Journal: Oncology Letters

Article Title: BLID is a drug-responsive target of FOXO3a and multi-omics analysis reveals survival mechanisms and therapeutic vulnerabilities in BLID-deficient breast cancer cells

doi: 10.3892/ol.2025.15155

Figure Lengend Snippet: RT-qPCR validation of changes in expression of a subset of genes following BLID knockdown. RT-qPCR analysis of several genes in (A) MCF-7 and (B) MDA-MB-231 cells. β-Actin served as the internal control. (A) The y-axis title of the middle graph is identical to the y-axis title of the left graph. (B) The y-axis title of the middle-left graph is identical to the y-axis title of the far-left graph. *P<0.05 and **P<0.01 (BLID shRNA vs. the corresponding Scr shRNA group), n=3. shRNA, short hairpin RNA; Scr, scramble control; Ctl, control; RT-qPCR, reverse transcription-quantitative PCR; BLID, BH-3 like motif containing inducer of cell death.

Article Snippet: In addition, Stealth siRNA negative control (scramble) with the same supplier's proprietary sequence designed to minimize sequence homology to any known vertebrate transcript (cat. no. 12935300, Thermo Fisher Scientific, Inc; Invitrogen) was used.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing, Knockdown, Control, shRNA, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: CD44 silencing results in reduced sTN58 binding. (A, E) Cis-Pt-R (A) and BT-549 (E) cells were left untreated or transfected with si-CD44 or siRNA ctrl. At 48 h post-transfection, cells were harvested, and cell lysates prepared and immunoblotted with CD44 Ab. Equal loading was confirmed by immunoblot with anti-α-tubulin antibody. Molecular weights of protein markers are reported. (B, F) The histogram depicts the densitometric ratio of CD44 expression to α-tubulin. Values are shown relative to the untreated control, arbitrarily set to 1. ∗ P < 0.05, ∗∗ P < 0.01 relative to siRNA ctrl. (C, G) Binding of CD44-PE Ab ( left ) and Alexa 647-sTN58 ( right ) to Cis-Pt-R (C) and BT-549 (G) cells following 48 h transfection with si-CD44 (green) and siRNA ctrl (gray). (D, H) The histogram shows gMFI of si-CD44-transfected cells treated with sTN58 aptamer or CD44 Ab, normalized to the gMFI of untreated cells, and expressed as percentage with respect to siRNA ctrl-transfected cells. ∗∗∗∗ P < 0.0001 relative to siRNA ctrl. (I) Immunoblot analysis of CD44 and the housekeeping protein α-tubulin. The molecular weights of protein markers are reported. (J) The histogram shows the relative fold-change in CD44 expression levels compared to α-tubulin, normalized to MDA-MB-231 target cells, whose expression level is arbitrarily set to 1. ∗ P < 0.05, ∗∗∗ P < 0.001 relative to MDA-MB-231. (K) Flow cytometry analyses of Cis-Pt-R, MCF 10A, THP-1 and HS-5 cells treated with Alexa 647-sTN58. (L) Quantification of the gMFI of Alexa 647-sTN58-treated cells normalized to the gMFI of the untreated cells. ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 relative to untreated cells; ns, no significant. In B, D, F, H, J, L, bars depict mean ± SD of at least two independent experiments.

Article Snippet: Scrambled non-targeting siRNA (siRNA ctrl, Qiagen) was used as a negative control.

Techniques: Binding Assay, Transfection, Western Blot, Expressing, Control, Flow Cytometry

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: sTN58 aptamer and CD44 Ab colocalize with integrin β1 Ab on Cis-Pt-R cells. (A) Following 5 min incubation at RT with 2 μM Alexa 647-sTN58, Cis-Pt-R cells were stained with integrin β1 Ab, visualized by confocal microscopy, and photographed. (B) Cell lysates from Cis-Pt-R cells left untreated or treated for 48 h with 100 nM siRNA ctrl or si-ITGB1 were analyzed by immunoblotting with integrin β1, CD44 and anti-α-tubulin antibodies. Molecular weights of protein markers are reported. (C) The histogram shows the protein expression/α-tubulin ratio based on the densitometric signals. Values are shown relative to untreated samples, arbitrarily set to 1. (D) Binding of integrin β1-APC-Cy7 Ab ( left ) and Alexa 647-sTN58 ( right ) to Cis-Pt-R cells following 48 h transfection with siRNA ctrl (gray) and si-ITGB1 (pink). (E) The histogram shows gMFI of si-ITGB1-transfected cells treated with Alexa 647-sTN58 or integrin β1-APC-Cy7 Ab, normalized to the gMFI of untreated cells, and expressed as percentage with respect to siRNA ctrl-transfected cells. Bars depict mean ± SD of two independent experiments. ∗∗∗ P < 0.001; ns, no significant. (F) Confocal microscopy analyses of A431 cells treated with sTN58 and stained with integrin β1 Ab, as in A, or stained with CD44-PE and integrin β1 antibodies. Alexa 647-SCR was used as a negative control. In A, F, aptamers and CD44-PE Ab are visualized in red, integrin β1 Ab in green and nuclei in blue. All digital images were captured at the same setting to allow direct comparison of staining patterns. Magnification 63×, 1.0× digital zoom, scale bar = 10 μm. Co-localization results appear yellow in the merged images. Arrowheads indicate some co-localization points between sTN58 and integrin β1 Ab (Overlap Coefficient, 0.80). (G) Flow cytometry analyses of A431 cells treated with CD44-PE Ab, Alexa 647-sTN58 and integrin β1-APC-Cy7 Ab. (H) Quantification of the gMFI of sTN58-, CD44 PE- and integrin β1-APC-Cy7-treated cells normalized to the gMFI of the untreated cells. Bars depict mean ± SD of three independent experiments. ∗∗∗∗ P < 0.0001 relative to untreated cells; ns, no significant.

Article Snippet: Scrambled non-targeting siRNA (siRNA ctrl, Qiagen) was used as a negative control.

Techniques: Incubation, Staining, Confocal Microscopy, Western Blot, Expressing, Binding Assay, Transfection, Negative Control, Comparison, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Strengthening monocarboxylate transporters by adiponectin receptor agonist ameliorates diabetic peripheral neuropathy

doi: 10.1186/s12964-025-02326-5

Figure Lengend Snippet: AdipoRon modulates AdipoR1 and AdipoR2 expression in human Schwann cells (HSCs). Human Schwann cells (HSCs) were transfected with adipoR1 or adipoR2 siRNA and cultured in high glucose and palmitate medium, with or without AdipoRon treatment. ( A ) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin B. Quantitative analyses of protein expression levels are shown ( n = 3). ( B ) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with MBP (red, myelin marker) are shown with corresponding quantitative analyses ( n = 6). ( C ) Representative western blot ( n = 3) and IF images ( n = 6) for LDHA and LDHB expression are shown with quantitative analyses. ( D ) Quantitative measurements of intracellular lactate, AMP, and ATP levels, including the ATP/AMP ratio, are shown ( n = 5). ( E ) Representative double immunofluorescence staining images of MCT4 (green) and MitoSOX (red) in HSCs under different conditions ( n = 6). Data are presented as the mean ± SD. * P < 0.05, ** P < 0.01, and # P < 0.001, compared with other groups. Scale bars in (B, C and E) represent 20 μm

Article Snippet: The cells were then transfected with 50 nM of target-specific siRNA ( AdipoR1 siRNA: 5’-GGACAACGACUAUCUGCUACATT-3’, AdipoR2 siRNA: 5’-CCAACUGGAUGGUACACGA-3’) or scrambled siRNA (5’-CCUACGCCACCAAUUUCGU-3’, Bioneer, Daejeon, Korea) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) with Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, Cell Culture, Western Blot, Staining, Marker, Double Immunofluorescence Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Strengthening monocarboxylate transporters by adiponectin receptor agonist ameliorates diabetic peripheral neuropathy

doi: 10.1186/s12964-025-02326-5

Figure Lengend Snippet: Effect of AdipoRon on signaling pathways in ND7/23 cells (murine DRG neuronal cell). ND7/23 cells were transfected with adipoR1 or adipoR2 siRNA and cultured in low- or high-glucose (HG) and palmitate (PA) medium, with or without AdipoRon treatment. ( A ) Representative western blot images showing the expression of AdipoR1, AdipoR2, CaMKKα and β, pLKB1/total LKB1, pAMPK/total AMPK, p-mTOR/total mTOR, PGC-1α, PPARα, Bax/Bcl-2, LC3II/LC3I ratios, MCT1, MCT2, MCT4, GAPDH, cytoplasmic Nrf2, nuclear Nrf2, and lamin ( B ). Quantitative analyses of protein expression levels in each group are shown ( n = 3). (B) Representative images of double IF staining for MCT1, MCT2, and MCT4 (green) with NF (white, axonal marker) are shown with corresponding quantitative analyses ( n = 6). ( C ) Representative western blot ( n = 3) and double IF images ( n = 6) for LDHA (red) and LDHB (green) expression, with quantitative analyses. ( D ) Quantitative measurements of intracellular lactate, AMP, and ATP levels, and ATP/AMP ratio ( n = 5). ( E ) Representative sections stained with Fluo-4AM (green, intracellular Ca ++ ), DHE (red, cellular oxidative stress), MitoSox (red, mitochondrial oxidative stress), TUNEL (green, apoptosis), and LC3 (green, autophagy). Quantitative analyses of positive areas and density are shown for each group. Data are presented as the mean ± SD ( n = 6). ( F ) Representative double IF staining images of MCT1, MCT2, MCT4 (green) and MitoSOX (red) in ND7/23 cells under different conditions ( n = 6). (G) Quantitative measurements of intracellular lactate, ATP and AMP concentrations and ATP/AMP ratios in SCs and ND7/23 cells in low glucose (LG), high glucose and palmitate (HG + PA), and high glucose and palmitate treated with adiopoRon (HG + PA + adipoR), respectively ( n = 5). * P < 0.05, ** P < 0.01, and # P < 0.001, compared with other groups. Scale bars in (B, C, E and F) represent 5 μm

Article Snippet: The cells were then transfected with 50 nM of target-specific siRNA ( AdipoR1 siRNA: 5’-GGACAACGACUAUCUGCUACATT-3’, AdipoR2 siRNA: 5’-CCAACUGGAUGGUACACGA-3’) or scrambled siRNA (5’-CCUACGCCACCAAUUUCGU-3’, Bioneer, Daejeon, Korea) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) with Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions.

Techniques: Protein-Protein interactions, Transfection, Cell Culture, Western Blot, Expressing, Staining, Marker, TUNEL Assay