Journal: Life Science Alliance

Article Title: The nucleocytoplasmic translocation of HINT1 regulates the maturation of cell density

doi: 10.26508/lsa.202503215

Figure Lengend Snippet: (A) Endogenously expressed HINT1 in HeLa cells localizes to the nucleus at low cell density, and to both the nucleus and cytoplasm at high density. Notably, nuclear localization of HINT1 at high density is more prominent in HeLa cells compared with HEK293A, hsSKM, and MEF cells . HINT1 was stained with rabbit anti-HINT1 antibody followed by anti-rabbit IgG Alexa Fluor Plus 488 antibody. Nuclear DNA was stained with Hoechst 33342. Actin filaments were stained with phalloidin Alexa Fluor 568. Scale: 20 μm. (B) Correlation coefficient of HINT1 staining and Hoechst nuclear staining was calculated and plotted (n = 100).

Article Snippet: Human skeletal muscle (hsSKM) and MEF cells were purchased from the ATCC.

Techniques: Staining

Journal: Life Science Alliance

Article Title: The nucleocytoplasmic translocation of HINT1 regulates the maturation of cell density

doi: 10.26508/lsa.202503215

Figure Lengend Snippet: (A) HEK293A, hsSKM, and MEF cells were seeded at high density and treated with or without 5 μM of latrunculin B for 60 min. The cells were stained as shown in . Scale: 20 μm. (B) HEK293A, hsSKM, and MEF cells were seeded at high density and treated with or without 50 μM of blebbistatin for 180 min. The cells were stained as shown in . Scale: 20 μm.

Article Snippet: Human skeletal muscle (hsSKM) and MEF cells were purchased from the ATCC.

Techniques: Staining

Journal: Life Science Alliance

Article Title: The nucleocytoplasmic translocation of HINT1 regulates the maturation of cell density

doi: 10.26508/lsa.202503215

Figure Lengend Snippet: (A) Cells (HEK293A, hsSKM, and MEF) were treated with or without 10 μM ivermectin, an importin α/β1 complex inhibitor, for 6 h, followed by staining as shown in . (B) Cells (HEK293A, hsSKM, and MEF) were treated with or without 20 μM importazole, an importin β inhibitor, for 24 h at low density, and then stained as shown in . Scale: 20 μm.

Article Snippet: Human skeletal muscle (hsSKM) and MEF cells were purchased from the ATCC.

Techniques: Staining

Journal: Life Science Alliance

Article Title: The nucleocytoplasmic translocation of HINT1 regulates the maturation of cell density

doi: 10.26508/lsa.202503215

Figure Lengend Snippet: (A) hsSKM cells were treated with or without 10 nM of leptomycin B for 24 h at both low and high densities, then stained with rabbit anti-HINT1 antibody followed by anti-rabbit IgG Alexa Fluor Plus 488 antibody. Nuclear DNA was stained with Hoechst 33342. Actin filaments were stained with phalloidin Alexa Fluor 568. Scale: 20 μm. (B) Correlation coefficient of HINT1 staining and Hoechst nuclear staining was calculated and plotted. Similar results were obtained with HEK293A and MEF cells . (C) WT and HINT1-KO HEK293A cells were treated with or without 10 nM of leptomycin B for 24 h at high densities and stained with fluorogenic membrane dye to demarcate the cell boundaries. Scale: 20 μm. (D) Values represent the cross-sectional area of each cell as a dot (n = 100). The percentages are values used for convenience when seeding cells and do not represent the actual cell density. However, for 100%, the cell count was measured and cultured in such a way that the cells would reach confluency by the time of adding leptomycin B.

Article Snippet: Human skeletal muscle (hsSKM) and MEF cells were purchased from the ATCC.

Techniques: Staining, Membrane, Cell Counting, Cell Culture

Journal: Life Science Alliance

Article Title: The nucleocytoplasmic translocation of HINT1 regulates the maturation of cell density

doi: 10.26508/lsa.202503215

Figure Lengend Snippet: (A) MARCKS phosphorylation is dependent on cell density in HEK293A, hsSKM, and MEF cells. Cells were cultured overnight at low and high densities, and the phosphorylation levels of MARCKS were assessed by Western blotting using a phospho-MARCKS (Ser167/170) polyclonal antibody. Total MARCKS and actin levels were also measured via Western blotting as internal controls. Quantitative analysis of MARCKS expression level and phosphorylation levels in HEK293 cells, hsSKM, and MEF cells at low and high densities. The data were normalized using the band intensity from HEK293 cells at low densities. n = 4. Statistical analysis was conducted using one-way ANOVA. * P < 0.05; ** P < 0.01. (B) Cytoplasmic forced expression of HINT1 reduces MARCKS phosphorylation. HEK293A cells were seeded at low density, transfected with either WT HINT1-HA or mutant R24A/K25A HINT1-HA, incubated for 24 h, and subsequently lysed in SDS sample buffer. Phospho-MARCKS, total MARCKS, and actin levels were analyzed via Western blotting. Quantitative analysis of MARCKS phosphorylation levels in HEK293 cells, with or without transfection of WT HINT1-HA or mutant R24A/K25A HINT1-HA (right panel). The data were normalized using the band intensity from nontransfected cells. n = 3. Statistical analysis was conducted using a paired t test. * P < 0.05. (C) MARCKS phosphorylation level increases in HINT1 KO cells. WT and HINT1 KO cells were cultured overnight at low density, and subsequently lysed in SDS sample buffer. Phospho-MARCKS, total MARCKS, and actin levels were analyzed via Western blotting. Quantitative analysis of MARCKS phosphorylation levels in WT and HINT1 KO cells (right panel). The data were normalized using the band intensity from WT cells. n = 3. Statistical analysis was conducted using a paired t test. * P < 0.05. (D) Proposed model illustrates how HINT1 translocation is regulated by cell density and how this process contributes to the disruption of actin SFs, leading to a rounded, confined cell morphology. At low density, HINT1 predominantly localizes in the nucleus, where it binds to chromatin to regulate gene expression, thereby promoting cell proliferation and development. Cell–cell contact alone is insufficient to induce HINT1 translocation to the cytoplasm. At confluency, cells initiate the export of HINT1 to the cytoplasm, a process mediated by exportin 1. As cell density increases, more HINT1 is actively transported to the cytoplasm. In the cytoplasm, HINT1 inhibits phosphorylation of MARCKS, which influences actin remodeling, resulting in a rounded and compact cell morphology. This translocation also suppresses gene expression, leading to a halt in proliferation and promoting contact inhibition of proliferation. The subcellular localization of HINT1 appears to be cell type–dependent, which may explain why some cells form SFs, whereas others do not in situ. Source data are available for this figure.

Article Snippet: Human skeletal muscle (hsSKM) and MEF cells were purchased from the ATCC.

Techniques: Phospho-proteomics, Cell Culture, Western Blot, Expressing, Transfection, Mutagenesis, Incubation, Translocation Assay, Disruption, Gene Expression, Inhibition, In Situ