Journal: bioRxiv

Article Title: Hyperexcitability in Alzheimer’s Disease triggers a compensatory neuroprotective response via TREK1

doi: 10.1101/2025.10.16.682816

Figure Lengend Snippet: a, Representative immunofluorescence images showing increased VGLUT1 intensity in the hippocampus of 3xTg mice 15 days after intrahippocampal injection with TREK1 shRNA lentivirus compared with vehicle-injected controls. b, Representative images showing decreased VGAT intensity under the same TREK1 knockdown conditions. c, Quantification of VGLUT1 fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 67–76 sections; ****p < 0.0001; unpaired t-test). d, Quantification of VGAT fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 60–63 sections; ****p < 0.0001; unpaired t-test). e, Quantification of Excitatory/inhibitory (E/I) ratio, calculated as VGLUT1/VGAT intensity, is markedly elevated in TREK1 knockdown mice compared with vehicle injected controls (n = 3– 4; **p < 0.01; unpaired t-test). f, Representative immunofluorescence images demonstrating enhanced Aβ deposition in the hippocampus of TREK1 knockdown 3xTg mice compared with vehicle-injected controls. g, Quantification of Aβ fluorescence intensity in 3xTg mice after TREK1 knockdown (n = 50 sections; *p < 0.05; unpaired t-test). h, Quantification showing a significant decrease in MAP2 intensity in the hippocampus of 3xTg mice injected with TREK1 shRNA compared with scrambled (Sc) shRNA controls (n = 159–166 sections; ****p < 0.0001; unpaired t-test). i, Schematic representation illustrating the effects of TREK1 knockdown on excitatory/inhibitory balance. Data are expressed as mean ± SEM from 3–4 mice per group.

Article Snippet: Primary antibodies- chicken MAP2 (1:1000, Invitrogen, PA1-10005), rabbit TREK1 (1:100, Alomone Labs, #APC-047), Rabbit CTCF (1:100, Invitrogen, #MA5-88115), Mouse AC1 (1:50, Santa Cruz, #SC- 365350), Mouse AC8 (1:50, Santa Cruz, #SC-377442) and rabbit MAP2 (1:150, Cell Signalling Technology, #8707S) were diluted in PBST and applied overnight at 4°C.

Techniques: Immunofluorescence, Injection, shRNA, Knockdown, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Dietary Lauric Acid Suppresses Inflammation, Cholestasis, Hepatocyte Injury, and Senescence in 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine-induced Inflammatory Cholangiopathy

doi: 10.1016/j.jcmgh.2026.101731

Figure Lengend Snippet: Effect of lauric acid on expression of inflammation, senescence, and oxidative stress genes and biomarkers in hepatocytes isolated from experimental mouse groups. qRT-PCR-mRNA analysis in hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( A ) Genes involved in inflammation and oxidative stress. ( B ) Senescence genes. ( C ) Mitochondrial fatty acid oxidation and oxidative stress genes. ( D ) β-gal staining ( yellow arrow ) as a senescence marker in overnight cultured primary hepatocytes isolated from chow, DDC, LA, and DDC/ LA mice. Bar graph showing the number of senescent (β-gal + ) cells per microscopic field in liver sections from chow, DDC, DDC/LA, and LA groups. ( E ) Immunoblotting and quantification of β-gal in fresh hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( F ) Serum hydrogen peroxide levels and CCL2 concentrations (ELISA), and lipid peroxides, nitrite, and malondialdehyde levels from isolated hepatocytes. ( G–H ) Co-culture of human cholangiocytes H69 ( upper wells ) and Huh7 cells was conducted. H69 cells were incubated with and without DDC overnight in the presence or absence of DLPC. ( G ) mRNA expression by qPCR was measured in Huh7 cells ( bottom wells ) for CCL2, CDKN1A, NR0B2, and ABCB11 and ( H ) in H69 cells for CCL2, CDKN1A, KRT19, CTGF, and TNF . ( I ) Primary mouse hepatocytes were incubated with CCL2 in the presence or absence of DLPC overnight, and mRNA expression by qPCR was analyzed for senescence genes Cdkn1a and Cdkn1b . ( J ) β-gal staining ( yellow arrow = senescent cells) of cultured Huh7 cells incubated with CCL2 overnight in the presence or absence of DLPC. Bar graph showing the number of senescent (β-gal + ) Huh7 cells after treatment with CCL2 alone or in combination with DLPC. CCL2 treatment markedly increased cellular senescence, which was inhibited by DLPC. ( K ) Confocal fluorescence microscopy of primary cultured hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P16 ( red ), and nuclear DAPI ( blue ) from showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. ( L ) Confocal fluorescence microscopy of primary mouse hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P21/WAF/CIP ( red ) and nuclear DAPI ( blue ) also showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. For ( G–I ), data points represent replicates in 3 independent experiments. For ( D and J ), photomicrographs are shown that are representative of 3 separate experiments. For ( K and L ), immunofluorescent images are shown that are representative of 3 separate experiments. ( M ) Western analysis of pSTAT1 protein, total STAT1, and actin expression in hepatocytes isolated from the mouse groups, including quantification of integrated density values (IDVs). ( N ) ChIP assay of hepatocytes isolated from mouse groups for STAT1 binding to the promoter region of Cdkn1b using a STAT1-specific antibody. ( O ) Western analysis of pSTAT1 protein expression in cultured primary mouse hepatocytes exposed to CCL2 overnight in the presence or absence of DLPC, including quantification of IDVs of immunoblots. Statistical analysis was performed by 1-way ANOVA with Tukey’s correction for multiple comparisons. a P < .05 vs all other groups; b P < .05 vs DDC; b P < .05 vs CCL2.

Article Snippet: Phospho-STAT1 , 9167 , 1:1000 , Cell Signaling.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Staining, Marker, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Incubation, Fluorescence, Microscopy, Binding Assay

Journal: bioRxiv

Article Title: Aurora A kinase activation contributes to the fibrotic phenotype in Systemic Sclerosis through primary cilia shortening

doi: 10.64898/2026.03.13.711548

Figure Lengend Snippet: (A) Representative immunofluorescence images of primary cilia in healthy control (HC), systemic sclerosis (SSc), and VEDOSS (very early SSc) dermal fibroblasts under basal conditions (CTR) or following 24 h TGFβ stimulation. Acetylated α-tubulin marks the axoneme (red) and nuclei are stained with DAPI (blue). (B) Quantification of primary cilium length in HC, SSc, and VEDOSS fibroblasts under basal conditions. n=3 donors per group, a minimum of 100 cilia were quantified per sample. (C) Time course of TGFβ-induced cilia shortening in HC and SSc fibroblasts. n=3 donors per group, a minimum of 100 cilia were quantified per sample. (D) Quantification of cilium length in HC and SSc fibroblasts subjected to a 24 h TGFβ pulse followed by washout and recovery in 0.5% serum for 24 and 48 h. n=3 experimental repeats. (E) Quantification of cilium length in HC and SSc fibroblasts after sequential serum starvation, TGFβ exposure, and two passages in 10% serum. n=3 experimental repeats. (F) Cilium length in HC and SSc fibroblasts treated with the TGFβ receptor inhibitor SD208 in the presence or absence of exogenous TGFβ. n=3 experimental repeats. (G) Representative western blot of pSMAD3, total SMAD3, and βactin in HC and SSc fibroblasts treated with TGFβ and/or SD208. n=3 donors per group. (H) Quantification of cilium length in HC and SSc fibroblasts cultured in full growth medium in the presence of DMSO (CTR) or SD208 for 2 passages. n=3 experimental repeats. All data panels were analysed by ANOVA (ns, non-significant; ** P<0.01; *** P<0.001; ****P<0.0001).

Article Snippet: Proteins were transferred onto Hybond nitrocellulose membranes (Amersham) and probed with antibodies specific for αSMA (Abcam ab7817), β-Actin (Sigma A5441), CAV1 (Santa Cruz sc894), p53 (Santa Cruz sc126), acetylated-α-Tubulin (Cell Signaling 5335), MLC2 (Cell Signaling 8505), pMLC2 (Ser19) (Cell Signaling 3674), ppMLC2 (Ser19, Thr18) (Cell Signaling 3674), SMAD3 (Cell Signalling 9523), pSMAD3 (Abcam ab52903).

Techniques: Immunofluorescence, Control, Staining, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Aurora A kinase activation contributes to the fibrotic phenotype in Systemic Sclerosis through primary cilia shortening

doi: 10.64898/2026.03.13.711548

Figure Lengend Snippet: (A) Top: representative western blot showing the effect of SMAD3 siRNA (siSMAD3) on total SMAD3 protein level and on TGFβ-induced SMAD3 phosphorylation in healthy control (HC) and systemic sclerosis (SSc) fibroblasts, compared to scrambled siRNA (siSCR). Bottom: Densitometric quantification of SMAD3 in control (siSCR) and siSMAD3-treated HC and SSc fibroblasts (n=3 individual donors per group). (B) Quantification of cilium length in HC and SSc fibroblasts treated with siSCR or siSMAD3 and stimulated for 24 h with TGFβ. n=3 individual donors per group. (C) Top: representative western blots showing pMLC2, ppMLC2, and total MLC2, in response to treatments TGFβ with or without KD025 in three HC donor and three SSc donor fibroblast lines. Bottom: Densitometric quantification of pMLC2 normalised to MLC2 in the same condition. (D) Cilium length in HC and SSc fibroblasts treated with the ROCK2 inhibitor KD205 ± TGFβ. Mean cilia length was measured in three HC donor and three SSc donor fibroblast lines treated as indicated. All data panels were analysed by ANOVA (ns, non-significant; * P<0.05; ****P<0.0001).

Article Snippet: Proteins were transferred onto Hybond nitrocellulose membranes (Amersham) and probed with antibodies specific for αSMA (Abcam ab7817), β-Actin (Sigma A5441), CAV1 (Santa Cruz sc894), p53 (Santa Cruz sc126), acetylated-α-Tubulin (Cell Signaling 5335), MLC2 (Cell Signaling 8505), pMLC2 (Ser19) (Cell Signaling 3674), ppMLC2 (Ser19, Thr18) (Cell Signaling 3674), SMAD3 (Cell Signalling 9523), pSMAD3 (Abcam ab52903).

Techniques: Western Blot, Phospho-proteomics, Control

Journal: Cell Death & Disease

Article Title: Androgen receptor-dependent DRAM1 activation drives autophagic resistance to BRAF inhibitors in BRAFV600-mutant melanoma

doi: 10.1038/s41419-026-08547-x

Figure Lengend Snippet: A AR, LC3II and p62 protein level in A375 and WM793 cells with or without AR overexpression. B TEM analysis of autophagosomes and autolysosomes in A375 EV and A375 AR-OE cells (scale bars: 0.5 μm). A autophagosomes, AL autolysosomes, M: mitochondria and N nucleus. Manually counted. Mean ± SEM, n = 30, two-tailed Student’s t test, ** p < 0.01. C A375 EV and A375 AR-OE cells stably expressing TF-LC3 were imaged using fluorescence microscopy (Scale bars: 10 μm). White boxed regions in the panels are enlarged. Representative images of autolysosomes (red dots) and autophagosomes (yellow dots) are shown. Manually counted. n = 30, two tailed Student’s t test, ** p < 0.01, *** p < 0.001. Following treatment with DAB (0.5 μM) and/or 3-MA (2.5 mM), A375 EV and A375 AR OE cells were analyzed for D protein expression, E cell proliferation and F colony formation assays as indicated. WM793R cells transfected with si-AR or si-scramble were treated with DAB (0.5 μM) and/or Rapa (5 nM), G protein expression, H cell proliferation and I colony formation assays were measured as indicated. Mean ± SEM, n = 3, one-way ANOVA and Tukey’s post-hoc test. ** p < 0.01. For E , H EV(DAB) vs AR OE(DAB) ** p = 0.0018, AR OE(DAB) vs AR OE(DAB + 3MA) ** p = 0.0052, si-scramble (DAB) vs si-AR(DAB) ** p = 0.0037, si-AR(DAB) vs si-AR(DAB+Rapa) ** p = 0.0014.

Article Snippet: Antibodies were used as follows: AR (#5153), β-actin (#4970), Lamin B (#17416), LC3 (#12741) and p62 (#23214) were brought from Cell Signaling Technology.

Techniques: Over Expression, Two Tailed Test, Stable Transfection, Expressing, Fluorescence, Microscopy, Transfection

Journal: Cell Death & Disease

Article Title: Androgen receptor-dependent DRAM1 activation drives autophagic resistance to BRAF inhibitors in BRAFV600-mutant melanoma

doi: 10.1038/s41419-026-08547-x

Figure Lengend Snippet: DRAM1, LC3 and p62 protein expression were measured in A A375 and WM793 cells transfected with Flag-DRAM1, B A375R and WM793R cells transfected with si-DRAM1 or si-scramble C A375 and WM793 AR-OE cells transfected with si-DRAM1. D A375 cells expressing TF-LC3, AR-OE-TF-LC3 and AR-OE-TF-LC3 cells transfected with si-DRAM1 were imaged by fluorescence microscopy (Scale bars: 10 μm). White boxed regions in the panels are enlarged. Representative images of autolysosomes (red dots) and autophagosomes (yellow dots) are shown. Manually counted. Mean ± SEM, n = 30, two tailed Student’s t test, ** p < 0.01, *** p < 0.001. E , F cell proliferation and colony formation assays were measured in cells from ( D ) treated with DAB as indicated. Mean ± SEM, n = 3, one-way ANOVA and Tukey’s post-hoc test. ** p < 0.01. G , H WM793R cells transfected with/without si-DRAM1 were incubated with DAB (0.5 μM), and cell proliferation and colony formation assays were measured. Mean ± SEM, n = 3, two-tailed Student’s t test, ** p < 0.01. E EV vs AR-OE ** p = 0.0017, AR-OE + si-scramble vs AR-OE+si-DRAM1 ** p = 0.0032, G DAB + si-scramble vs DAB + si-DRAM1 ** p = 0.0047.

Article Snippet: Antibodies were used as follows: AR (#5153), β-actin (#4970), Lamin B (#17416), LC3 (#12741) and p62 (#23214) were brought from Cell Signaling Technology.

Techniques: Expressing, Transfection, Fluorescence, Microscopy, Two Tailed Test, Incubation

Journal: Acta Pharmaceutica Sinica. B

Article Title: A cisplatin prodrug-based self-assembling ozone delivery nanosystem sensitizes radiotherapy in triple-negative breast cancer

doi: 10.1016/j.apsb.2025.03.020

Figure Lengend Snippet: Biodistribution and cytotoxicity of PtF and PFD@PtF in vitro . (A) Subcellular biodistribution of fluorescein isothiocyanate (FITC) labeled PtF nanoparticles in SUM149 and 4T1 cells showed by confocal laser scanning microscopy images. Blue, 4′,6-diamidino-2-phenylindole (DAPI); Red, wheat germ agglutinin (WGA) 647; Green, FITC-labeled PtF. Scale bar = 50 μm. (B) The cellular internalization of NileRed-labeled PtF in SUM149 and 4T1 cells is detected by flow cytometry. (C) Co-localization of NileRed-labeled PtF nanoparticles with clathrin, caveolin-1 (CAV-1), and lysosomal associated membrane protein 1 (LAMP1) in SUM149 and 4T1 cells presented by confocal laser scanning microscopy images. Blue, DAPI; Red, NileRed-labeled PtF; Green, clathrin, caveolin-1, and LAMP1. Scale bar = 20 μm. (D–H) MTT survival assays of SUM149, 4T1, MCF-10A, EG7, and Jurkat cells after treatment with indicated regimens. Irradiation dose, 20 Gy. Data are presented as mean ± SD ( n = 6). (D) Two-way ANOVA; PtF vs. Cisplatin ( P = 1.175 × 10 −10 ); R + PtF vs. R + cisplatin ( P = 4.118 × 10 −6 ). (E) Two-way ANOVA; PtF vs. Cisplatin ( P = 5.1 × 10 −14 ); R + PtF vs. R + cisplatin ( P = 5 × 10 −14 ). (F) Two-way ANOVA; PtF vs. Cisplatin ( P = 3.747 × 10 −11 ); R + PtF vs. R + cisplatin ( P = 7.779 × 10 −11 ). (G) Two-way ANOVA; PtF vs. Cisplatin ( P = 1.8 × 10 −14 ). (H) Two-way ANOVA; PtF vs. Cisplatin ( P = 4.982 × 10 −4 ). (I–M) The IC 50 of indicated regimens against SUM149, 4T1, MCF-10A, EG7, and Jurkat cells. Data are presented as mean ± SD ( n = 3). (I) Two-way ANOVA; PtF vs. Cisplatin ( P = 4.585 × 10 −7 ); R + PtF vs. R + cisplatin ( P = 2.086 × 10 −6 ). (J) Two-way ANOVA; PtF vs. Cisplatin ( P = 6.17 × 10 −5 ); R + PtF vs. R + cisplatin ( P = 1.546 × 10 −5 ). (K) Two-way ANOVA; PtF vs. Cisplatin ( P = 7.514 × 10 −5 ); R + PtF vs. R + cisplatin ( P = 1.196 × 10 −4 ). (L) Two-way ANOVA; PtF vs. Cisplatin ( P = 1.646 × 10 −5 ); R + PtF vs. R + cisplatin ( P = 4.547 × 10 −3 ). (M) Two-way ANOVA; PtF vs. Cisplatin ( P = 1.03 × 10 −3 ). (N) The tumor-selective index (SI) of indicated regimens recorded by the ratio of the IC 50 against epithelial cells or immune cells to the IC 50 against TNBC cells. Data are presented as mean ± SD ( n = 3).

Article Snippet: Subsequently, the cell slides were treated with primary antibodies targeting clathrin (Cell Signaling Technology, #4796S, Danvers, MA, USA), caveolin-1 (Cell Signaling Technology, #3267S), and LAMP1 (lysosome tracker, Abcam, ab208943, Cambridge, UK, mouse use; Cell Signaling Technology, #9091T, human use) at 4 °C overnight.

Techniques: In Vitro, Labeling, Confocal Laser Scanning Microscopy, Flow Cytometry, Membrane, Irradiation