Journal: Clinical and experimental medicine

Article Title: AHNAK2 confers 5-fluorouracil resistance in colorectal cancer via activation of the AKT/GSK-3β signaling axis.

doi: 10.1007/s10238-025-01682-3

Figure Lengend Snippet: Fig. 4 AHNAK2 promoted the migration and invasion of 5-FU-resistant CRC cells. LoVo/5-FU and HCT116/5- FU cells were transfected with shAHNAK2 and LvAHNAK2 for 48 h, respectively. A–B Cell migration was determined in transfected cells by wound heal- ing assay. C–D Transwell assay was performed to assess cell migration and invasion in trans- fected cells above. Each value represents the mean ± SD from three independent experiments. ***p < 0.001, compared with shNC; ##p < 0.01,###p < 0.001, compared with Lv-NC

Article Snippet: Two human CRC cell lines, LoVo (CCL-229) and HCT116 (CRL-3487) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), along with normal colonic epithelial NCM460 cells from Bowers Type Culture Collection (NCM460, BTCC, Beijing, China).

Techniques: Migration, Transfection, Transwell Assay

Journal: Clinical and experimental medicine

Article Title: AHNAK2 confers 5-fluorouracil resistance in colorectal cancer via activation of the AKT/GSK-3β signaling axis.

doi: 10.1007/s10238-025-01682-3

Figure Lengend Snippet: Fig. 5 Mechanistic role of AHNAK2 in 5-FU resistance via AKT/GSK-3β signaling. LoVo/5-FU and HCT116/5- FU cells were transfected with shAHNAK2 and LvAHNAK2 for 48 h, respectively. The protein levels of PCNA, CDK4, cleaved caspase-3, and E-cadherin were determined in transfected LoVo/5-FU (A) and HCT116/5-FU (B) cells using western blot analysis. The protein levels of p-AKT, AKT, p-GSK-3β and GSK-3β were determined in transfected LoVo/5-FU (C) and HCT116/5- FU (D) cells using western blot analysis

Article Snippet: Two human CRC cell lines, LoVo (CCL-229) and HCT116 (CRL-3487) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), along with normal colonic epithelial NCM460 cells from Bowers Type Culture Collection (NCM460, BTCC, Beijing, China).

Techniques: Transfection, Western Blot

Journal: Bioscience Reports

Article Title: Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

doi: 10.1042/bsr20120085

Figure Lengend Snippet: Figure 1 Baf increases respiration in HCT116 cells through mitochondrial uncoupling (A) The O2 profiles in WT and SCO2 −/ −HCT116 cells treated with 0.25 μM Baf at 6 % hypoxia. Cells were loaded with an O2 sensing probe NanO2 and iO2 levels were monitored for 5 h. In control samples respiration was blocked with AA (10 μM). (B) Treatment with Baf partially depolarizes the mitochondrial membrane potential (m) in WT cells, as monitored by TMRM staining (20 nM in solution). (C) In SCO2 −/ −cells the m is reduced, as compared with WT. (D) A reduction in the m in SCO2 −/ −cells is not associated with a decrease in the mitochondrial size, as follows from the comparative analysis of MTG staining and VDAC1 protein levels. Bar represents 20 μm. Asterisks indicate statistical significance.

Article Snippet: Briefly, HCT116 cells were seeded on Ibidi chambers, grown and treated as described above, washed with PBS, fixed with 3.7 % PFA (paraformaldehyde) in PBS, quenched with 50 mM NH4Cl, permeabilized with 0.25 % TX100, blocked with 5 % BSA, incubated with anti-HIF-1α (Novus Biologicals) primary and fluorescent anti-mouse secondary antibodies conjugated with Alexa Fluor555 dye (Invitrogen), counterstained with DAPI, mounted with ProLong® Gold and air-dried.

Techniques: Control, Membrane, Staining

Journal: Bioscience Reports

Article Title: Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

doi: 10.1042/bsr20120085

Figure Lengend Snippet: Figure 2 Stabilization of HIF-1α and HIF-2α in HCT116 cells upon Baf treatment (0.25 μM) is associated with mitochon- drial uncoupling (A) Western blot analysis demonstrates significant elevation in HIF-1α and HIF-2α levels in HCT116 WT cells treated with Baf for 5 h at 6 % O2. This effect is abolished by inhibiting mitochondrial respiration with AA. In the presence of DMOG, the level of HIF-1α and HIF-2α stabilization is similar in all samples, including those which are treated with Baf/AA. In the presence of CMA, HIF-1α and HIF-2α levels in WT cells are also increased significantly, yet to a lesser degree than upon Baf treatment. (B) Cellular ATP levels are reduced in WT cells treated with Baf/AA for 5 h at 6 % O2, regardless the presence of DMOG. Treatment with Baf in galactose ( + ) medium causes dramatic decrease in the ATP level. (C) The levels of PHD1, PHD3, VHL and ATP6V0C proteins are similar in WT and SCO2 −/ −cells, while the content of PHD2 in SCO2 −/ −cells is significantly lower than in WT control. Asterisks indicate statistically significant difference (∗P < 0.01 and ∗∗P < 0.001, Student’s t test) between the experimental samples and mock control (DMSO). Hash in (A) shows a significant difference between the effects of Baf and CMA.

Article Snippet: Briefly, HCT116 cells were seeded on Ibidi chambers, grown and treated as described above, washed with PBS, fixed with 3.7 % PFA (paraformaldehyde) in PBS, quenched with 50 mM NH4Cl, permeabilized with 0.25 % TX100, blocked with 5 % BSA, incubated with anti-HIF-1α (Novus Biologicals) primary and fluorescent anti-mouse secondary antibodies conjugated with Alexa Fluor555 dye (Invitrogen), counterstained with DAPI, mounted with ProLong® Gold and air-dried.

Techniques: Western Blot, Control

Journal: Bioscience Reports

Article Title: Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

doi: 10.1042/bsr20120085

Figure Lengend Snippet: Figure 3 Nuclear localization and functional activity of HIF-1α in the HCT116 cells treated with Baf at 6 % O2 (A, B) Immunofluorescence analysis shows significant increase in nuclear HIF-1α staining in WT cells treated with Baf for 5 h. No effect of Baf or CMA on the nuclear HIF-1α accumulation is observed in SCO2 −/ −cells. Treatment with DMOG causes translocation of HIF-1 into the nuclei of both WT and SCO2 −/ −in an O2-independent manner. Nuclei are counterstained with DAPI. (C) Quantitative analysis of the data shown in (A, B) is performed by measuring HIF-1-specific fluorescence in the DAPI-positive regions. (D) DNA-binding analysis using TransAM® HIF-1 kit demonstrates an increase in HIF-1-HRE interaction in WT cells upon Baf treatment. (E) Treatment with Baf activates ‘hypoxia-specific’ gene expression in WT cells, as demonstrated using RT–PCR analysis. Data are presented as the ratio between mRNA levels in treated and non-treated cells. (F) Western blot analysis shows that PHD2 protein level is increased in WT cells treated with Baf and DMOG; however no increase in PHD2 levels in SCO2 −/ −cells is observed. Asterisks indicate statistically significant difference between the experimental samples and mock control (DMSO).

Article Snippet: Briefly, HCT116 cells were seeded on Ibidi chambers, grown and treated as described above, washed with PBS, fixed with 3.7 % PFA (paraformaldehyde) in PBS, quenched with 50 mM NH4Cl, permeabilized with 0.25 % TX100, blocked with 5 % BSA, incubated with anti-HIF-1α (Novus Biologicals) primary and fluorescent anti-mouse secondary antibodies conjugated with Alexa Fluor555 dye (Invitrogen), counterstained with DAPI, mounted with ProLong® Gold and air-dried.

Techniques: Functional Assay, Activity Assay, Staining, Translocation Assay, Binding Assay, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control