Journal: iScience

Article Title: RSK1-SRF signaling axis drives fibroblast activation and pulmonary fibrosis: Genetic causality and therapeutic targeting

doi: 10.1016/j.isci.2026.115495

Figure Lengend Snippet: RSK1 is a causal risk factor for IPF and is upregulated in fibrotic lungs (A–D) Mendelian randomization (MR) analysis evaluating the causal association between RSK1 expression quantitative trait loci (eQTLs) and IPF risk, using the inverse variance weighted (IVW), MR Egger, weighted median, and weighted mode methods. (B and C) Scatter plot and weight plot for the IVW method, respectively. (D) Association strength between RSK1 eQTLs and IPF. (E) Boxplot of RPS6KA1 expression in lung tissues from IPF patients and healthy controls ( GSE70866 ; ∗ p < 0.05, as indicated). (F) Immunoblot analysis of phosphorylated RSK1 ( p -RSK1) and total RSK1 in mouse lungs on days 7 and 14 after bleomycin (BLM) challenge. (G) Densitometric quantification of (F); β-actin serves as the loading control. (H) Representative immunohistochemistry (IHC) staining images of p -RSK1 in mouse lungs during fibrosis progression. Scale bars, 100 μm. (I) Quantification of p -RSK1 IHC staining shown as positive area (%). (J and K) Representative IHC staining images of (J) RSK1 and (K) p -RSK1 in human IPF and normal lung tissues. Scale bars, 100 μm. (L) Immunofluorescence co-staining of p -RSK1 (green) with F4/80, E-cadherin (E-Cad), and fibronectin (FN) (red) in saline- and BLM-treated mouse lungs; nuclei are stained with DAPI (blue). Scale bars, 100 μm. Data are presented as the mean ± SD. For tissue-based assays, n denotes independent animals ( n = 6 per group). Image quantification was performed as described in ; technical sampling was not counted toward n . Statistical tests are described in . ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; # p ≥ 0.05.

Article Snippet: Bleomycin hydrochloride , MedChemExpress , Cat#HY-17565A.

Techniques: Expressing, Western Blot, Control, Immunohistochemistry, Immunofluorescence, Staining, Saline, Sampling

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: Phloridzin dose-dependently mitigated the effect of bleomycin on BALF LDH activity. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; LDH: lactate dehydrogenase; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Activity Assay, Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: Phloridzin dose-dependently exhibited antioxidant effects in bleomycin-treated animals. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; GSH: reduced glutathione; MDA: malondialdehyde; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: Phloridzin dose-dependently abrogated the effect of bleomycin on the inflammatory markers in the lung tissues. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; IL-1β: interleukin-1 beta; NF-κB: nuclear factor kappa B; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: Phloridzin dose-dependently ameliorated the effect of bleomycin on TGF-β1 levels in the lung tissues. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; NS: non-significant; PZ: phloridzin; TGF-β1: transforming growth factor beta 1.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: A photomicrograph demonstrating the immunohistochemical staining of the lung tissue specimens for cleaved caspase-3 (x200, scale bar=40 µm) from A) and B) the control group and PZ-alone treated group respectively showing minimal positive immune expression of cleaved caspase-3 (Arrow); C) the group treated with bleomycin alone showing strong positive immune expression of cleaved caspase-3 (Arrows); D) the bleomycin group treated with phloridzin 60 mg/kg/day showing moderate positive immune expression of cleaved caspase-3 (Arrows); E) the bleomycin group treated with phloridzin 120 mg/kg/day showing mild positive immune expression of cleaved caspase-3 (Arrows); and F) The percentage effect of phloridzin (60 and 120 mg/kg/day, p.o) on the immune expression of cleaved caspase-3 in the lung tissues of bleomycin-treated rats. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: A photomicrograph demonstrating the immunohistochemical staining of the lung tissue specimens for beclin-1 (×200, scale bar = 40 µm) from A) and B) the control group and PZ-alone treated group respectively showing strong positive immune expression of beclin-1 (Arrows); C) the group treated with bleomycin alone showing minimal positive immune expression of beclin-1 (Arrows); D) the bleomycin group treated with phloridzin 60 mg/kg/day showing moderate positive immune expression of beclin-1 (Arrows); E) the bleomycin group treated with phloridzin 120 mg/kg/day showing strong positive immune expression of beclin-1 (Arrows); and F) The percentage effect of phloridzin (60 and 120 mg/kg/day, p.o) on the immune expression of beclin-1 in the lung tissues of bleomycin-treated rats. Values are expressed as mean ± standard deviation. Number of animals = 15 rats in each group. One-way analysis of variance (ANOVA) was used to assess the differences between the different groups, and then Tukey's multiple comparisons post-hoc test was employed. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Standard Deviation

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: A photomicrograph of hematoxylin and eosin-stained sections (×100, scale bar = 100 µm) from the lung tissues of A) and B) the control group and PZ-alone treated group respectively showing normal lung morphological structure with intact alveoli (Thin arrows) and bronchioles (Thick arrows) with normal blood vessels (BV); C) and D) the group treated with bleomycin alone showing significant perturbation of the lung architecture as evidenced by destruction of the interalveolar septa (Thin arrows), distortion of the walls of the bronchi (Thick arrow), perinuclear vacuolation with pyknotic nuclei in the epithelial lining of the bronchioles (wavy green arrows), massive inflammatory cellular infiltration (Arrow heads), severe congestion of the vascular bed (V), with massive interstitial hemorrhage (IH); E) and F) bleomycin group treated with phloridzin (60 mg/kg/day) showing significant decline in the alveolar walls' destruction (Thin arrows), moderate inflammatory cellular infiltration (Arrow head), moderate vascular congestion (V), and significant decrease in the interstitial haemorrhage (IH); G) and H) bleomycin group treated with phloridzin (120 mg/kg/day) showing apparently normal alveoli (Thin arrows) with significant diminution of the inflammatory cellular infiltration (Arrow head) and minimal vascular congestion (V) with restoration of the bronchiolar wall (Thick arrow).

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Staining, Control

Journal: Toxicology Reports

Article Title: Phloridzin mitigates bleomycin-elicited lung fibrosis in Wistar rats: The interplay between antioxidant defenses, inflammatory processes, transforming growth factor beta 1, and autophagy

doi: 10.1016/j.toxrep.2026.102267

Figure Lengend Snippet: A photomicrograph of Masson’s trichrome stained sections (×200, scale bar = 100 µm) from the lung tissues of A) and B) the control group and PZ-alone treated group respectively showing minimal collagen fibers deposits around the bronchioles and the pulmonary blood vessels (Arrows); C) the group treated with bleomycin alone showing massive deposition of the collagen fibers around the alveoli, bronchioles, and pulmonary blood vessels in addition to the interalveolar septa (Arrows); D) bleomycin group treated with phloridzin (60 mg/kg/day) exhibiting a significant decline in the deposition of the collagen fibers around the pulmonary blood vessels and the alveoli, and in the interalveolar septa (Arrows); E) bleomycin group treated with phloridzin (120 mg/kg/day) exhibiting minimal deposition of the collagen fibers around the pulmonary blood vessels and the alveoli, in addition to the interalveolar septa (Arrows), and F) The effect of phloridzin (60 and 120 mg/kg/day, p.o) on the fibrosis score in the lung tissues harvested from bleomycin-treated rats. Values were expressed as median (Interquartile range, IQR) and compared using Kruskal-Wallis followed by Dunn's test. Number of animals = 15 rats in each group. A p-value less than 0.05 was considered statistically significant. BLM: bleomycin; NS: non-significant; PZ: phloridzin.

Article Snippet: Bleomycin, a white powder with a purity of ≥ 98%, a product of Celon Laboratories Ltd., Hyderabad, Telangana, India (CAS # 9041–93–4).

Techniques: Staining, Control

Journal: Redox Biology

Article Title: SIRT3-mediated deacetylation of FoxM1 prevents pulmonary fibrosis via modulating the activation of pulmonary fibroblasts

doi: 10.1016/j.redox.2026.104108

Figure Lengend Snippet: FoxM1 is highly expressed in fibroblasts of fibrotic lung tissues. (A, B) qPCR (n = 9) and Western blot (n = 6) analysis of the expression of FoxM1 in normal and IPF lung tissues. ∗ P < 0.05. (C) qPCR analysis of the mRNA levels of FoxM1 in the lung tissues from BLM-treated mice. n = 3, ∗ P < 0.05. (D) Western blot analysis of the protein levels of FoxM1, CTHRC1, α-SMA, and Collagen I in the lung tissues from bleomycin (BLM)-treated mice. n = 3, ∗ P < 0.05. (E) The Pearson's correlation analysis of COL1A1 expression with FoxM1 expression based on the RNA-seq results of GSE24206 from GEO database. (F) The Pearson's correlation analysis of Ashcroft score with FoxM1 expression in the lung tissues from BLM-treated mice. (G) Representative images of co-immunostaining for α-SMA and FoxM1 in IPF lung tissues. White arrows indicate double-positive cells. (H) Representative images of co-immunostaining for α-SMA and FoxM1 in the lung tissues from BLM-treated mice. White arrows indicate double-positive cells. (I) Western blot analysis of FoxM1 expression in pulmonary fibroblasts isolated from mice subjected to BLM treatment. n = 3, ∗ P < 0.05. (J) Western blot analysis of FoxM1, CTHRC1, α-SMA, and Collagen I expression in pulmonary fibroblasts treated with TGF-β1. n = 3, ∗ P < 0.05. All data were presented as the means ± SEM. Paired t -test (A, B, I) and one-way ANOVA with Tukey's post-hoc test (C, D, J) were used for statistical analysis.

Article Snippet: For the induction of pulmonary fibrosis, a single dose of bleomycin (BLM, Nippon Kayaku, Tokyo, Japan) at 5 mg/kg, dissolved in 50 μl of sterile saline, was administered intratracheally to the treatment group.

Techniques: Western Blot, Expressing, RNA Sequencing, Immunostaining, Isolation

Journal: Redox Biology

Article Title: SIRT3-mediated deacetylation of FoxM1 prevents pulmonary fibrosis via modulating the activation of pulmonary fibroblasts

doi: 10.1016/j.redox.2026.104108

Figure Lengend Snippet: Enhanced FoxM1 expression is responsible for the resistance of fibroblasts to FasL-mediated apoptosis . (A) Caspase 3 activity analysis in pulmonary fibroblasts isolated from bleomycin (BLM)-treated mice following FasL treatment. (B) Cell viability assessment using the CCK-8 assay in pulmonary fibroblasts isolated from BLM-treated mice after FasL treatment. (C) Caspase 3 activity analysis in TGF-β1-treated pulmonary fibroblasts, along with or without FasL treatment. (D) Cell viability assessment using the CCK-8 assay in TGF-β1-treated pulmonary fibroblasts, along with or without FasL treatment. (E) Western blot analysis of FoxM1 expression in pulmonary fibroblasts transfected with or without LV-FoxM1. (F) Western blot analysis of FoxM1 expression in pulmonary fibroblasts transfected with or without FoxM1 siRNA (si-FoxM1). (G, H) Caspase 3 activity and cell viability assessment in pulmonary fibroblasts transfected with or without LV-FoxM1, following with or without FasL treatment. (I, J) Caspase 3 activity and cell viability assessment in pulmonary fibroblasts transfected with or without si-FoxM1, following with or without FasL treatment. All data (n = 3, ∗ P < 0.05) were presented as the means ± SEM. Paired t -test was used for statistical analysis.

Article Snippet: For the induction of pulmonary fibrosis, a single dose of bleomycin (BLM, Nippon Kayaku, Tokyo, Japan) at 5 mg/kg, dissolved in 50 μl of sterile saline, was administered intratracheally to the treatment group.

Techniques: Expressing, Activity Assay, Isolation, CCK-8 Assay, Western Blot, Transfection

Journal: Redox Biology

Article Title: SIRT3-mediated deacetylation of FoxM1 prevents pulmonary fibrosis via modulating the activation of pulmonary fibroblasts

doi: 10.1016/j.redox.2026.104108

Figure Lengend Snippet: Impairing nuclear translocation of FoxM1 suppresses fibroblast activation and protects mice from bleomycin-induced pulmonary fibrosis. (A) Western blot analysis was performed to assess the nuclear expression levels of FoxM1 in pulmonary fibroblasts isolated from BLM-treated mice. n = 3, ∗ P < 0.05. (B) Western blot analysis of nuclear FoxM1, CTHRC1, α-SMA, and Collagen I expression in TGF-β1-treated pulmonary fibroblasts accompany with or without RCM-1 treatment. n = 3, ∗ P < 0.05. (C, D) EdU assay for the proliferation of TGF-β1-treated pulmonary fibroblasts accompany with or without RCM-1 treatment. n = 3, ∗ P < 0.05. (E) Hematoxylin–eosin (H&E) and masson's trichrome staining for the lung tissues from BLM-treated mice injected with or without RCM-1. (F, G) The ashcroft score (n = 6, ∗ P < 0.05.) and hydroxyproline contents (n = 6, ∗ P < 0.05.) in the lung tissues of BLM-treated mice injected with or without RCM-1. (H) The survival of BLM-treated mice injected with or without RCM-1. n = 18. (I) Western blot analysis of FoxM1, CTHRC1, α-SMA, and Collagen I expression in the lung tissues from BLM-treated mice injected with or without RCM-1. n = 3, ∗ P < 0.05. All data were presented as the means ± SEM. Paired t -test (A) and one-way ANOVA with Tukey's post-hoc test (B, D, F–I) were used for statistical analysis.

Article Snippet: For the induction of pulmonary fibrosis, a single dose of bleomycin (BLM, Nippon Kayaku, Tokyo, Japan) at 5 mg/kg, dissolved in 50 μl of sterile saline, was administered intratracheally to the treatment group.

Techniques: Translocation Assay, Activation Assay, Western Blot, Expressing, Isolation, EdU Assay, Staining, Injection

Journal: Redox Biology

Article Title: SIRT3-mediated deacetylation of FoxM1 prevents pulmonary fibrosis via modulating the activation of pulmonary fibroblasts

doi: 10.1016/j.redox.2026.104108

Figure Lengend Snippet: Acetylation of FoxM1 is required for the activation of pulmonary fibroblasts. (A, B) Western blot analysis of FoxM1 expression in the cytoplasm and nucleus of CHX-treated pulmonary fibroblasts along with or without MG132 treatment at indicated time. n = 3, ∗ P < 0.05. (C) Western blot analysis of FoxM1 expression in CHX-treated pulmonary fibroblasts along with or without TGF-β1 treatment. n = 3, ∗ P < 0.05. (D) Western blot analysis of the acetylation levels of FoxM1 in pulmonary fibroblasts isolated from bleomycin (BLM)-treated mice. n = 3, ∗ P < 0.05. (E) Western blot analysis of the acetylation levels of FoxM1 in pulmonary fibroblasts treated with or without TGF-β1. n = 3, ∗ P < 0.05. (F, G) Western blot analysis of FoxM1 acetylation, CTHRC1, α-SMA, and Collagen I expression in pulmonary fibroblasts treated with TSA (50 nM), or NAM (1 mM) for 24 h, or not. n = 3, ∗ P < 0.05. All data were presented as the means ± SEM. Paired t -test (D, E) and one-way ANOVA with Tukey's post-hoc test (B, C, G) were used for statistical analysis.

Article Snippet: For the induction of pulmonary fibrosis, a single dose of bleomycin (BLM, Nippon Kayaku, Tokyo, Japan) at 5 mg/kg, dissolved in 50 μl of sterile saline, was administered intratracheally to the treatment group.

Techniques: Activation Assay, Western Blot, Expressing, Isolation

Journal: Redox Biology

Article Title: SIRT3-mediated deacetylation of FoxM1 prevents pulmonary fibrosis via modulating the activation of pulmonary fibroblasts

doi: 10.1016/j.redox.2026.104108

Figure Lengend Snippet: Sirt3-dependent deacetylation of FoxM1 regulates the stability of FoxM1. (A) The Pearson's correlation analysis of COL1A1 expression with SIRTs expression based on the RNA-seq results of GSE2052 from GEO database. (B) Western blot analysis of SIRT3 expression in the lung tissues from bleomycin (BLM)-treated mice. n = 3, ∗ P < 0.05. (C) Western blot analysis of the acetylation levels of FoxM1 in SIRT3 flox/flox mice intratracheally injected with AAV-Cre. n = 3, ∗ P < 0.05. (D) Western blot analysis was performed to assess the acetylation status of FoxM1 in pulmonary fibroblasts following transfection with Sirt3 siRNA (si-Sirt3). n = 3, ∗ P < 0.05. (E) Western blot analysis of FoxM1 expression in CHX-treated pulmonary fibroblasts transfected with or without si-Sirt3. n = 3, ∗ P < 0.05. (F) Western blot analysis of CTHRC1, α-SMA and Collagen I expression in pulmonary fibroblasts transfected with or without si-Sirt3. n = 3, ∗ P < 0.05. (G) EdU assay for the proliferation of pulmonary fibroblasts transfected with or without si-Sirt3. n = 3, ∗ P < 0.05. (H) Western blot analysis of FoxM1 acetylation, CTHRC1, α-SMA, and Collagen I expression in TGF-β1-treated pulmonary fibroblasts transfected with or without LV-Sirt3. n = 3, ∗ P < 0.05. All data were presented as the means ± SEM. Paired t -test (B-D, F, G) and one-way ANOVA with Tukey's post-hoc test (E, H) were used for statistical analysis.

Article Snippet: For the induction of pulmonary fibrosis, a single dose of bleomycin (BLM, Nippon Kayaku, Tokyo, Japan) at 5 mg/kg, dissolved in 50 μl of sterile saline, was administered intratracheally to the treatment group.

Techniques: Expressing, RNA Sequencing, Western Blot, Injection, Transfection, EdU Assay

Journal: Redox Biology

Article Title: SIRT3-mediated deacetylation of FoxM1 prevents pulmonary fibrosis via modulating the activation of pulmonary fibroblasts

doi: 10.1016/j.redox.2026.104108

Figure Lengend Snippet: Nicotinamide riboside protects mice from bleomycin-induced pulmonary fibrosis via activation of SIRT3. (A) Western blot analysis of CTHRC1, SIRT3, FoxM1, α-SMA and Collagen I expression in TGF-β1-treated pulmonary fibroblasts accompany with or without NR treatment. n = 3, ∗ P < 0.05. (B) Cell viability assessment using the CCK-8 assay in pulmonary fibroblasts treated as in A n = 6, ∗ P < 0.05. (C) The survival of BLM-treated mice oral gavaged with or without NR. n = 18. (D) Hematoxylin–eosin (H&E) and masson's trichrome staining for the lung tissues from mice treated as in C. (E) The ashcroft score of mice treated as in C n = 6, ∗ P < 0.05. (F) Western blot analysis of CTHRC1, SIRT3, FoxM1, α-SMA and Collagen I expression in the lung tissues from mice treated as in C n = 3, ∗ P < 0.05. (G) Western blot analysis of CTHRC1, SIRT3, FoxM1, α-SMA and Collagen I expression in pulmonary fibroblasts isolated from the lung tissues of mice treated as in C n = 3, ∗ P < 0.05. All data were presented as the means ± SEM. One-way ANOVA with Tukey's post-hoc test was used for statistical analysis.

Article Snippet: For the induction of pulmonary fibrosis, a single dose of bleomycin (BLM, Nippon Kayaku, Tokyo, Japan) at 5 mg/kg, dissolved in 50 μl of sterile saline, was administered intratracheally to the treatment group.

Techniques: Activation Assay, Western Blot, Expressing, CCK-8 Assay, Staining, Isolation