Journal: Cancer Biology & Therapy

Article Title: Fibroblast-associated TPM2 links cell-matrix remodeling to EMT-Notch signaling and gemcitabine resistance in intrahepatic cholangiocarcinoma

doi: 10.1080/15384047.2026.2725344

Figure Lengend Snippet: Functional validation of TPM2-associated gemcitabine response in patient-derived intrahepatic cholangiocarcinoma organoids. (A) Representative brightfield images of patient-derived intrahepatic cholangiocarcinoma organoids and hematoxylin and eosin (H&E) staining of organoids and matched primary tumor tissues from two cases. Scale bars, 100 μm. (B) Representative immunofluorescence staining showing AFP (red) and CK19 (green) in organoids (left panels), and ALB (red) in corresponding organoid samples (right panels). Nuclei are counterstained with DAPI (blue). Scale bars, 100 μm. (C) Dose-response curves showing gemcitabine sensitivity of two intrahepatic cholangiocarcinoma organoid models. Cell viability was assessed after treatment with serial concentrations of gemcitabine. Data are representative of three independent biological experiments. (D) Representative radiologic images obtained before treatment and after two cycles of hepatic arterial infusion chemotherapy (HAIC) with gemcitabine plus oxaliplatin (GEMOX) from the patients corresponding to ICCO1 and ICCO2 organoids. Yellow dashed outlines delineate the radiologically visible intrahepatic tumor lesions, and yellow arrows highlight the corresponding lesions to facilitate comparison of tumor changes before and after treatment. (E) Representative western blot validation of TPM2 knockout in the relatively gemcitabine-resistant ICCO2 organoid model using two independent sgRNAs targeting TPM2. β -Tubulin was used as a loading control. Data are representative of three independent biological experiments. (F) Representative brightfield images and corresponding Calcein-AM/PI fluorescence images of control and TPM2-knockout ICCO2 organoids following 5-day treatment with DMSO or 10 μM gemcitabine. Calcein-AM marks viable cells, whereas PI marks dead cells. Scale bars, 50 μm. (G) Quantification of Calcein-AM-positive and PI-positive areas in ICCO2 organoids following 5-day treatment with DMSO or 10 μM gemcitabine. At least 10 organoids per group were quantified in each independent biological experiment. Data are presented as mean ± SEM from three independent biological experiments. (H) Quantification of Calcein-AM-positive organoid area following 5-day treatment with DMSO or 10 μM gemcitabine. At least 20 organoids per group were quantified in each independent biological experiment. Data are presented as mean ± SEM from three independent biological experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Equal volumes of lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with antibodies against TPM2 (#A3096; ABclonal; 1:1000), Notch1 (#A19090; ABclonal; 1:1000), HES1 (#A0925; ABclonal; 1:1000) and β -Tubulin (#A12289; ABclonal; 1:5000).

Techniques: Functional Assay, Biomarker Discovery, Derivative Assay, Organoids, Brightfield, Staining, Immunofluorescence, Sensitivity, Comparison, Western Blot, Knock-Out, Control, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: Fibroblast-associated TPM2 links cell-matrix remodeling to EMT-Notch signaling and gemcitabine resistance in intrahepatic cholangiocarcinoma

doi: 10.1080/15384047.2026.2725344

Figure Lengend Snippet: TPM2 depletion suppresses EMT/ECM-related programs and Notch signaling in intrahepatic cholangiocarcinoma organoids. (A) Representative immunofluorescence staining of CK19, TPM2, and Vimentin in control and TPM2-knockout ICCO2 organoids. CK19 marks cholangiocytic epithelial tumor cells, TPM2 denotes the knockout target, and Vimentin serves as an EMT-associated marker. Nuclei were counterstained with DAPI. Scale bar, 20 μm. (B, C) Quantification of TPM2-positive area (B) and Vimentin-positive area (C) in control and TPM2-knockout ICCO2 organoids. At least 20 fields of view per group were quantified in each independent biological experiment. Positive areas were quantified within organoid regions using ImageJ under identical imaging and thresholding settings. Data are presented as mean ± SEM. *** P < 0.001. (D) RT-qPCR analysis of Notch pathway genes, EMT-associated markers, and ECM-related genes in control and TPM2-knockout ICCO2 organoids. Relative mRNA expression was normalized to GAPDH and is shown relative to the control group. Experiments were performed using three independent biological replicates, with three technical replicates per biological replicate. (E) Representative western blot analysis showing reduced Notch1 and HES1 expression after TPM2 knockout in ICCO2 organoids. β -Tubulin was used as a loading control. Data are representative of three independent biological experiments. (F) Proposed model illustrating the convergence of fibroblast-associated and tumor-cell-intrinsic TPM2 programs on cell-matrix/cytoskeletal remodeling, Notch-EMT signaling, and gemcitabine tolerance. Solid arrows indicate relationships directly supported by the experimental data in the corresponding context, whereas dashed arrows indicate inferred, indirect, or biologically plausible relationships that require further validation.

Article Snippet: Equal volumes of lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with antibodies against TPM2 (#A3096; ABclonal; 1:1000), Notch1 (#A19090; ABclonal; 1:1000), HES1 (#A0925; ABclonal; 1:1000) and β -Tubulin (#A12289; ABclonal; 1:5000).

Techniques: Organoids, Immunofluorescence, Staining, Control, Knock-Out, Marker, Imaging, Quantitative RT-PCR, Analysis, Expressing, Western Blot, Cytoskeletal, Biomarker Discovery

Journal: Bioactive Materials

Article Title: Melatonin-incorporated brain extracellular matrix hydrogel enhances NSCs mitochondrial metabolism to promote neuroregeneration via the AMPK-PGC-1α-NRF1/TFAM axis after spinal cord injury

doi: 10.1016/j.bioactmat.2026.04.006

Figure Lengend Snippet: Melatonin dose modulates NSCs lineage commitment, viability, oxidative stress, and mitochondrial membrane potential at day 5. (A) Representative immunofluorescence images of TUJ1 with Nestin in NSCs cultured within the cell-laden MT/BEM matrix and exposed to melatonin (0, 25, 50, 75, 100 μM) for 5 days (scale bar, 50 μm). (B) Representative images of GFAP with Nestin under the same conditions (scale bar, 50 μm). (C) Representative images of Olig2 with Nestin (scale bar, 50 μm). (D) Percentages of TUJ1(+), GFAP (+), and Olig2(+) cells relative to total nuclei (DAPI) (n = 10 fields/group). (E) RT-qPCR of TUJ1, GFAP, and Olig2 normalized to GAPDH and expressed as fold change versus control (ΔΔCt) (n = 6). (F) Live/Dead staining (Calcein AM/EthD-1) at day 5. (G) Western blots of TUJ1 and GFAP with GAPDH loading control. (H) Densitometry of TUJ1/GAPDH and GFAP/GAPDH (n = 3 independent experiments). (I) ROS staining by DCFH-DA with Rosup as the positive control. (J) Quantification of ROS fluorescence intensity (n = 10 fields/group, compared to control). (K) JC-1 staining of mitochondrial membrane potential (ΔΨm) with CCCP as the positive control for depolarization. (L) JC-1 red/green ratio (n = 10 fields/group, compared to control). Statistical analysis: Data are presented as mean ± SD. one-way ANOVA with Holm–Sidak's multiple comparisons for multi-group datasets (D, E, J, L); unpaired two-tailed t -test for the two-group comparison (H). Significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Following blocking, membranes were incubated overnight at 4 °C with the following primary antibodies: GFAP (1:1000; #80788, CST, USA), TUJ1 (1:1000; #YA586, MCE, China), GAPDH (1:1000; #2118, CST, USA), phospho-ACC (Ser79) (1:1000; #11818, CST, USA), Acetyl-CoA Carboxylase (C83B10) (1:1000; #3676, CST, USA), Total OXPHOS Rodent WB Antibody Cocktail (1:1000; #ab110413, Abcam, UK), phospho-AMPKα (Thr172) (1:1000; #2535, CST, USA), and total AMPKα (1:1000; #2532, CST, USA).

Techniques: Membrane, Immunofluorescence, Cell Culture, Quantitative RT-PCR, Control, Staining, Western Blot, Positive Control, Fluorescence, Two Tailed Test, Comparison

Journal: Bioactive Materials

Article Title: Melatonin-incorporated brain extracellular matrix hydrogel enhances NSCs mitochondrial metabolism to promote neuroregeneration via the AMPK-PGC-1α-NRF1/TFAM axis after spinal cord injury

doi: 10.1016/j.bioactmat.2026.04.006

Figure Lengend Snippet: Three-dimensional immunofluorescence and transcriptomic profiling of melatonin-treated NSCs. (A) Representative 3D confocal reconstructions of NSCs networks cultured for 5 days in BEM or MT/BEM hydrogels, immunostained for Nestin, TUJ1, GFAP and OLIG2 with DAPI nuclear counterstain. Scale bar: 50 μm. (B-C) Quantification of neurite outgrowth showing total neurite length (μm) (B) and neurite filament area (μm 2 ) (C) per field of view. (D) Quantification of astroglial differentiation expressed as GFAP + area (% of ROI). (E) Quantification of oligodendroglial lineage commitment expressed as OLIG2 + cells (% of DAPI + nuclei). Data are presented as mean ± SD. Statistical significance was assessed using an unpaired two-tailed t -test; ∗p < 0.05, ∗∗p < 0.01 versus NSCs@BEM.

Article Snippet: Following blocking, membranes were incubated overnight at 4 °C with the following primary antibodies: GFAP (1:1000; #80788, CST, USA), TUJ1 (1:1000; #YA586, MCE, China), GAPDH (1:1000; #2118, CST, USA), phospho-ACC (Ser79) (1:1000; #11818, CST, USA), Acetyl-CoA Carboxylase (C83B10) (1:1000; #3676, CST, USA), Total OXPHOS Rodent WB Antibody Cocktail (1:1000; #ab110413, Abcam, UK), phospho-AMPKα (Thr172) (1:1000; #2535, CST, USA), and total AMPKα (1:1000; #2532, CST, USA).

Techniques: Immunofluorescence, Cell Culture, Two Tailed Test

Journal: Bioactive Materials

Article Title: Melatonin-incorporated brain extracellular matrix hydrogel enhances NSCs mitochondrial metabolism to promote neuroregeneration via the AMPK-PGC-1α-NRF1/TFAM axis after spinal cord injury

doi: 10.1016/j.bioactmat.2026.04.006

Figure Lengend Snippet: Molecular validation of neural repair and mechanism activation in spinal cord tissue. Western blot and qPCR analyses of spinal cord tissue lysates from Sham, SCI, BEM, NSCs@BEM, and NSCs@MT/BEM groups. (A) Representative Western blots for the neuronal marker TUJ1 and the glial scar marker GFAP. (B) Representative Western blots for phosphorylated AMPK (p-AMPK), phosphorylated ACC (p-ACC), and their respective total proteins. (C) Representative Western blots for the five oxidative phosphorylation (OXPHOS) complex subunits. (D) Densitometric quantification of TUJ1 and GFAP protein levels. (E) Densitometric quantification of the p-AMPK/total AMPK and p-ACC/total ACC ratios. (F) Densitometric quantification of OXPHOS complex protein levels. (G) Relative mRNA expression of neural markers (TUJ1, GFAP, Olig2) and key mitochondrial biogenesis regulators (Ppargc1a, Tfam) determined by qPCR. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Holm–Sidak's multiple comparisons test. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Article Snippet: Following blocking, membranes were incubated overnight at 4 °C with the following primary antibodies: GFAP (1:1000; #80788, CST, USA), TUJ1 (1:1000; #YA586, MCE, China), GAPDH (1:1000; #2118, CST, USA), phospho-ACC (Ser79) (1:1000; #11818, CST, USA), Acetyl-CoA Carboxylase (C83B10) (1:1000; #3676, CST, USA), Total OXPHOS Rodent WB Antibody Cocktail (1:1000; #ab110413, Abcam, UK), phospho-AMPKα (Thr172) (1:1000; #2535, CST, USA), and total AMPKα (1:1000; #2532, CST, USA).

Techniques: Biomarker Discovery, Activation Assay, Western Blot, Marker, Phospho-proteomics, Expressing