Journal: Development (Cambridge, England)

Article Title: H3K36me3 modification by SETD2 is essential for Col11a2 and Sema3e transcription to maintain dentinogenesis in mice

doi: 10.1242/dev.204352

Figure Lengend Snippet: Setd2 knockdown diminishes H3K36me3 occupancy at Col11a2 and Sema3e loci, accompanied by their transcriptional downregulation in the odontoblasts. (A) Heat maps showing the normalized read density of H3K36me3 CUT&Tag signals in the odontoblast-like cells with Setd2 knockdown compared with control cells transfected with scramble siRNA spanning 5 kb upstream of the transcription start site (TSS) to 5 kb downstream of the transcription end site (TES) ( n =2). (B) Venn diagram showing the number of genes with differential expression in RNA-seq, the number of genes with decreased occupancy of H3K36me3 in CUT&Tag-seq, and the overlapping genes (e.g. Col11a2 and Sema3e ). (C) GO analysis of the overlapping genes in B showing the enriched biological process terms. (D) Uniform manifold approximation and projection plots using the dataset illustrate cell clustering of P3.5 mouse molar cells and the presence of the transcripts for Sema3e and Col11a2 in the odontoblast cluster. Dashed circles indicate the cluster of odontoblasts. (E) H3K36me3 CUT&Tag-seq results showing its peaks at the gene loci of Col11a2 and Sema3e in the odontoblast-like cells with or without Setd2 knockdown. (F) CUT&Tag-qPCR analysis of H3K36me3 occupancy at Col11a2 and Sema3e gene loci, using isotype IgG as a negative control to assess non-specific binding. The quantification data are presented as mean±s.d. and were analyzed by two-tailed unpaired Student's t -test ( n =3). (G) The odontoblasts in the molars of control and Dmp1 -Cre ;Setd2 fl/fl mice were collected using laser-capture microdissection. RNAs were extracted, followed by RT-qPCR to validate the changes in the mRNA levels of Col11a2 and Sema3e . The quantification data are presented as mean±s.d. and were analyzed by two-tailed unpaired Student's t -test ( n =3). CTR, control mice; ns, not significant; Scr, scramble. *** P <0.001; **** P <0.0001.

Article Snippet: The concentrations of primary antibodies applied were as follows: DMP1 (1:200; Abclonal), DSPP (1:200; Novus Biologicals), p-AKT1 (1:200; Immunoway), SEMA3E (1:200; R&D Systems) and COL11A2 (1:200; Immunoway) (see ).

Techniques: Knockdown, Control, Transfection, Quantitative Proteomics, RNA Sequencing, Negative Control, Binding Assay, Two Tailed Test, Laser Capture Microdissection, Quantitative RT-PCR

Journal: Development (Cambridge, England)

Article Title: H3K36me3 modification by SETD2 is essential for Col11a2 and Sema3e transcription to maintain dentinogenesis in mice

doi: 10.1242/dev.204352

Figure Lengend Snippet: SEMA3E and COL11A2 promote activation of AKT1, thus regulating odontoblast differentiation. (A) IF and the quantitative analysis of the fluorescence intensity of COL11A2, SEMA3E and p-AKT1 in the odontoblast layer of mouse molars. The quantification data are presented as mean±s.d. and were analyzed by two-tailed unpaired Student's t -test ( n =3). ** P <0.01; *** P <0.001; **** P <0.0001. (B) mDPCs were obtained from control and Dmp1 -Cre ;Setd2 fl/fl mice and cultured in DM for 5 days, followed by western blot assays of COL11A2, SEMA3E, collagen I, DSPP, DMP1, AKT1 and p-AKT1. β-Actin was used as a loading control. (C) mDPCs were transfected with siRNAs targeting Setd2 , Sema3e and Col11a2 alone or in combination, followed by western blot analysis of collagen I, DSPP, DMP1, AKT1, p-AKT1, COL11A2 and SEMA3E. β-Actin served as a loading control. CTR, control mice; Scr, scramble.

Article Snippet: The concentrations of primary antibodies applied were as follows: DMP1 (1:200; Abclonal), DSPP (1:200; Novus Biologicals), p-AKT1 (1:200; Immunoway), SEMA3E (1:200; R&D Systems) and COL11A2 (1:200; Immunoway) (see ).

Techniques: Activation Assay, Fluorescence, Two Tailed Test, Control, Cell Culture, Western Blot, Transfection

Journal: Development (Cambridge, England)

Article Title: H3K36me3 modification by SETD2 is essential for Col11a2 and Sema3e transcription to maintain dentinogenesis in mice

doi: 10.1242/dev.204352

Figure Lengend Snippet: Schematic illustrating the role of SETD2 in regulating odontoblast differentiation and dentinogenesis. SETD2 catalyzes the trimethylation of H3K36. SETD2-mediated H3K36me3 is correlated with the expression of Col11a2 and Sema3e , upregulation of which facilitates AKT1 activation, promoting odontoblast differentiation and dentin formation.

Article Snippet: The concentrations of primary antibodies applied were as follows: DMP1 (1:200; Abclonal), DSPP (1:200; Novus Biologicals), p-AKT1 (1:200; Immunoway), SEMA3E (1:200; R&D Systems) and COL11A2 (1:200; Immunoway) (see ).

Techniques: Expressing, Activation Assay

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: Analysis of Sema3E and Plexin D1 levels in IPF Patients. A) ELISA analysis comparing Sema3E levels in the plasma of patients with IPF ( n = 63) and control subjects ( n = 24). B) Representative western blot analysis depicting Sema3E expression in plasma samples from IPF patients. C) Representative western blot analysis illustrating Sema3E and Plexin D1 expression in lung tissue samples from control subjects ( n = 10) and IPF patients ( n = 12). D–G) Correlation analysis between Sema3E levels and clinical parameters including (D) FVC%pred, (E) DLCO%pred, (F) TLC%pred, and (G) FEV1%pred in IPF patients ( n = 32). H) Co‐immunostaining of Sema3E and α‐SMA in lung sections from patients with IPF( n = 3) and control subjects( n = 3). I) Co‐immunostaining of Plexin D1 and α‐SMA in lung sections from patients with IPF( n = 3) and control subjects( n = 3). Nuclei were stained blue with Diamidino‐2‐phenylindole dihydrochloride (DAPI), and images were captured under original magnification ×400. Data are presented as the mean ± SEM. Statistical analyses were performed using unpaired t‐tests and Pearson's correlation. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significance levels. (FVC%pred: Forced Vital Capacity % predicted; TLC%pred: Total Lung Capacity % predicted; DLCO%pred: Diffusing Capacity of the Lung for Carbon Monoxide % predicted; FEV1%pred: Forced Expiratory Volume in 1 second % predicted.).

Article Snippet: Antibodies for Sema3E (AF3239, R&D Systems, USA), Plexin D1(AF4160, R&D Systems, USA), Fibronectin(15613‐1‐AP, Proteintech, China), Collagen Type I(66761‐1, Proteintech, China), α‐SMA(119 245, CST, USA), Phospho‐HER2/ErbB2 (2243, CST, USA), Phospho‐AKT(4060, CST, USA), Phospho‐p44/42 MAPK(4370, CST, USA), ErbB2(18299‐1‐AP, Proteintech, China), AKT (4691, CST, USA), p44/42 MAPK(4695, CST, USA), GADPH(AC001, Abclonal, China), Phospho‐SMAD2(# 8828, CST, USA), Phospho‐SMAD3(#9520, CST, USA), SMAD2/3(#3102, CST, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, Western Blot, Expressing, Immunostaining, Staining

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: Analysis of Sema3E and Plexin D1 levels in mice with BLM induction. A) Co‐immunostaining of Sema3E and α‐SMA in lung sections from the BLM‐induced mouse model ( n = 3) and saline controls ( n = 3). B) Co‐immunostaining of Plexin D1 ( n = 3) and α‐SMA ( n = 3) in lung sections from the BLM‐induced mouse model and saline controls. Nuclei were counterstained blue with DAPI, and images were captured at an original magnification of ×400. C) Western blot analysis depicting the expression levels of Sema3E and Plexin D1 in lung homogenates from saline‐treated ( n = 5) and BLM‐induced ( n = 5) mouse models. D) ELISA analysis comparing Sema3E levels in the plasma of the saline‐treated mice ( n = 5) and BLM‐induced mouse model( n = 5). Data are presented as the mean ± SEM. Statistical analyses were performed using unpaired t‐tests. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies for Sema3E (AF3239, R&D Systems, USA), Plexin D1(AF4160, R&D Systems, USA), Fibronectin(15613‐1‐AP, Proteintech, China), Collagen Type I(66761‐1, Proteintech, China), α‐SMA(119 245, CST, USA), Phospho‐HER2/ErbB2 (2243, CST, USA), Phospho‐AKT(4060, CST, USA), Phospho‐p44/42 MAPK(4370, CST, USA), ErbB2(18299‐1‐AP, Proteintech, China), AKT (4691, CST, USA), p44/42 MAPK(4695, CST, USA), GADPH(AC001, Abclonal, China), Phospho‐SMAD2(# 8828, CST, USA), Phospho‐SMAD3(#9520, CST, USA), SMAD2/3(#3102, CST, USA).

Techniques: Immunostaining, Saline, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: The impact of Sema3E‐Plexin D1 on the differentiation, proliferation, and migration of fibroblasts. A) Western blot analysis of Sema3E expression in primary human lung fibroblasts (PHLFs) following stimulation with different concentrations of TGFβ1 for 48 h. B) Measurement of Sema3E concentration in culture media by ELISA. PHLFs were stimulated with TGF‐β1, and Sema3E concentration was assessed after 48 h. C,D) Western blot and RT‐qPCR analysis of Fibronectin, Col1a1, and α‐SMA protein and mRNA expression in Sema3E siRNA or Scrambled siRNA‐treated PHLFs following TGF‐β1 induction. E,F) Western blot and RT‐qPCR analysis of Fibronectin, Col1a1, and α‐SMA protein and mRNA expression in PlexinD1 siRNA or Scrambled siRNA‐treated PHLFs following TGF‐β1 induction. G) Representative results for EdU staining in Sema3E siRNA or Scrambled siRNA‐treated PHLFs. H) Representative results for Transwell assay in Sema3E siRNA or Scrambled siRNA‐treated PHLFs. I) Representative results for EdU staining in Plexin D1 siRNA or Scrambled siRNA‐treated PHLFs. J) Representative results for Transwell assay in Plexin D1 siRNA or Scrambled siRNA‐treated PHLFs. Images were captured under original magnification ×200. Data are presented as the mean ± SEM of three independent experiments. Statistical analyses were performed using unpaired t‐tests and one‐way ANOVA test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies for Sema3E (AF3239, R&D Systems, USA), Plexin D1(AF4160, R&D Systems, USA), Fibronectin(15613‐1‐AP, Proteintech, China), Collagen Type I(66761‐1, Proteintech, China), α‐SMA(119 245, CST, USA), Phospho‐HER2/ErbB2 (2243, CST, USA), Phospho‐AKT(4060, CST, USA), Phospho‐p44/42 MAPK(4370, CST, USA), ErbB2(18299‐1‐AP, Proteintech, China), AKT (4691, CST, USA), p44/42 MAPK(4695, CST, USA), GADPH(AC001, Abclonal, China), Phospho‐SMAD2(# 8828, CST, USA), Phospho‐SMAD3(#9520, CST, USA), SMAD2/3(#3102, CST, USA).

Techniques: Migration, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Staining, Transwell Assay

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: P61‐Sema3E promotes differentiation, proliferation, and migration of fibroblasts through Plexin D1. A) Verification of two recombinant proteins, P87‐Sema3E and P61‐Sema3E, by western blot. B) Western blot analysis of the levels of Fibronectin, Col1a1, and α‐SMA in primary human lung fibroblasts (PHLFs) after stimulation with different concentrations of P61‐Sema3E for 48 h. C) Representative results for EdU staining in PHLFs treated with different concentrations of P61‐Sema3E for 48 h. D) Representative results for Transwell assay in PHLFs treated with different concentrations of P61‐Sema3E for 48 h. E) Western blot analysis of Fibronectin, Col1a1, and α‐SMA expression in PlexinD1 siRNA or Scrambled siRNA‐treated PHLFs following P61‐Sema3E induction. F) Representative results for EdU staining in PlexinD1 siRNA or Scrambled siRNA‐treated PHLFs following P61‐Sema3E stimulation for 48 h. G) Representative results for Transwell assay in Plexin D1 siRNA or Scrambled siRNA‐treated PHLFs following P61‐Sema3E stimulation for 48 h. Images were captured under original magnification ×200. Data are presented as the mean ± SEM of three independent experiments. Statistical analyses were performed using unpaired t‐tests and one‐way ANOVA test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies for Sema3E (AF3239, R&D Systems, USA), Plexin D1(AF4160, R&D Systems, USA), Fibronectin(15613‐1‐AP, Proteintech, China), Collagen Type I(66761‐1, Proteintech, China), α‐SMA(119 245, CST, USA), Phospho‐HER2/ErbB2 (2243, CST, USA), Phospho‐AKT(4060, CST, USA), Phospho‐p44/42 MAPK(4370, CST, USA), ErbB2(18299‐1‐AP, Proteintech, China), AKT (4691, CST, USA), p44/42 MAPK(4695, CST, USA), GADPH(AC001, Abclonal, China), Phospho‐SMAD2(# 8828, CST, USA), Phospho‐SMAD3(#9520, CST, USA), SMAD2/3(#3102, CST, USA).

Techniques: Migration, Recombinant, Western Blot, Staining, Transwell Assay, Expressing

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: P61‐Sema3E produces a pro‐fibrotic effect through the activation of ErbB2. A) Western blot analysis of the levels of ErbB2, P‐ErbB2, AKT, P‐AKT, ERK1/2, and P‐ERK1/2 in primary human lung fibroblasts (PHLFs) after stimulation with different concentrations of P61‐Sema3E for 2 h. B) Western blot analysis of ErbB2, P‐ErbB2, AKT, P‐AKT, ERK1/2, and P‐ERK1/2 expression in PHLFs treated with PlexinD1 siRNA or Scrambled siRNA following P61‐Sema3E induction. C) Co‐immunoprecipitation analysis of Plexin D1 and ErbB2, FLAG‐tagged Plexin D1 plasmid were transfected into PHLFs. D) Co‐immunoprecipitation analysis of Plexin D1 and ErbB2, PHLFs were treated with P61‐Sema3E for 2 h. E) Western blot analysis of Fibronectin, Col1a1, and α‐SMA expression in PHLFs treated with the ErbB2 inhibitor Lapatinib or PBS following P61‐Sema3E stimulation for 48 h. Data are represented as the mean ± SEM of three independent experiments. Statistical analyses were performed using one‐way ANOVA test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies for Sema3E (AF3239, R&D Systems, USA), Plexin D1(AF4160, R&D Systems, USA), Fibronectin(15613‐1‐AP, Proteintech, China), Collagen Type I(66761‐1, Proteintech, China), α‐SMA(119 245, CST, USA), Phospho‐HER2/ErbB2 (2243, CST, USA), Phospho‐AKT(4060, CST, USA), Phospho‐p44/42 MAPK(4370, CST, USA), ErbB2(18299‐1‐AP, Proteintech, China), AKT (4691, CST, USA), p44/42 MAPK(4695, CST, USA), GADPH(AC001, Abclonal, China), Phospho‐SMAD2(# 8828, CST, USA), Phospho‐SMAD3(#9520, CST, USA), SMAD2/3(#3102, CST, USA).

Techniques: Activation Assay, Western Blot, Expressing, Immunoprecipitation, Plasmid Preparation, Transfection

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: Downregulation of Sema3E attenuates BLM‐induced pulmonary fibrosis. A,B) Histological analysis of lung fibrosis severity in mice following BLM induction. (A) Representative images of lung sections stained with H&E, Masson's trichrome, and Sirius red to assess fibrosis severity. (B) Bar graph showing the quantitative mean score of fibrosis severity. Samples include AAV9‐NC saline mice ( n = 5), AAV9‐ShSema3E saline mice ( n = 5), AAV9‐NC BLM mice ( n = 5), and AAV9‐ShSema3E BLM mice ( n = 5). C) Quantification of hydroxyproline contents in AAV9‐NC mice and AAV9‐ShSema3E mice after BLM challenge. D,E) Western blot and RT‐qPCR analysis of Fibronectin, Col1a1, and α‐SMA protein and mRNA expression in lung homogenates from the mentioned mouse groups. F) Western blot analysis of ErbB2, P‐ErbB2, AKT, P‐AKT, ERK1/2, and P‐ERK1/2 expression in lung homogenates from the mentioned mouse groups. Samples include AAV9‐NC saline mice ( n = 3), AAV9‐ShSema3E saline mice ( n = 3), AAV9‐NC BLM mice ( n = 3), and AAV9‐ShSema3E BLM mice ( n = 3). Data are represented as the mean ± SEM. Statistical analyses were performed using one‐way ANOVA tests. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Antibodies for Sema3E (AF3239, R&D Systems, USA), Plexin D1(AF4160, R&D Systems, USA), Fibronectin(15613‐1‐AP, Proteintech, China), Collagen Type I(66761‐1, Proteintech, China), α‐SMA(119 245, CST, USA), Phospho‐HER2/ErbB2 (2243, CST, USA), Phospho‐AKT(4060, CST, USA), Phospho‐p44/42 MAPK(4370, CST, USA), ErbB2(18299‐1‐AP, Proteintech, China), AKT (4691, CST, USA), p44/42 MAPK(4695, CST, USA), GADPH(AC001, Abclonal, China), Phospho‐SMAD2(# 8828, CST, USA), Phospho‐SMAD3(#9520, CST, USA), SMAD2/3(#3102, CST, USA).

Techniques: Staining, Saline, Western Blot, Quantitative RT-PCR, Expressing

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: Sema3E deficiency in fibroblasts protects mice from BLM‐induced lung injury and fibrosis. A,B) Representative lung sections from Sema3E‐C and Sema3E‐CKO mice treated with saline or BLM, stained with H&E, Masson's Trichrome, and Sirius Red (Panel A, left). Panel B (right) shows quantitative fibrosis scores. Each group consisted of five mice ( n = 5). C) Quantification of hydroxyproline contents in Sema3E‐CKO and Sema3E‐C mice after BLM challenge. D,E) Western blot and RT‐qPCR analyses of Fibronectin, Col1a1, and α‐SMA protein and mRNA levels in lung homogenates from each group ( n = 5). F) Western blot analysis of ErbB2, P‐ErbB2, AKT, P‐AKT, ERK1/2, and P‐ERK1/2 in lung homogenates from the same groups of three mice( n = 3). Data are expressed as the mean ± SEM. Statistical significance was determined by one‐way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Antibodies for Sema3E (AF3239, R&D Systems, USA), Plexin D1(AF4160, R&D Systems, USA), Fibronectin(15613‐1‐AP, Proteintech, China), Collagen Type I(66761‐1, Proteintech, China), α‐SMA(119 245, CST, USA), Phospho‐HER2/ErbB2 (2243, CST, USA), Phospho‐AKT(4060, CST, USA), Phospho‐p44/42 MAPK(4370, CST, USA), ErbB2(18299‐1‐AP, Proteintech, China), AKT (4691, CST, USA), p44/42 MAPK(4695, CST, USA), GADPH(AC001, Abclonal, China), Phospho‐SMAD2(# 8828, CST, USA), Phospho‐SMAD3(#9520, CST, USA), SMAD2/3(#3102, CST, USA).

Techniques: Saline, Staining, Western Blot, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: Furin inhibitor suppresses differentiation, proliferation, and migration of fibroblasts in IPF. A) Western blot analysis of Furin levels in primary human lung fibroblasts (PHLFs) exposed to varying concentrations of TGF‐β1 for 48 h. B) Western blot analysis of P61‐Sema3E, Fibronectin, Col1a1, and α‐SMA expression in PHLFs treated with different concentrations of the Furin inhibitor Hexa‐D‐arginine following TGF‐β1 induction for 48 h. C) Representative images of EdU staining in PHLFs treated with Hexa‐D‐arginine after TGF‐β1 induction for 48 h. D) Representative images from the Transwell assay in PHLFs treated with Hexa‐D‐arginine following TGF‐β1 induction for 48 h. Images were captured at ×200 magnification. E,G) Histological assessment of lung fibrosis severity in mice following BLM induction. (E) Left panel: Representative lung sections stained with H&E, Masson's trichrome, and Sirius Red to evaluate fibrosis severity. (G) Right panel: Bar graph quantifying fibrosis severity scores (Saline: n = 5, BLM: n = 5, BLM + Hexa‐D‐arginine: n = 5). F) Schematic representation of the experimental design. Mice received intratracheal instillation of 2 mg kg −1 BLM and were administered intraperitoneal injections of Hexa‐D‐arginine starting on day 7 until the end of the experiment. H) Quantification of hydroxyproline content in lung tissues. I) Western blot analysis of Fibronectin, Col1a1, and α‐SMA protein expression in lung homogenates from the indicated mouse groups (Saline: n = 5, BLM: n = 5, BLM + Hexa‐D‐arginine: n = 5). Data are presented as the mean ± SEM from three independent experiments. Statistical significance was determined using one‐way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: Antibodies for Sema3E (AF3239, R&D Systems, USA), Plexin D1(AF4160, R&D Systems, USA), Fibronectin(15613‐1‐AP, Proteintech, China), Collagen Type I(66761‐1, Proteintech, China), α‐SMA(119 245, CST, USA), Phospho‐HER2/ErbB2 (2243, CST, USA), Phospho‐AKT(4060, CST, USA), Phospho‐p44/42 MAPK(4370, CST, USA), ErbB2(18299‐1‐AP, Proteintech, China), AKT (4691, CST, USA), p44/42 MAPK(4695, CST, USA), GADPH(AC001, Abclonal, China), Phospho‐SMAD2(# 8828, CST, USA), Phospho‐SMAD3(#9520, CST, USA), SMAD2/3(#3102, CST, USA).

Techniques: Migration, Western Blot, Expressing, Staining, Transwell Assay, Saline