human foreskin fibroblasts hff  (ATCC)

Journal: Advanced Biotechnology

Article Title: EYA4 promotes cellular senescence by enhancing P21 transcription through interaction with SIX2

doi: 10.1007/s44307-026-00109-8

Figure Lengend Snippet: EYA4 is upregulated in senescent primary human diploid fibroblasts. A Expression levels of EYA4 in 12 tissues from young (20 ~ 49 years) and elderly (≥ 60 years) individuals based on RNA-seq data obtained from the GTEx database. The sample size was between 31 and 384 across tissues. Mean values ± SEM were shown. The y-axis was log₂-transformed to improve visualization of differences across a wide range. B SA-β-gal staining of replicative senescent (PD > 50) or control (~ 25PD) HFF-1 cells. C RT-qPCR analysis of P21 and EYA4 in HFF-1 cells described in ( B ). D Immunoblot analysis of p21 and EYA4 in HFF-1 cells described in ( B ). E–G Same as B – D , respectively, for stress-induced senescent HFF-1 cells. To induce cellular senescence, HFF-1 cells (~ 25 PD) were treated by bleomycin (BLM, 2.5 μM) or saline (Ctl) for 3 days. H – M Same as ( B – G ), respectively, for BJ fibroblasts under the same conditions. Scale bars, 100 μm. All values are means ± SEM of three independent experiments. Two-tailed unpaired Student’s t-test was used to determine the statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: HEK293T (ATCC Cat# CRL-3216, RRID: CVCL_0063), HeLa (ATCC Cat# CCL-2, RRID: CVCL_0030), HFF-1 (ATCC Cat# SCRC-1041, RRID: CVCL_3285), and BJ (ATCC Cat# CRL-2522, RRID: CVCL_3653) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, RNA Sequencing, Transformation Assay, Staining, Control, Quantitative RT-PCR, Western Blot, Saline, Two Tailed Test

Journal: Advanced Biotechnology

Article Title: EYA4 promotes cellular senescence by enhancing P21 transcription through interaction with SIX2

doi: 10.1007/s44307-026-00109-8

Figure Lengend Snippet: EYA4 deficiency alleviates replicative cellular senescence in primary human diploid fibroblasts. A EYA4 knockdown efficiency. Young (~ 25PD) and pre-replicative senescent (~ 45PD) HFF-1 cells were transfected with scramble siRNA (NC) or siEYA4 , and RT-qPCR was performed to detect knockdown efficiency 72 h later. B Immunoblot analysis of p21 and EYA4 in cells described in ( A ). C RT-qPCR analysis of P21 in cells described in ( A ). D SA-β-gal staining to assess senescence in cells described in ( A ). E Quantification of ( D ). The percentage of SA-β-gal positive cells was calculated (n ≥ 100 × three repeats). F EdU incorporation assay of cells described in (A). Scale bars, 100 μm. G Quantification of ( F ). The percentage of EdU positive cells was calculated (n ≥ 100 × three repeats). All values are means ± SEM of three independent experiments. Two-tailed unpaired Student’s t-test was used to determine the statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: HEK293T (ATCC Cat# CRL-3216, RRID: CVCL_0063), HeLa (ATCC Cat# CCL-2, RRID: CVCL_0030), HFF-1 (ATCC Cat# SCRC-1041, RRID: CVCL_3285), and BJ (ATCC Cat# CRL-2522, RRID: CVCL_3653) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Western Blot, Staining, Two Tailed Test

Journal: Advanced Biotechnology

Article Title: EYA4 promotes cellular senescence by enhancing P21 transcription through interaction with SIX2

doi: 10.1007/s44307-026-00109-8

Figure Lengend Snippet: EYA4 deficiency alleviates stress-induced senescence in primary human diploid fibroblasts. A EYA4 knockdown efficiency. HFF-1 cells (~ 25PD) were transfected with scramble siRNA (NC) or siEYA4 , and then treated by bleomycin (BLM, 2.5 μM) or saline (Ctl) for 3 days after siRNA transfection. RT-qPCR was performed to detect knockdown efficiency. B Immunoblot analysis of p21 and EYA4 in cells described in ( A ). C RT-qPCR of P21 in cells described in ( A ). D SA-β-gal staining to assess senescence in cells described in ( A ). E Quantification of ( D ). The percentage of SA-β-gal positive cells was calculated (n ≥ 100 × three repeats). F EdU incorporation assay of cells described in ( A ). Scale bars, 100 μm. G Quantification of ( F ). The percentage of EdU positive cells was calculated (n ≥ 100 × three repeats). All values are means ± SEM of three independent experiments. Two-tailed unpaired Student’s t-test was used to determine the statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: HEK293T (ATCC Cat# CRL-3216, RRID: CVCL_0063), HeLa (ATCC Cat# CCL-2, RRID: CVCL_0030), HFF-1 (ATCC Cat# SCRC-1041, RRID: CVCL_3285), and BJ (ATCC Cat# CRL-2522, RRID: CVCL_3653) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Transfection, Saline, Quantitative RT-PCR, Western Blot, Staining, Two Tailed Test

Journal: iScience

Article Title: Enhanced cell contact and planarian tissue extract increase DNA replication and viability for neoblast cultivation

doi: 10.1016/j.isci.2026.115274

Figure Lengend Snippet: The feeder cell layer promotes DNA replication in neoblasts (A) Schematic diagram and representative images showing SiRNeoblasts seeded on HFF1 and MEF feeder layer, respectively. Scale bars, 50 μm. (B) Cell morphology of SiRNeoblasts cultured on HFF1 or MEF feeder layer for 1, 3, and 7 days. Scale bars, 50 μm. (C) Relative F-ara-EdU + cell ratio of SiRNeoblasts cultured in the plastic flat plate, HFF1, and MEF feeder layer conditions for 3 days. Three wells were assayed in each group. Data are represented as mean ± SEM. Binomial GLM calculates p values. ∗∗∗∗ p < 0.0001. The ratio was calculated as (total number of F-ara-EdU + cells)/(initial number of seeded cells). (D) Heatmap shows expression of genes related to nucleoside metabolism in cells without culture or cells cultured in the plastic flat plate, HFF1, and MEF feeder layer conditions for 1 day. Each group contains three replicates. See also , , and .

Article Snippet: Human: HFF1 , ATCC , SCRC-1041.

Techniques: Cell Culture, Expressing

Journal: iScience

Article Title: Enhanced cell contact and planarian tissue extract increase DNA replication and viability for neoblast cultivation

doi: 10.1016/j.isci.2026.115274

Figure Lengend Snippet: Planarian extract improves cell viability (A and B) Relative PI + dead cell ratio of SiRNeoblasts cultured in the plastic flat plate, the U-bottom plate, HFF1, and MEF feeder layer conditions for 3 days (A) and 7 days (B), respectively. Three wells were assayed in each group. Data are represented as mean ± SEM. All other experimental groups were compared to the plastic flat plate group to assess statistical significance. One-way ANOVA with the Tukey test calculates adjusted p values. ∗∗0.001 < p < 0.01, ∗∗∗∗ p < 0.0001. n.s., no significance. (C) Workflow of preparing planarian extract. (D) Relative PI + dead cell ratio of SiRNeoblasts cultured for 1 and 3 days under five conditions. These five conditions include the plastic flat plate with or without planarian extract, the U-bottom plate with or without planarian extract, and HFF1 feeder layer. Three wells were assayed in each group. Data are represented as mean ± SEM. One-way ANOVA with the Tukey test calculates adjusted p values. ∗0.01 < p < 0.05, ∗∗0.001 < p < 0.01, ∗∗∗0.0001 < p < 0.001. n.s., no significance. (E) Relative F-ara-EdU + cell ratio of SiRNeoblasts cultured for 1 day under five conditions. Three wells were assayed in each group. Data are represented as mean ± SEM. Binomial GLM calculates p values. ∗∗∗∗ p < 0.0001. The ratio was calculated as (total number of F-ara-EdU + cells)/(initial number of seeded cells). (F) Relative piwi-1 + cell ratio of SiRNeoblasts cultured for 7 days under six conditions. The differences among these six conditions lie in three factors: aggregate formation, the addition of extract or not, and the duration of extract exposure. Three wells were assayed in each group. Data are represented as mean ± SEM. One-way ANOVA with the Tukey test calculates adjusted p values. ∗0.01 < p < 0.05, ∗∗0.001 < p < 0.01, n.s., no significance. The ratio was calculated as (total number of piwi-1 + cells)/(initial number of seeded cells). (G) Relative dead cell ratio of SiRNeoblasts cultured for 7 days under six conditions. Three wells were assayed in each group. Data are represented as mean ± SEM. One-way ANOVA with the Tukey test calculates adjusted p values. ∗0.01 < p < 0.05, ∗∗∗0.0001 < p < 0.001, ∗∗∗∗ p < 0.0001. See also .

Article Snippet: Human: HFF1 , ATCC , SCRC-1041.

Techniques: Cell Culture

Journal: iScience

Article Title: Enhanced cell contact and planarian tissue extract increase DNA replication and viability for neoblast cultivation

doi: 10.1016/j.isci.2026.115274

Figure Lengend Snippet: The feeder cell layer promotes DNA replication in neoblasts (A) Schematic diagram and representative images showing SiRNeoblasts seeded on HFF1 and MEF feeder layer, respectively. Scale bars, 50 μm. (B) Cell morphology of SiRNeoblasts cultured on HFF1 or MEF feeder layer for 1, 3, and 7 days. Scale bars, 50 μm. (C) Relative F-ara-EdU + cell ratio of SiRNeoblasts cultured in the plastic flat plate, HFF1, and MEF feeder layer conditions for 3 days. Three wells were assayed in each group. Data are represented as mean ± SEM. Binomial GLM calculates p values. ∗∗∗∗ p < 0.0001. The ratio was calculated as (total number of F-ara-EdU + cells)/(initial number of seeded cells). (D) Heatmap shows expression of genes related to nucleoside metabolism in cells without culture or cells cultured in the plastic flat plate, HFF1, and MEF feeder layer conditions for 1 day. Each group contains three replicates. See also , , and .

Article Snippet: Mitotically inactivated HFF1 (ATCC, SCRC-1041) and MEF (embryonic day 13.5) cells treated with 10 μg/mL Mitomycin C (Sigma, M5353) for 3 hours were recovered one day prior to seeding planarian cells.

Techniques: Cell Culture, Expressing

Journal: iScience

Article Title: Enhanced cell contact and planarian tissue extract increase DNA replication and viability for neoblast cultivation

doi: 10.1016/j.isci.2026.115274

Figure Lengend Snippet: Planarian extract improves cell viability (A and B) Relative PI + dead cell ratio of SiRNeoblasts cultured in the plastic flat plate, the U-bottom plate, HFF1, and MEF feeder layer conditions for 3 days (A) and 7 days (B), respectively. Three wells were assayed in each group. Data are represented as mean ± SEM. All other experimental groups were compared to the plastic flat plate group to assess statistical significance. One-way ANOVA with the Tukey test calculates adjusted p values. ∗∗0.001 < p < 0.01, ∗∗∗∗ p < 0.0001. n.s., no significance. (C) Workflow of preparing planarian extract. (D) Relative PI + dead cell ratio of SiRNeoblasts cultured for 1 and 3 days under five conditions. These five conditions include the plastic flat plate with or without planarian extract, the U-bottom plate with or without planarian extract, and HFF1 feeder layer. Three wells were assayed in each group. Data are represented as mean ± SEM. One-way ANOVA with the Tukey test calculates adjusted p values. ∗0.01 < p < 0.05, ∗∗0.001 < p < 0.01, ∗∗∗0.0001 < p < 0.001. n.s., no significance. (E) Relative F-ara-EdU + cell ratio of SiRNeoblasts cultured for 1 day under five conditions. Three wells were assayed in each group. Data are represented as mean ± SEM. Binomial GLM calculates p values. ∗∗∗∗ p < 0.0001. The ratio was calculated as (total number of F-ara-EdU + cells)/(initial number of seeded cells). (F) Relative piwi-1 + cell ratio of SiRNeoblasts cultured for 7 days under six conditions. The differences among these six conditions lie in three factors: aggregate formation, the addition of extract or not, and the duration of extract exposure. Three wells were assayed in each group. Data are represented as mean ± SEM. One-way ANOVA with the Tukey test calculates adjusted p values. ∗0.01 < p < 0.05, ∗∗0.001 < p < 0.01, n.s., no significance. The ratio was calculated as (total number of piwi-1 + cells)/(initial number of seeded cells). (G) Relative dead cell ratio of SiRNeoblasts cultured for 7 days under six conditions. Three wells were assayed in each group. Data are represented as mean ± SEM. One-way ANOVA with the Tukey test calculates adjusted p values. ∗0.01 < p < 0.05, ∗∗∗0.0001 < p < 0.001, ∗∗∗∗ p < 0.0001. See also .

Article Snippet: Mitotically inactivated HFF1 (ATCC, SCRC-1041) and MEF (embryonic day 13.5) cells treated with 10 μg/mL Mitomycin C (Sigma, M5353) for 3 hours were recovered one day prior to seeding planarian cells.

Techniques: Cell Culture