Journal: ADMET & DMPK

Article Title: Anti-inflammatory potential of plant-derived extracellular vesicles from Solanum nigrum L. integrated in gelatine-dopamine hydrogel on RAW 264.7 and MC3T3 cells

doi: 10.5599/admet.3149

Figure Lengend Snippet: Interleukin-6 (IL-6) concentration by (A) RAW 264.7 and (B) MC3T3 cells LPS induction and PDEV (2.5, 5 and 10 μg mL -1 ) treatment released from Gel-Dop hydrogel. Data presented as mean ± standard deviation (*** p < 0.001, **** p <0,0001)

Article Snippet: IL-6 concentration was measured using a mouse IL-6 ELISA kit (BT Lab) according to the kit protocol.

Techniques: Concentration Assay, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Expression of adiponectin, AdipoR1 and APPL1 is downregulated in aged mouse plasma and myocardial tissue. (A) Relative expression levels of p16 and p21 in myocardial tissues of young mice (aged 2 months) and aged mice (aged 15 months) were detected by western blotting. (B) Relative mRNA expression levels of adiponectin, AdipoR1 and APPL1 in the plasma of young mice and aged mice were detected by reverse transcription-quantitative PCR. (C) Relative expression levels of adiponectin, AdipoR1 and APPL1 in myocardial tissues of young mice and aged mice were analyzed using western blot analysis. **P<0.01, ***P<0.001 vs. young mice. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Clinical Proteomics, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Expression of adiponectin, AdipoR1 and APPL1 is downregulated in D-gal-treated cardiomyocytes. (A) Relative expression levels of p16 and p21 in H9c2 cells treated with 2.5, 5 and 10 g/l of D-gal were analyzed using western blot analysis. *P<0.05, **P<0.01, ***P<0.001 vs. control. Transfection with Ov-adiponectin promotes the expression of AdipoR1 and APPL1 in D-gal-treated cardiomyocytes. (B) Relative mRNA expression of adiponectin in H9c2 cells before and after transfection with Ov-adiponectin plasmid was analyzed via RT-qPCR. ***P<0.001 vs. control; ### P<0.001 vs. D-gal + Ov-NC. (C) Relative protein expression of adiponectin in H9c2 cells before and after transfection with Ov-adiponectin plasmid was analyzed using RT-qPCR. ***P<0.001 vs. control; ## P<0.01 vs. D-gal + Ov-NC. (D) Relative mRNA expression levels of AdipoR1 and APPL1 in H9c2 cells before and after transfection with Ov-adiponectin plasmids were detected using RT-qPCR. ***P<0.001 vs. control; ### P<0.001 vs. D-gal + Ov-NC. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; D-gal, D-galactose; Ov-, overexpression.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Over Expression

Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1. (A and B) Relative expression levels of AdipoR1 in D-gal-treated H9c2 cells with Ov-adiponectin before and after transfection with si-AdipoR1 and si-APPL were measured using RT-qPCR. **P<0.01, ***P<0.001 vs. D-gal + Ov-adiponectin. (C and D) Relative expression levels of APPL1 in D-gal-treated H9c2 cells with Ov-adiponectin before and after transfection with si-AdipoR1 and si-APPL were measured using RT-qPCR. ***P<0.001 vs. D-gal + Ov-adiponectin. (E) Cellular senescence in the different groups was detected using SA-β-gal staining. (F) Relative expression levels of p16 and p21 in the different groups were detected using western blot analysis. ***P<0.001 vs. control; ## P<0.01, ### P<0.001 vs. D-gal + Ov-NC; $ P<0.05 vs. D-gal + Ov-adiponectin + negative-NC. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1; Ov-, overexpression; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; si-, small interfering RNA; D-gal, D-galactose; SA-β-gal, senescence-associated β-galactose.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Staining, Western Blot, Control, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Small Interfering RNA

Journal: BMC Biotechnology

Article Title: Modified gateway system for double shRNA expression and Cre/lox based gene expression

doi: 10.1186/1472-6750-11-24

Figure Lengend Snippet: Successful shRNA/cDNA targeting using designed entry vectors . A) pENTR constructs targeting exon 2 of p16 were transfected into 293T cells. RNA was isolated 48 hours post transfection and the mRNA expression level was determined using Q-RT-PCR. The average represents fold gene expression level relative to pENTRhU6hH1 transfected 293T cells (Students t-test; p < 0.01). B) pENTR constructs for CCND1 and/or CCND3 shRNA expression were recombined with plko.puro.DEST. Western blot shows the successful knockdown of CCND1/CCND3 in Panc1 cells. C) pENTR constructs for p16 shRNA expression were recombined with plko.YFP.DEST. Western blot shows p16 knockdown in H6c7 cells transfected with p16 shRNA constructs. D) pENTR.Cre mediated Cdk4 overexpression in 293T cells transiently transfected with pENTR.Cdk4.ON. The average represents fold gene expression level relative to control vector cell line f) Luciferase activity downregulation by Cre protein in stable HeLa cells overexpressing Luc2 mediated by plko.dsRed2.Luc2.ON lentivirus.

Article Snippet: Immunoblotting was performed using whole protein extracts and probed with the following antibodies against cyclin D3 (BD Biosciences, San Jose, CA), cyclin D1 (BD Biosciences), p16 INK4a (Cell Signalling, Boston, MA) and p14 ARF (NeoMarkers, Freemont, CA), GAPDH (Abcam, Cambridge, MA), and secondary mouse or rabbit conjugated IgG-horseradish peroxidase (Cell Signalling).

Techniques: shRNA, Construct, Transfection, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Western Blot, Knockdown, Over Expression, Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Schematics of the experimental strategy to measure senescence markers (p21, p16 and SA-βGal activity) and SASP cytokines levels in mice lungs at 10 and 20 days p.i. Day 1 CFU= 55. (b) %p21+p16+ and (c) %SA-βGal+ (CellEvent Senescence green+) cells out of all live lung (uninfected and Mtb- infected mice) and Spleen ( Mtb- infected mice) cells at 10 days p.i. as determined by multicolor flow cytometry. (d) %p21+p16+ and (e) %SA-βGal+ cells out of different lung-cell types at 10 days p.i., as determined by multicolor flow cytometry. Gating for p16⁺, p21⁺, and SA-βGal⁺ events for each cell type were established using reference cells from Mtb -infected WT B6 mice (set at 1 %). (AMs: Alveolar macrophages, IMs: Interstitial macrophages) (f) Normalized concentration of SASP-cytokines in lung homogenates at 20 dpi and 4 wpi as measured by a LEGENDplex™ Mouse Inflammation Panel (13-plex). The data are means ± SEM. Each data point represents a mouse (n=11-12). (two-way ANOVA with Tukey’s (b-e) or Dunnett’s (f) multiple comparisons test).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Activity Assay, Infection, Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Representative H&E-stained images of Mtb -infected mice lungs treated with Vehicle or drugs, and respective ImageJ-based quantification. Yellow arrow indicates necrotic granuloma in Vehicle treated B6.Sst1S mice at 5 wpi. The data are means ± SEM. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. Square represents necrotic granuloma formation in the lung. (b) Representative γH2A.X-Immunohistochemistry images of mice after lungs vehicle/ drugs treatment, and respective Violin plots to show ImageJ quantification of γH2A.X-stained area. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. (c) %p21+βGal+ and %p16+βGal+ subpopulation observed in all live lung cells of Mtb -infected mice (n= 5-8). The data are means ± SEM. Each data point represents a mouse. Statistical analysis was calculated by two-way ANOVA with Dunnett’s multiple comparisons test. Bubble plot to show %p21+βGal+ and %p16+βGal+ subpopulation in different lung cell types in Mtb -infected (d) B6.Sst1S mice (n= 8) and (e) WT B6 old mice (n= 5) at indicated time point and treatment groups. The size of the bubble indicates %subpopulation out of all of particular cell types and color is adjusted p values relative to Veh group. Statistics are calculated by one-way ANOVA with Tukey’s multiple comparisons test ( p > 0.15: ns).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Staining, Infection, Two Tailed Test, Immunohistochemistry

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: Senescence markers in primary human and mouse VSMCs undergoing senescence. ( A ) % EdU + in cultured human VSMCs (Control), after 24h treatment with 500 nM doxorubicin (Dox 1d), after an additional 21 days recovery in control conditions (control 21d) or after doxorubicin (Dox 1d+ 21d), or at replicative senescence (RS). ( B – D ) mRNA levels of Lamin B1, p16, and p21 in cell populations described in ( A ) relative to control (1d) cells. ( E ) Western blot for Lamin B1, p16, p21, and p53 for cells treated in ( A ). n = 6–8 human VSMC isolates. ( F and G ) EdU + % ( F ) or SAβG + % ( G ) of mouse p16-3MR VSMCs treated increasing concentrations of Doxorubicin for 1 day followed by 7 days recovery vs. vehicle control. ( I – K ) qPCR for Lamin B1, IL6, p16, or p21 mRNA expression for cells treated in ( F ). ( L ) Western blot of mouse cells as treated in ( F ) for Lamin B1, p16, or p21. n = 3–8 mouse VSMC isolates. Data are means (SD), one-way ANOVA with correction for multiple comparisons ( A ) or unpaired Student’s t -test vs. Control 1d ( B – D ) or vs. Vehicle (Dox 0 nM) ( F – K ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Cell Culture, Control, Western Blot, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: p16/Cdkn2a is detected in VSMCs in mouse atherosclerotic plaques. ( A and B ) UMAP plots showing scRNA-seq profiles of unsorted aortic cells from Myh11Cre ERt2+ /Confetti + mice ( A ), or sorted Confetti + VSMCs from atherosclerotic plaque and media of fat-fed Myh11Cre ERt2 /Confetti + /ApoE −/− mice ( B ). Log-transformed expression levels of Myh11 and p16/Cdkn2a are shown alongside e-Cadherin/Cdh5 and Pdgfrα ( A ) or Cd68 and Ly6a/Sca1 ( B ) using a scale from white to dark red. Insets show high power regions of clusters 6, 8, and 9 and expression of p16 in ( B ). Feature plots show log-normalized expression levels. ( C ) % cells in each cluster with detectable expression of p16/cdkn2a after 14 weeks or 18 weeks or high-fat feeding, or combined.

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Transformation Assay, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: GCV treatment of p16-3MR mice does not affect atherosclerosis, but induces inflammation. ( A ) Aortic root plaques in ApoE→ApoE, p16→ApoE, ApoE→p16, or p16→p16 mice + GCV, or p16→p16 mice + saline, stained with Masson’s trichrome, TUNEL, or Mac3. Scale bar = 300 µm. High power inset shows apoptotic cell and nuclear debris from outlined area. ( B ) Plaque area for mice in ( A ). ( C and D ) Number of TUNEL + cells/aortic root plaque ( C ) or %Mac3 + cells ( D ) for mice in ( A ). ( E – G ) Relative mRNA expression for p16, IL18, or TNFα in experimental mice. Data are means (SD) n = 5–10 mice. One-way ANOVA with correction for multiple comparisons ( B – D ) or Kruskal–Wallis H test followed by Dunn’s multiple comparisons test ( E – G ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Saline, Staining, TUNEL Assay, Expressing

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: ABT-263 (Navitoclax) selectively reduces senescent VSMCs. ( A and B ) Photomicrographs ( A ) or quantification ( B ) of mouse VSMCs stained for SAβG, as replicating control cells or after dox1 + 7 days treatment, or each group ±1 µM ABT-263 treatment for 48 h. ( C ) Western blot and quantification for p16 in cells treated in ( A and B ). ( D ) Fold change in mRNA expression compared with control replicating cells for p16 and a range of SASP cytokines against the housekeeping gene HMBS. Data are means (SD), n = 4–5. Unpaired Student’s t -test. ( E ) Mouse macrophages cultured for 28 days, then treated with 1 µM ABT-263 for 48 h and stained for SAβG. Data are means (SD), n = 3. Unpaired Student’s t -test.

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Staining, Control, Western Blot, Expressing, Cell Culture

Journal: Cardiovascular Research

Article Title: Efficacy and limitations of senolysis in atherosclerosis

doi: 10.1093/cvr/cvab208

Figure Lengend Snippet: ABT-263 reduces atherosclerosis, but not local SASP cytokine expression. ( A ) ORO staining of mouse descending aorta treated with control (vehicle) or ABT-263, and quantification of %ORO area ( n = 11–14). Scale bar = 3 mm. ( B ) Masson’s trichrome histochemistry of aortic root atherosclerotic plaque from mice treated in ( A ). Panels below show high power view of outlined area. Arrow shows necrotic core. Scale bar = 200 µm. ( C – E ) Aortic root plaque area/total area ( C ) Cap area ( D ), or Core area ( E ) for mice in ( A ). n = 11–13. ( F ) qPCR for relative expression of p16 or SASP cytokines in aortic arches of experimental mice against the housekeeping gene HMBS ( n = 7). Data are means (SD), n = 10. Unpaired Student’s t -test ( A, C – E ) or Mann–Whitney U test ( F ).

Article Snippet: Sections were incubated with either primary antibodies: p16 (20 μg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy 3 -conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature.

Techniques: Expressing, Staining, Control, MANN-WHITNEY

Journal: Advanced Healthcare Materials

Article Title: A Human Kidney Tubuloid Model of Repeated Cisplatin‐Induced Cellular Senescence and Fibrosis for Drug Screening

doi: 10.1002/adhm.202501795

Figure Lengend Snippet: Tubuloids exhibit cellular senescence and release inflammatory cytokines following repeated cisplatin treatment. A) Experimental design for repeated cisplatin treatment. B) Representative images of SA‐β‐GAL staining of tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. C) Immunostaining of p16 in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. D) Western blotting of p16 in tubuloids exposed to cisplatin (0.2 and 2.0 µg mL −1 ) 5 times repeatedly or control. E) Statistical analysis of p16 expression (n = 4). F) Western blotting of p21 in tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. G) Statistical analysis of p21 expression (n = 5). H) Western blotting of p53 and Lamin B1 in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. I) Statistical analysis of p53 expression (n = 3). J) Statistical analysis of Lamin B1 expression (n = 3). K) Cell Cycle Analysis in tubuloids exposed to cisplatin (0.2 µg mL −1 ) 5 times repeatedly or control. L) Immunostaining of an inflammatory cytokine, IL‐1β, in tubuloids exposed to cisplatin (2.0 µg mL −1 ) 5 times repeatedly or control. Scale bars: 100 µm. M,N) Quantitative PCR of IL‐1β (M) and IL‐6 (N) in tubuloids exposed to cisplatin (0.2 and 2.0 µg mL −1 ) 5 times repeatedly or control (n = 3). Statical analysis was applied to delta‐delta Ct value, not fold increase. All data are presented as means ± standard errors (SEs). Statistical significance is presented by * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies were also used, including rabbit anti‐p16‐INK4A polyclonal antibody (1:200) (Proteintech), mouse anti‐p21 antibody (1:100) (sc‐6247, Santa Cruz Biotechnology, TX, USA), rabbit anti‐Lamin B1 polyclonal antibody (1:1000) (12987‐1‐AP, Proteintech), rabbit anti‐p53 polyclonal antibody (1:1000) (10442‐1‐AP, Proteintech), rabbit anti‐KIM‐1 antibody (1:400) (PA5‐20244, Thermo‐Fisher Scientific, MA, USA), rabbit anti‐caspase‐3 antibody (1:200) (Cell Signaling Technology), and rabbit anti‐Vimentin antibody (1:1000) (#5741, Cell Signaling Technology, MA, USA).

Techniques: Staining, Control, Immunostaining, Western Blot, Expressing, Cell Cycle Assay, Real-time Polymerase Chain Reaction