F) Osimertinib sensitivity in si-CCL2 cells was determined by MTT assay. Data represent the mean ± SD from three independent biological replicates (***p < 0.001, Student’s t-test). (G) Caspase-9 activity in HCC827-CCL2 cells after osimertinib treatment (***p < 0.001). (H) Caspase-9 activity in CCL2-knockdown cells following 500 nM osimertinib exposure for 24 h (**p < 0.01; ***p < 0.001, Student’s t-test). (I) Flow cytometry analysis of apoptosis (Annexin V/PI) in HCC827/gef cells. The bar chart displays the percentage of apoptotic cells derived from three independent experiments (**p < 0.01 compared with vehicle control, Student’s t-test ). (J, K) Western blot analysis of cleaved PARP and α-tubulin in CCL2-knockdown cells treated with osimertinib. Representative blots are shown from three independent experiments. (L) Cell viability of H1975/AZD-18 cells treated with osimertinib in the presence or absence of the CCL2-neutralizing antibody carlumab was assessed by MTT assay. Data represent the mean ± SD from three independent experiments (***p < 0.001, Student’s t-test).

Article Snippet: HCC827-CCL2 cells were generated by transfecting them with a plasmid carrying human CCL2 cDNA (SC118317, Origene, Rockville, MD).

Techniques: Quantitative RT-PCR, MTT Assay, Transfection, Activity Assay, Knockdown, Flow Cytometry, Derivative Assay, Control, Western Blot

Journal: Frontiers in Oncology

Article Title: The STAT3–ZEB1 axis contributes to CCL2-mediated resistance to osimertinib in lung cancer

doi: 10.3389/fonc.2026.1699471

Figure Lengend Snippet: CCL2 overexpression attenuates the antitumor efficacy of osimertinib and shortens survival in xenograft models. (A) Growth curves of HCC827-CCL2 and HCC827-mock xenografts in SCID mice (n = 6 per group). Tumor volumes were measured every 4 days and expressed as mean ± standard error (SE). ns, not significant. (B) Kaplan–Meier survival curves comparing mice bearing HCC827-CCL2 and mock xenografts. No significant difference was observed between groups (log-rank test, p > 0.05). (C) Tumor growth of mice treated with osimertinib (0.5 mg/kg/day) for 32 days (n = 6 per group). Data are presented as mean ± SE (*p < 0.05, Student’s t-test ). (D) Kaplan–Meier survival analysis of osimertinib-treated xenograft-bearing mice (HCC827-CCL2 vs mock). Survival was defined by tumor volume <400 mm³. Statistical significance was determined by the log-rank (Mantel–Cox) test (p = 0.019).

Article Snippet: HCC827-CCL2 cells were generated by transfecting them with a plasmid carrying human CCL2 cDNA (SC118317, Origene, Rockville, MD).

Techniques: Over Expression

Journal: Frontiers in Oncology

Article Title: The STAT3–ZEB1 axis contributes to CCL2-mediated resistance to osimertinib in lung cancer

doi: 10.3389/fonc.2026.1699471

Figure Lengend Snippet: CCL2 activates STAT3 and ERK. (A) Spearman’s correlation coefficients were calculated between CCL2 expression and the enrichment scores of MSigDB Hallmark gene sets derived from TCGA-LUAD transcriptomes. Only significantly correlated pathways (FDR < 0.01) are shown. (B) Western blot analysis of phosphorylated STAT3 and ERK1/2 in HCC827-CCL2 cells compared with vector control. CCL2 overexpression led to increased phosphorylation of STAT3 and ERK1/2, while p-AKT levels remained unchanged. (C) Phosphorylated STAT3 and ERK1/2 in HCC827/gef-si-CCL2 cells compared with control transfectants. Knockdown of CCL2 reduced p-STAT3 and p-ERK1/2 levels. α-tubulin served as the loading control. Representative blots are shown from three independent biological experiments.

Article Snippet: HCC827-CCL2 cells were generated by transfecting them with a plasmid carrying human CCL2 cDNA (SC118317, Origene, Rockville, MD).

Techniques: Expressing, Derivative Assay, Western Blot, Plasmid Preparation, Control, Over Expression, Phospho-proteomics, Knockdown

Journal: Frontiers in Oncology

Article Title: The STAT3–ZEB1 axis contributes to CCL2-mediated resistance to osimertinib in lung cancer

doi: 10.3389/fonc.2026.1699471

Figure Lengend Snippet: Targeting ZEB1 reverses osimertinib resistance in lung cancer cells. (A) Western blot analysis of EMT-related transcription factors in HCC827-CCL2 overexpression cells compared with vector control. ZEB1 expression levels increased upon CCL2 overexpression. Representative blots are shown from three independent experiments. (B) RT-qPCR and (C) Western blot analysis confirming ZEB1 knockdown efficiency in HCC827-CCL2 cells. Data represent the mean ± SD from three independent biological experiments (***p < 0.001, Student’s t -test). (D) MTT assay showing that ZEB1 knockdown restored osimertinib sensitivity in HCC827-CCL2 cells. Data represent the mean ± SD from four independent biological experiments, each performed in triplicate (***p < 0.001, Student’s t -test). (E) RT-qPCR and (F) Western blot analysis confirming ZEB1 knockdown efficiency in H1975/AZD-18 cells. Data represent the mean ± SD from three independent biological experiments (***p < 0.001, Student’s t -test). (G) MTT assay showing that ZEB1 knockdown restored osimertinib sensitivity in H1975/AZD-18 cells. Data represent the mean ± SD from four independent biological experiments, each performed in triplicate (***p < 0.001, Student’s t -test).

Article Snippet: HCC827-CCL2 cells were generated by transfecting them with a plasmid carrying human CCL2 cDNA (SC118317, Origene, Rockville, MD).

Techniques: Western Blot, Over Expression, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Knockdown, MTT Assay

Journal: Frontiers in Oncology

Article Title: The STAT3–ZEB1 axis contributes to CCL2-mediated resistance to osimertinib in lung cancer

doi: 10.3389/fonc.2026.1699471

Figure Lengend Snippet: Blocking CCR2–STAT3/ERK signaling restores osimertinib sensitivity (A) Western blot analysis of CCR2 expression in HCC827-CCL2 cells after transfection with CCR2 siRNA. Representative blots are shown from three independent biological experiments. (B) MTT assay showing reduced viability of HCC827-CCL2-si-CCR2 cells treated with osimertinib (48 h). Data represent the mean ± SD from three independent biological experiments, each performed in triplicate (***p < 0.001, Student’s t-test ). (C) Caspase-9 activity increased in CCR2-knockdown cells upon osimertinib treatment, measured by luminescent Caspase-Glo 9 assay. Data represent the mean ± SD from three independent experiments (***p < 0.001 vs. si-scramble, Student’s t-test ). (D) Western blot analysis of downstream signaling proteins (p-STAT3, p-ERK1/2, ZEB1) in HCC827-CCL2 cells treated with CCR2 antagonist INCB3344. Representative blots are shown from three independent experiments. (E) Combined treatment with osimertinib and INCB3344 decreased cell viability in HCC827-CCL2 cells (MTT assay). Data represent the mean ± SD from three independent biological replicates (*p < 0.05, Student’s t-test ). (F) INCB3344 enhanced caspase-9 activity upon osimertinib treatment. Data represent the mean ± SD from three independent biological experiments (**p < 0.01, Student’s t-test ). (G) HCC827-CCL2 cells treated with STAT3 inhibitor (S3I201, 40 μM, 24 h) were analyzed by Western blot for p-STAT3, p-ERK1/2, and ZEB1. Representative blots are shown from three independent experiments. (H) MTT assay showing that STAT3 inhibition sensitized HCC827-CCL2 cells to osimertinib treatment. Data represent the mean ± SD from three independent experiments (**p < 0.01, Student’s t-test ). (I) Tumor volume of H1975/AZD-18 xenografts treated with vehicle, osimertinib (1mg/kg/day), S3I201 (5mg/kg/2day), or a combination of both drugs for 16days (n = 5 in each subgroup). Data are presented as mean ± SE (*p < 0.05, Student’s t-test) . (J) Chromatin immunoprecipitation (ChIP) assay demonstrating STAT3 binding to the ZEB1 promoter in H1975/AZD-18 cells. Enrichment of ZEB1 promoter fragments was quantified by qPCR and normalized to IgG control. Data represent the mean ± SD from three independent experiments (*p < 0.05, Student’s t-test). (K) The expression of p-STAT3, p-ERK1/2, STAT3, ERK, and ZEB1 in HCC827-CCL2 cells with ERK inhibitor selumetinib was measured using Western blot analysis. Representative blots are shown from three independent experiments. (L) HCC872-CCL2 cells were treated with osimertinib and selumetinib treatment alone or in combination for 48 h; cell viability was determined using MTT assays. Data represent the mean ± SD from three independent experiments (**p < 0.001, Student’s t-test ).

Article Snippet: HCC827-CCL2 cells were generated by transfecting them with a plasmid carrying human CCL2 cDNA (SC118317, Origene, Rockville, MD).

Techniques: Blocking Assay, Western Blot, Expressing, Transfection, MTT Assay, Activity Assay, Knockdown, Inhibition, Chromatin Immunoprecipitation, Binding Assay, Control