Journal: Molecular Medicine

Article Title: The protein kinase IKK epsilon shapes the melanoma tumor microenvironment by modulating angiogenesis and the T-cell immunity

doi: 10.1186/s10020-026-01651-w

Figure Lengend Snippet: T-cell migration and activity. A T-cell migration towards a FCS gradient or conditioned medium from B16BL6 melanoma cells. Pan T-cells were isolated from the spleens of wildtype (WT) and IKKε KO mice and plated into cell culture insert filled with T-cell medium containing 0.1% FCS. The bottom chamber was filled either with medium containing 0.1% FCS (neg ctrl), 10% FCS (pos ctrl) or conditioned medium from B16BL6 cells without FCS (B16). Cell migration of different T-cell subtypes to the lower channel was determined by FACS analysis. Cell numbers were normalized to counting beads and negative controls. n = 3–6. One-way ANOVA with Tukey´s multiple comparisons test, * P < 0.05, ** P < 0.01. B — D Effects of co-culture of B16BL6 melanoma cells with CD8⁺ and γδ-T-cells, respectively, derived from the spleens of wildtype (WT) and IKKε KO mice. B Cell proliferation determined by WST assay, B16BL6 monocultures were set as 1 for better comparison. One-way ANOVA with Tukey´s multiple comparisons test, * P < 0.05, **P < 0.01 significant difference in comparison to B16BL6 monoculture ( C ) IFN-γ levels analyzed by cytokine bead array (CBA) and ( D ) granzyme B release determined by ELISA. n = 4–10/group. C and D The T-cell monocultures of wildtype and IKKε knock-out mice were set as 1 for better comparison. Student´s t-test, * P < 0.05, significant difference in comparison to wildtype, # P < 0.05, ## P < 0.01, ### P < 0.001 significant difference in comparison to T-cell monocultures

Article Snippet: Granzyme B, Cxcl1, Cxcl2 and VEGF protein levels were determined from cell and tumor protein lysates using Mouse Granzyme B ELISA Kit (Invitrogen/Thermo Fisher Scientific, Dreieich, Germany #BMS6079), Mouse CXCL1/KC Quantikine ELISA Kit (#MKC00B), Mouse CXCL2/MIP-2 DuoSet ELISA (#DY452) and Mouse VEGF Quantikine ELISA Kit (#MMVOO-1) (all R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions.

Techniques: Migration, Activity Assay, Isolation, Cell Culture, Analysis, Counting, Co-culture Assay, Derivative Assay, WST Assay, Comparison, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Molecular Medicine

Article Title: The protein kinase IKK epsilon shapes the melanoma tumor microenvironment by modulating angiogenesis and the T-cell immunity

doi: 10.1186/s10020-026-01651-w

Figure Lengend Snippet: Regulation of cytokines and angiogenesis markers in the tumor. A OLINK proteomic analysis of inflammatory cytokines and chemokines in the tumors of wildtype and IKKε KO mice at the end of the experiment. Heat map and Volcano Plot of significantly different regulations. n = 4/group ( B ) Cxcl1 and Cxcl2 ELISA. Cxcl1: WT n = 8, IKKε-KO n = 6; Cxcl2: WT n = 6, IKKε-KO n = 5. C Determination of CD31-positive cells as vessel markers in the tumors. Left panel: FACS analysis, n = 6 per group. Right panel: immunofluorescence images and quantitative analysis, n = 4–5 tumors per group; 3–5 areas per tumor were evaluated. CD31-positive areas were normalized to wildtype controls to facilitate comparison. D MMP-9 expression as assessed by Western blot and zymography, n = 5–6 per group. E HIF-1α expression as determined by immunofluorescence, the pictures show on representative stain from 6 independent analyses, the diagram shows the densitometric analysis of all experiments, scale bar represents 100 µm. F VEGF expression analyzed by ELISA and Western Blot, n = 3/group. Student´s t-test, * P < 0.05, ** P < 0.01

Article Snippet: Granzyme B, Cxcl1, Cxcl2 and VEGF protein levels were determined from cell and tumor protein lysates using Mouse Granzyme B ELISA Kit (Invitrogen/Thermo Fisher Scientific, Dreieich, Germany #BMS6079), Mouse CXCL1/KC Quantikine ELISA Kit (#MKC00B), Mouse CXCL2/MIP-2 DuoSet ELISA (#DY452) and Mouse VEGF Quantikine ELISA Kit (#MMVOO-1) (all R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions.

Techniques: Analysis, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Comparison, Expressing, Western Blot, Zymography, Staining

Journal: Molecular Medicine

Article Title: The protein kinase IKK epsilon shapes the melanoma tumor microenvironment by modulating angiogenesis and the T-cell immunity

doi: 10.1186/s10020-026-01651-w

Figure Lengend Snippet: Early regulation of cytokines and angiogenesis markers in the tumor. A Cxcl1 ELISA, ( B ) MMP-9 expression (Western Blot and zymography), ( C ) HIF-1α expression as determined by immunofluorescence. The pictures show a representative stain from 4–6 independent analyses, the diagram shows the densitometric analysis of all experiments, scale bar represents 100 µm. D FACS analysis of CD31. + -cells as vessel markers in the tumors, ( n = 6/group). E VEGF expression analyzed by ELISA and Western Blot, ( F ) p65 expression and activity in nuclear extracts as determined by Western Blot and transcription factor ELISA. n = 3–5/group. Student´s t-test, * P < 0.05, ** P < 0.01

Article Snippet: Granzyme B, Cxcl1, Cxcl2 and VEGF protein levels were determined from cell and tumor protein lysates using Mouse Granzyme B ELISA Kit (Invitrogen/Thermo Fisher Scientific, Dreieich, Germany #BMS6079), Mouse CXCL1/KC Quantikine ELISA Kit (#MKC00B), Mouse CXCL2/MIP-2 DuoSet ELISA (#DY452) and Mouse VEGF Quantikine ELISA Kit (#MMVOO-1) (all R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Zymography, Immunofluorescence, Staining, Analysis, Activity Assay

Journal: Molecular Medicine

Article Title: The protein kinase IKK epsilon shapes the melanoma tumor microenvironment by modulating angiogenesis and the T-cell immunity

doi: 10.1186/s10020-026-01651-w

Figure Lengend Snippet: Angiogenesis assay in HUVEC with and without depletion of IKKε by siRNA. A IKKε mRNA and protein expression in HUVEC cells after treatment with IKKε siRNA or a scrambled negative control as determined by PCR and Western Blot analysis, PCR: n = 3–4/group, Western Blot ( n = 2/group) ( B ) Tube formation assay, left side: microscopic pictures, right side: analysis of the total mesh area of naïve HUVEC, or HUVEC treated with NC and IKKε siRNA. n = 6–7/group. The scale bars represent 100 µm. C Spheroid sprout assay, left side: microscopic pictures, right side: analysis of cumulative sprout length (CSL) in spheroids of naïve HUVEC ( n = 15 spheroids), HUVEC treated with VEGF ( n = 38 spheroids) HUVEC-NC ( n = 25 spheroids) and HUVEC-IKKε siRNA ( n = 38 spheroids). Data were generated in 5 independent biological experiments. The scale bars represent 100 µm. One Way ANOVA with Tukey´s multiple comparisons test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Granzyme B, Cxcl1, Cxcl2 and VEGF protein levels were determined from cell and tumor protein lysates using Mouse Granzyme B ELISA Kit (Invitrogen/Thermo Fisher Scientific, Dreieich, Germany #BMS6079), Mouse CXCL1/KC Quantikine ELISA Kit (#MKC00B), Mouse CXCL2/MIP-2 DuoSet ELISA (#DY452) and Mouse VEGF Quantikine ELISA Kit (#MMVOO-1) (all R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions.

Techniques: Angiogenesis Assay, Expressing, Negative Control, Western Blot, Analysis, Tube Formation Assay, Spheroids, Generated

Journal: International Journal of Nanomedicine

Article Title: Levofloxacin-Catalase-PLGA Nanoparticles Potentiate Sonodynamic Therapy to Alleviate BCG-Induced Tuberculous Granulomas via PI3K/Akt/VEGF Pathway Suppression in Rats

doi: 10.2147/IJN.S628763

Figure Lengend Snippet: Microenvironmental features of 3D tuberculous spheroids. ( A ) Bright-field imaging of spheroid formation (scale bars: 360 μm (first three panels), 20 μm (right)); ( B ) Confocal immunofluorescence of HIF-1α and VEGF in control and tuberculous spheroids (scale bar: 50 μm); ( C ) Intracellular H 2 O 2 levels (n = 3); ( D and E ) qRT-PCR quantification of HIF-1α ( D ) and VEGF ( E ) mRNA expression in tuberculous spheroids and cell spheroids (n = 3); ( F and G ) Western blot ( F ) and grayscale analysis ( G ) of HIF-1α and VEGF protein levels by Image J (n = 3). *p < 0.05, **p < 0.01,* **p < 0.001.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against PI3K (CST, USA, 1:1000), p-PI3K (CST, USA, 1:1000), Akt (CST, USA, 1:1000), p-Akt (CST, USA, 1:1000), HIF-1α (Abcam, UK, 1:1000), VEGF (Proteintech, China, 1:1000), Bax (Proteintech, China, 1:1000), and Bcl-2 (Proteintech, China, 1:1500).

Techniques: Spheroids, Imaging, Confocal, Immunofluorescence, Control, Quantitative RT-PCR, Expressing, Western Blot, Analysis

Journal: International Journal of Nanomedicine

Article Title: Levofloxacin-Catalase-PLGA Nanoparticles Potentiate Sonodynamic Therapy to Alleviate BCG-Induced Tuberculous Granulomas via PI3K/Akt/VEGF Pathway Suppression in Rats

doi: 10.2147/IJN.S628763

Figure Lengend Snippet: US + LEV@CAT-NPs treatment downregulates HIF-1α/VEGF expression in BCG-induced tuberculous spheroids. ( A and B ) Immunofluorescence images of HIF-1α ( A ) and VEGF ( B ) in tuberculous spheroids following various treatments; scale bar: 100 μm. ( C and D ) Relative mRNA levels of HIF-1α and VEGF in tuberculous spheroids across different groups (n = 3). ( E ) Secreted VEGF concentration in the culture supernatant of tuberculous spheroids (n = 3).* **P < 0.001.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against PI3K (CST, USA, 1:1000), p-PI3K (CST, USA, 1:1000), Akt (CST, USA, 1:1000), p-Akt (CST, USA, 1:1000), HIF-1α (Abcam, UK, 1:1000), VEGF (Proteintech, China, 1:1000), Bax (Proteintech, China, 1:1000), and Bcl-2 (Proteintech, China, 1:1500).

Techniques: Expressing, Spheroids, Immunofluorescence, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Levofloxacin-Catalase-PLGA Nanoparticles Potentiate Sonodynamic Therapy to Alleviate BCG-Induced Tuberculous Granulomas via PI3K/Akt/VEGF Pathway Suppression in Rats

doi: 10.2147/IJN.S628763

Figure Lengend Snippet: Transcriptomic and functional validation that US + LEV@CAT-NPs suppress VEGF expression via PI3K/Akt axis inhibition. ( A ) KEGG enrichment plot from RNA-seq analysis identifying the PI3K/Akt pathway as one of the top enriched pathways; ( B and C ) Western blot analysis ( B ) and its quantitative results ( C ) confirming the downregulation of HIF-1α, VEGF, and reduced phosphorylation levels of PI3K and Akt; ( D and E ) Pharmacological inhibition with LY294002 demonstrating that VEGF suppression is dependent on PI3K/Akt signaling, as shown by representative blots ( D ) and quantitative analysis of p-PI3K/PI3K, p-Akt/Akt ratio and secreted VEGF levels ( E ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against PI3K (CST, USA, 1:1000), p-PI3K (CST, USA, 1:1000), Akt (CST, USA, 1:1000), p-Akt (CST, USA, 1:1000), HIF-1α (Abcam, UK, 1:1000), VEGF (Proteintech, China, 1:1000), Bax (Proteintech, China, 1:1000), and Bcl-2 (Proteintech, China, 1:1500).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Inhibition, RNA Sequencing, Analysis, Western Blot, Phospho-proteomics

Journal: International Journal of Nanomedicine

Article Title: Levofloxacin-Catalase-PLGA Nanoparticles Potentiate Sonodynamic Therapy to Alleviate BCG-Induced Tuberculous Granulomas via PI3K/Akt/VEGF Pathway Suppression in Rats

doi: 10.2147/IJN.S628763

Figure Lengend Snippet: Conditioned medium from US + LEV@CAT-NPs-treated tuberculous spheroids inhibits angiogenesis in HUVECs.( A ) Schematic diagram of the experimental workflow for evaluating the effects of conditioned media (CM) from US + LEV@CAT-NPs-treated tuberculous spheroids on HUVEC angiogenesis; ( B ) Representative images of tube formation by HUVECs cultured with different CM; scale bar: 50 μm; ( C ) Immunofluorescence staining of VEGF in HUVECs; scale bar: 20 μm; ( D and E ) Quantitative analysis of total tube length ( D ) and coverage area ( E ) for HUVEC tube‑formation assays (n = 3). Both tube length and coverage area were normalized to the CM‑Control group (set as 100%). Data are shown as mean ± SD. ( F ) qRT-PCR analysis of VEGF mRNA levels in HUVECs (n = 3). * P < 0.05, *** P < 0.001.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against PI3K (CST, USA, 1:1000), p-PI3K (CST, USA, 1:1000), Akt (CST, USA, 1:1000), p-Akt (CST, USA, 1:1000), HIF-1α (Abcam, UK, 1:1000), VEGF (Proteintech, China, 1:1000), Bax (Proteintech, China, 1:1000), and Bcl-2 (Proteintech, China, 1:1500).

Techniques: Spheroids, Cell Culture, Immunofluorescence, Staining, Analysis, Quantitative RT-PCR

Journal: International Journal of Nanomedicine

Article Title: Levofloxacin-Catalase-PLGA Nanoparticles Potentiate Sonodynamic Therapy to Alleviate BCG-Induced Tuberculous Granulomas via PI3K/Akt/VEGF Pathway Suppression in Rats

doi: 10.2147/IJN.S628763

Figure Lengend Snippet: Effects of different treatments on vascular structure and function, hypoxic microenvironment, and related signaling molecules in tuberculous granulomas in vivo. ( A ) Vascular density and maturity (pericyte coverage) assessed by immunofluorescence co-localization of α-SMA (green) and CD31 (red); scale bar: 50 μm; ( B ) Vascular leakage assessed by co-staining of 70 kDa FITC-dextran (green) and CD31 (red); scale bar: 100 μm; ( C ) Vascular perfusion assessed by co-localization of FITC-lectin (green) and CD31 (red); scale bar: 25 μm; ( D ) Immunohistochemical staining of HIF-1α; scale bar: 40 μm; ( E ) Immunofluorescence detection of hypoxic regions using pimonidazole (PIMO);scale bar: 100 μm; ( F ) Analysis of oxygen saturation (sO 2 ) in granulomas by photoacoustic imaging. Left panel: Representative photoacoustic imaging of granulomas; Right panel: Quantitative comparison of sO 2 levels in granulomas between the Control and US + LEV@CAT-NPs groups. ( G ) Semi-quantitative analysis of PIMO + reas in ( E ). ( H ) Immunohistochemical staining images of VEGF and VEGFR2; scale bar: 40 μm. ***P<0.001 .

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against PI3K (CST, USA, 1:1000), p-PI3K (CST, USA, 1:1000), Akt (CST, USA, 1:1000), p-Akt (CST, USA, 1:1000), HIF-1α (Abcam, UK, 1:1000), VEGF (Proteintech, China, 1:1000), Bax (Proteintech, China, 1:1000), and Bcl-2 (Proteintech, China, 1:1500).

Techniques: In Vivo, Immunofluorescence, Staining, Contrast Agent Perfusion, Immunohistochemical staining, Analysis, Imaging, Comparison, Control