Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: List of primers and corresponding annealing temperatures for RT 2 -PCR.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Sequencing

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 confers TMZ-resistance on GBM cells. ( A ) Confirmation of PDCD10 knockdown in lentiviral transduced U87 and T98g cells by RT 2 -PCR ( a ), western blot ( b ), and semi-quantitation of the blots ( c ). ev and sh: empty vector- and PDCD10 shRNA-transduced cells, respectively. IOD: integrated optical density. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev. ( B ) Knockdown of PDCD10 in GBM cells leads to a resistance to TMZ-induced cell death. U87 and T98g cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h. Thereafter, TMZ was washed-out. Remaining viable cells were cultured in the TMZ-free medium for 3 d, which is defined as the post-treatment phase. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). MTT assay was performed to determine the viability of cells at 72 h after TMZ treatment (treatment phase) and 3 d after washing-out of TMZ (post-treatment phase). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev; ##, p < 0.01; ###, p < 0.0001, compared with evC in the same phase; +++, p < 0.001, compared with corresponding group in treatment phase.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Western Blot, Quantitation Assay, Plasmid Preparation, shRNA, Cell Culture, Control, MTT Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 enhances cell viability after rechallenge with TMZ in regrown cells (RG) generated from the established acquired TMZ-resistant model. ( A ) Confirmation of PDCD10 knockdown in shU87-RG and shT98g-RG cells by RT 2 -PCR ( a ) and by FACS of respective transduced cells that expressed red-fluorescence protein (RFP) and green-fluorescence protein (GFP) ( b ). *, p < 0.05, compared with ev. ( B ) MTT assay in RG cells in treatment phase and post-treatment phase. MTT assay was performed with ev/shU87 and ev/shT98g cells that received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h (treatment phase) and 2 d and 4 d after washing-out TMZ (post-treatment phase, without reseeding). Control cells (C) received vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; ***, p < 0.001, compared with evRG C (72 h); ###, p < 0.001, compared with evRG-TMZ (72 h). ( C ) MTT assay in RG cells in a second post-treatment model with reseeding. ev/shU87-RG and ev/shT98g-RG cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ, respectively, for 72 h. Thereafter, TMZ–containing media and dead cells were washed-out, and the viable cells were harvested and reseeded at the same density, followed by 2, 4, and 6 d of culture in drug-free medium. A significantly more rapid regrowth was observed in both TMZ-treated shU87-RG and shT98g-RG cells, compared with the corresponding evRG cells after reseeding and culturing in drug-free media. *, p < 0.05; ***, p < 0.001, compared with corresponding evRG.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Generated, Fluorescence, MTT Assay, Control

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Overexpression of PDCD10 sensitizes GBM cells to TMZ treatment. ( A ) Confirmation of overexpression of PDCD10 in lentiviral transduced U87 and T98g cells by RT 2 -PCR ( a ) and western blot ( b ) and semi-quantitation of the blots ( c ). Western blotting with anti-V5 antibody distinguishes between the expression of transgenic C-terminal V5-tagged PDCD10 protein and endogenous protein. ev and ox: empty vector-transduced and PDCD10-overexpressing cells, respectively. IOD: integrated optical density. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev. ( B ) Overexpression of PDCD10 significantly reduces cell viability in a concentration-dependent manner after 72 h of TMZ treatment in both oxU87 ( a ) and oxT98g ( b ) cells. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with corresponding ev groups. ( C ) Overexpression of PDCD10 sensitizes GBM cells to TMZ treatment 72 h after TMZ treatment (treatment phase) and 3 d after washing-out TMZ (post-treatment phase). ev/oxU87 and ev/oxT98g cells received the treatment with 150 µM ( a ) and 300 µM ( b ) of TMZ for 72 h, respectively. Thereafter, TMZ-containing medium and dead cells were washed-out and the viable cells were further cultured in drug-free medium for 3 d followed by MTT assay. Control cells (C) were treated with vehicle DMSO (0.1% and 0.2% for U87 and T98g, respectively). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with corresponding ev groups; +++, p < 0.001, compared with corresponding evC in the treatment phase; #, p < 0.05, ###, p < 0.001, compared with corresponding evC in the same phase.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Over Expression, Western Blot, Quantitation Assay, Expressing, Transgenic Assay, Plasmid Preparation, Concentration Assay, Control, Cell Culture, MTT Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: DNA replication in response to TMZ treatment is dependent on PDCD10 expression. DNA replication was detected by EdU incorporation followed by FACS at 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and -culturing in drug-washout media (post-treatment phase). ev/shT98g-RG and ev/oxT98g cells received 500 and 300 µM TMZ, respectively. ( A , C ) Histograms of EdU-positive (EdU+) and -negative (EdU−) cell populations in ev/shT98g-RG and ev/oxT98g cells, respectively. ( B , D ) Bar graphs of EdU+/− populations based on the corresponding histograms in ( A , C ). Knockdown of PDCD10 leads to an increase in DNA replication in both the treatment and post-treatment phases of T98g-RG cells, whereas overexpression of PDCD10 suppresses DNA replication in response to TMZ treatment. The data are representative of at least three independent experiments.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Expressing, Knockdown, Over Expression

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Alteration in cell cycle checkpoints in response to TMZ treatment is dependent on PDCD10 expression. Cell cycle assay was performed by FACS after 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and -culturing in drug-washout media (post-treatment phase). ev/shT98g-RG and ev/oxT98g cells received 500 and 300 µM TMZ, respectively. ( A , C ) are representative of cell cycle histograms in ev/shT98g-RG and ev/oxT98g cells, respectively. DNA content-based cell cycle distributions were defined using FlowJo with the Dean–Jett–Fox algorithm and presented in histograms. Each cell cycle phase is shown in different colors: G0/G1- (blue), S- (yellow), and G2/M-phase (green). ( B , D ) Stacked bar graphs of the distribution of the cell population in the cell cycle based on the corresponding histograms in ( A , C ). Knockdown of PDCD10 leads to the escape of cells from G2/M arrest and increases the population in the S phase (DNA replication phase) in both the treatment and post-treatment phases, whereas overexpression does the opposite. The data are representative of at least three independent experiments.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Expressing, Cell Cycle Assay, Knockdown, Over Expression

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 in T98g-RG cells leads to deregulation of DNA damage response (DDR) genes. ev/shT98g-RG and ev/oxT98g cells received 300 µM of TMZ or vehicle (0.2% DMSO) treatment (no TMZ treatment). Cells were harvested for PCR detection of DDR genes after 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and culturing in drug-free media (post-treatment phase). ( A ) Expression of MGMT in T98g-RG ( a ) cells and ev/oxT98g ( b ) cells in the no treatment, treatment (300 µM, 72 h), and post-treatment (3 d after washing out) phases. ( B ) Western blot ( a ) and semi-quantitation of the blots ( b ) of the MGMT protein expression in ev/shT98g-RG and ev/oxT98g cells. ( C ) Expression of DDR genes ( MSH2 , MSH6, and PMS2 ) in T98g-RG cells in the no treatment ( a ), treatment ( b ), and post-treatment phases ( c ), respectively. ( D ) Expression of DDR genes in ev/oxT98g cells in the no treatment ( a ), treatment ( b ), and post-treatment phases ( c ), respectively. *, p < 0.05; **, p < 0.01 and ***, p < 0.001, compared with corresponding ev.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Expressing, Western Blot, Quantitation Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: PDCD10 expression determines the colony formation capacity of GBM cells. ev/shT98g-RG ( A ) and ev/oxT98g ( B ) cells received 300 µM of TMZ or vehicle (0.2% DMSO) treatment. Cells were harvested for colony formation assay in a 12-well plate in triplicate after 72 h of TMZ treatment (treatment phase) and at 3 d after TMZ-washing-out and culturing in drug-free media (post-treatment phase). The number of colonies was quantified after staining with 0.5% crystal violet using the ImageJ software (version 1.54j). Representative images of colony formation in ev/shT98g-RG and ev/oxT98g are shown in ( Aa ) and ( Ba ), respectively. Quantitative analysis of the colony numbers is presented in ( Ab ) and ( Bb ) for ev/shT98g-RG and ev/oxT98g cells, respectively. ***, p < 0.001, compared with ev; ##, p < 0.01 and ###, p < 0.001, compared with corresponding C; +++, p < 0.001, compared with corresponding group in the treatment phase.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Expressing, Colony Assay, Staining, Software

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Knockdown of PDCD10 enhances the self-renewal capacity of U87-RG cells and GSCs generated from the parental cell line U87-RG (U87-RG-GSCs), and increases the expression of stem cell markers in RG-GSCs. ( A ) Representative images of neurospheres derived from U87-RG cells and U87-RG-GSCs. Scale bar: 100 µm. ( B ) Quantitative analysis of neurospheres formation efficiency (SFE). ***, p < 0.001, compared with corresponding ev; ###, p < 0.001, compared with corresponding parental cells. ( C ) Knockdown of PDCD10 increases the mRNA expression of stemness genes in RG-GSCs. The expression of stem cell markers Nestin and KLF4 was detected by RT 2 -PCR in untreated U87-RG-GSCs, and in shU87-RG-GSCs treated with TMZ (150 µM) for 72 h. *, p < 0.05; ***, p < 0.001, compared with corresponding ev; #, p < 0.05; ###, p < 0.001, compared with evC; +, p < 0.05, compared with corresponding C. ( D ) Knockdown of PDCD10 enhances the viability of parental U87-RG cells and their GSC variants. U87-RG cells (left) and U87-RG-GSCs (right) received TMZ (150 M) or vehicle DMSO (0.1%) treatment for 72 h. Cell viability was detected after 72 h of treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with corresponding ev; #, p < 0.05; ##, p < 0.01, compared with evC; +, p < 0.05, ++, p < 0.01, compared with corresponding parental cells. ( E ) Knockdown of PDCD10 reduces the mRNA expression of MMR genes ( MSH2 , MSH6 , and PMS2 ) in U87-RG-GSCs. U87-RG-GSCs received treatment of 150 µM TMZ or vehicle (0.1% DMSO; no treatment). Cells were harvested for PCR detection of MMR genes after 72 h of treatment. Expression of MMR genes in non-treated ( Ea ) and TMZ-treated U87-RG-GSCs ( Eb ). *, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ev.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Generated, Expressing, Derivative Assay

Journal: Cells

Article Title: PDCD10 Is a Key Player in TMZ-Resistance and Tumor Cell Regrowth: Insights into Its Underlying Mechanism in Glioblastoma Cells

doi: 10.3390/cells13171442

Figure Lengend Snippet: Schematic summary of the role and mechanism of PDCD10 in acquired TMZ-resistance. Knockdown of PDCD10 (shPDCD10) in GBM cells significantly increased cell survival in response to TMZ treatment, and strongly promoted tumor cell regrowth in the post-treatment phase, which collectively accounted for acquired TMZ-resistance. Mechanism studies revealed that the loss of PDCD10 modulated the expression of DNA damage response genes (i.e., upregulating MGMT and downregulating MMR genes MSH2 , MSH6 , and PMS2 ), and altered the cell cycle process, as evidenced by the evasion of tumor cells from arrest at the G2/M phase, and the increase in tumor cells in the proliferating S phase. In addition, shPDCD10-GBM cells exhibited higher cell plasticity, as demonstrated by an increased capacity for colony formation and transformation of shPDCD10-GBM cells into GSC-like cells that expressed higher levels of the stem cell markers Nestin and KLF4. In support of these findings, overexpression of PDCD10 (oxPDCD10) induced contrary changes in the molecular and cell behaviors observed in shPDCD10-GBM cells, increasing the sensitivity of oxPDCD10-GBM cells to TMZ treatment and suppressing tumor cell regrowth after TMZ treatment. Our results indicate that PDCD10 plays a pivotal role in acquired TMZ-resistance and thus represents a promising target for perturbing TMZ-resistance and tumor recurrence.

Article Snippet: For constitutive knockdown in T98g cells (shT98g), a lentiviral shRNA vector for human PDCD10 (OriGene, Rockville, MD, USA, cat# TL302576) was used.

Techniques: Knockdown, Expressing, Transformation Assay, Over Expression

Journal: Journal of cellular and molecular medicine

Article Title: MicroRNA-425-5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10.

doi: 10.1111/jcmm.12742

Figure Lengend Snippet: Fig. 5 miR-425-5p regulates chemoresistance through PDCD10. (A) Western blot analysis of PDCD10 protein level in HCT116-R cells cotransfected with miR-425-5p inhibitor and scramble or PDCD10 siRNAs. b-actin was used as loading control. (B) Western Blot analysis of PDCD10 protein level in HCT116-R cells transfected with PDCD10 cDNA. b-actin was used as loading control. (C) Transient knockdown of PDCD10 reversed the chemosensitizing effects of miR-425-5p inhibitor in HCT116-R cells with 5-FU (5 lM) or OX (10 lM) treatments. Data represent the mean S.D., n = 2. *P < 0.05. 425 inh: miR-425-5p inhibitor; Scr: scramble miRNA; (D) Overexpression of PDCD10 phenocopied the effects of miR-425-5p inhi- bitor to sensitize HCT116-R to 5-FU (5 lM) or OX (10 lM). Data represent mean S.D., n = 3. *P < 0.05. (E) Caspase activity in control and PDCD10 overexpression cells treated with or without 5-FU and OX. Data represent mean S.D., n = 3. **P < 0.01.

Article Snippet: Transfection of miR-425-5p inhibitor, miR-425-5p mimic and its nonspecific control (Invitrogen, Hampton, VA, USA) were performed according to the manual provided with the siPORTM NeoFXTM Transfection Agent (Ambion, Grand Island, NE, USA). pLenti-C-Myc-DDK PDCD10 cDNA (RC221815L1) and two individual siRNAs (SR307707) targeting PDCD10 were obtained from Origene. siRNAs were transfected with Lipofectamine RNAi Max reagent (Invitrogen, Grand Island, NE, USA) as per manufacturer’s protocol. cDNA transfections were performed with Lipofectamine LTX reagent (Invitrogen) as per manufacturer’s protocol.

Techniques: Western Blot, Control, Transfection, Knockdown, Over Expression, Activity Assay

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: Cavernomas have clonal origins. Cdh5(PAC)- Cre-ER T2 /R26R- Confetti or Cdh5(PAC)- Cre-ER T2 / Ccm3 fl/fl /R26R- Confetti mice following tamoxifen induction of the four fluorescent proteins and of Ccm3 deletion at 1 day after birth, with analysis at day 8. a Representative images of vessels from retinas of Ccm3 +/+ (upper panel) or Ccm3 −/− (lower panel) mice stained for Isolectin B4. Ccm3 +/+ retina showed random expression of the four fluorophores to form a mosaic; the retina vasculature of Ccm3 −/− mice showed cerebral cavernous malformations and clonal regions composed of cells of the same colour. b Representative images of cerebellum from Ccm3 −/− mice with vessels stained for Podocalyxin (left panel) or Isolectin B4 (central and right panels). Small cavernomas are mostly formed by cells of the same colour, while larger cavernomas show a mixed composition. c Quantification of clonality of the lesions, divided according to small and large lesions on the basis of the longer diameter (cut-off, 60 μm). Lesions were considered clonal when composed of ≥80% cells of the same colour. Each dot represents the percentage of clonal lesions in an animal; data are means ± SE. Source data are provided as a Source Data file. d Representative tiling of the cerebellum from P8 Ccm3 −/− mice with vessels stained for Podocalyxin. e , f Three-dimensional (3D) reconstruction of representative ‘newly formed’, ‘small’, ‘large’ and ‘mulberry’ lesions. CGYR stands for CFP, GFP, YFP and RFP. Scale bars, 100 μm ( a , d , f ); 50 μm ( b ); 40 μm ( e ); white arrows point at lesions. See also Supplementary Movies – for animated 3D reconstruction of these lesions

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques: Staining, Expressing

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: The slow progression model of cerebral cavernous malformation (CCM) develops large lesions. A chronic model of CCM was generated by treating Cdh5(PAC)- Cre-ER T2 / Ccm3 f/f /R26R- Confetti mice with low-dose tamoxifen. a Scheme of treatment with tamoxifen at P2 and analysis at P8, P14 and P30. b Representative photographs of whole brains from chronic P8, P14 and P30 mice; scale bar: 100 μm. c Representative tiling of a cerebellum at P14 showing the distribution of lesions; upper panel shows a projection from a 1-mm-thick section; lower panels show three-dimensional reconstruction of corresponding regions. Lower left panel was rotated by 90°; vessels were stained for Podocalyxin; scale bars: 1000 μm lower magnification, 300 μm higher magnification. d Representative confocal image of P30 retina stained for Isolectin B4 (black vessels) showing large cavernomas at the front; scale bar: 500 μm lower magnification, 100 μm higher magnification. e Representative confocal images of P30 brain stained for Isolectin B4 (black vessels) showing cavernomas in the hippocampus and the olfactory bulb; 1000 μm lower magnification, 500 μm higher magnification. f , g Quantification of total number and mean area of lesions in chronic P8, P14 and P30 mice. Data are means ± SE; p < 0.001 among groups (one-way analysis of variance (ANOVA)); * p < 0.01 (Tukey’s post hoc test). h Lesions were divided into four groups according to their size, as shown. Data are means ± SE. * p < 0.001 among groups (one-way ANOVA); p < 0.01 (Tukey’s post hoc test); a total of 1698 lesions have been counted from 5 animals at P7, 4 animals at P14 and 9 at P30. Source data are provided as a Source Data file

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques: Generated, Staining

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: Cavernomas are composed of clones of Ccm3 −/− cells that attract Ccm3 +/+ cells. Representative images of retina ( a , b ) and cerebellum ( c ) from chronic Cdh5(PAC)- Cre-ER T2 / Ccm3 f/f /R26R- Confetti mice at P30. The vessels are counterstained with Isolectin B4 ( a , b ) or Podocalyxin ( c ) and are displayed as greyscale. d Three-dimensional reconstruction of mulberry cavernomas from P14 brain. The vessels are counterstained with Podocalyxin; L stands for Lumen. Scale bars, 500 μm ( a ); 100 μm ( b ); 50 μm ( c ), 100 μm ( d ). e – g Analysis of clones in normal vessels of Cdh5(PAC)- Cre-ER T2 /R26R- Confetti mice and normal vessels and lesions of Cdh5(PAC)- Cre-ER T2 / Ccm3 f/f /R26R- Confetti mice. e Distribution of the four fluorophores among clones. Data are means ± SE; n = 3 mice in each group. f Quantification of the number of cells that compose each clone. Data are means ± SE; n = 3 mice in each group. g Distribution of clone size. Clones were divided according to the number of cells they are composed of; each dot represents an animal. A total of 2371 clones were analysed from 3 mice in each group. Source data are provided as a Source Data file

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques: Clone Assay

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: Large cavernomas are mosaic. a Representative images of chronic Cdh5(PAC)- Cre-ER T2 / Ccm3 f/f mice co-stained for KLF4 (green) and either ERG1 or COLLAGEN IV (red). b Quantification of the percentage of KLF4-positive endothelial cells lining the cavernomas of chronic P8, P14 and P30 mice. Data are means ± SE; p < 0.001 among groups (one-way analysis of variance); * p < 0.01 (Tukey’s post hoc test); n = 3 mice in each group. Source data are provided as a Source Data file. c – h Representative images of brain sections co-stained for PECAM1 (red) and mesenchymal markers (green), showing small lesions from P8 mice and large cavernomas from P14 mice. White arrows, endothelial cells positive for mesenchymal markers within the lesions; yellow arrows, endothelial cells negative for mesenchymal markers in normal vessels; blue arrows, endothelial cells negative for mesenchymal markers within the lesions. Scale bars, 100 μm

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques: Staining

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: Ccm3 +/+ cells recruited into cavernomas undergo phenotypical modifications. a Representative images from chronic Cdh5(PAC)- Cre-ER T2 / Ccm3 f/f /R26-EYFP mice showing small lesion from P8 mice and large cavernoma from P14 mice cerebellum. b – f Brain sections immunostained for EYFP (green), PECAM1 (grey) and the mesenchymal markers KLF4 ( b ), ID1 ( c ), pSMAD3 ( d ), SCA1 ( e ) and αSMA (red) ( f ). White arrows, endothelial cells positive for EYFP in the cavernomas; yellow arrows, endothelial cells with no recombination in the cavernomas; blue arrows, endothelial cells in normal vessels. Scale bars, 100 μm

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques:

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: Ccm3 −/− cells form cavernomas and recruit Ccm3 +/+ cells in wild-type animals. a Endothelial cells were isolated from acute Cdh5(PAC)- Cre-ER T2 / Ccm3 fl/fl /R26R- Confetti at P7 and re-injected into the brains of adult wild-type mice. b Brain section showing the formation of aberrant vessels that comprise both Confetti -positive endothelial cells, which are Ccm3 −/− coming from the donor, as well as non-labelled Ccm3 +/+ endothelial cells coming from the host. c In the same way, endothelial cells were isolated from acute Cdh5(PAC)- Cre-ER T2 / Ccm3 fl/fl at P7 and re-injected into the brains of adult Cdh5(PAC)- Cre-ER T2 /R26R-Confetti mice treated with 2 mg of Tamoxifen 1 week before injection. d Brain section showing the formation of aberrant vessels, which comprise also Ccm3 +/+ Confetti -positive endothelial cells coming from the host. e Endothelial cells were isolated from acute Cdh5(PAC)- Cre-ER T2 / Ccm3 fl/fl at P7 and engrafted in a subcutaneous Matrigel plug into adult Cdh5(PAC)- Cre-ER T2 /R26R- Confetti mice. f Section of the plug showing the formation of aberrant vessels, which comprise both non-labelled Ccm3 −/− endothelial cells coming from the donor as well as Confetti -positive endothelial cells, thus Ccm3 +/+ coming from the host. White arrows, endothelial cells positive for Confetti ; yellow arrows, endothelial cells negative for Confetti . Scale bars, 100 μm

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques: Isolation, Injection

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: Ccm3 −/− cells recruit and induce endothelial-to-mesenchymal transition in Ccm3 +/+ cells. a , b Lung immortalised Ccm3 −/− endothelial cells expressing LifeAct-mCherry and Ccm3 +/+ cells expressing LifeAct-EGFP were co-cultured for 7 days, with spheroids formed, showing experimental scheme ( a ) and representative confocal images ( b ). c Non-labelled Ccm3 +/+ and LifeAct-mCherry expressing Ccm3 −/− cells were co-cultured for 7 days, with the spheroids formed detached by shaking the culture plate. The detached spheroids were collected and added to an already formed monolayer of LifeAct-EGFP Ccm3 +/+ cells (see also Supplementary Fig. for more details). d Representative confocal images of the basal and more apical levels of spheroids where Ccm3 −/− (red) have recruited Ccm3 +/+ cells (green). e Representative three-dimensional reconstructions of spheroids with Ccm3 +/+ cells (green) recruited. Scale bars 50 μm ( b , d ). f Quantification of migratory rates of Ccm3 +/+ endothelial cells assessed using the classical wound-healing assay. After the scratch, cells were incubated with medium conditioned by either Ccm3 +/+ cells (negative control) or Ccm3 −/− cells and without or with the BMP inhibitor DMH1. Data are means ± SE. p < 0.001 among groups (one-way analysis of variance (ANOVA)); * p < 0.01 (Tukey’s post hoc test); n = 3 independent experiments. g Labelled Ccm3 +/+ and Ccm3 −/− cells co-cultured as described above. After 7 days, spheroids were carefully detached, disaggregated and the cells separated by fluorescence-activated cell sorting (FACS). The cells that remained at the bottom were trypsinised and Ccm3 +/+ cells were separated by FACS. In parallel, Ccm3 −/− cells were cultured separately as positive control and processed together with spheroids. After FACS, gene expression was analysed by RT-qPCR. Data are means ± SE; p < 0.01 among groups (one-way ANOVA); * p < 0.01 (Tukey’s post hoc test); n = 4 independent experiments. h Ccm3 +/+ endothelial cells were incubated for 7 days with medium conditioned by either Ccm3 −/− cells or Ccm3 +/+ cells (negative control), and the gene expression was analysed by RT-qPCR. Data are means ± SE. * p < 0.01 (Student’s t test); n = 3 independent experiments. Source data are provided as a Source Data file

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques: Expressing, Cell Culture, Wound Healing Assay, Incubation, Negative Control, Fluorescence, FACS, Positive Control, Gene Expression, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: Endothelial progenitors are involved in cavernomas formation. a In situ hybridization for Procr using the RNAScope assay with chronic Ccm3 −/− cerebral cavernous malformation (CCM) mouse brains. Red, Procr probe; grey, endothelial cells stained with a combination of anti-COLLAGEN IV and anti-ERG1 antibodies. Black and green panels show endothelial cells with high expression of Procr, blue panel shows cells with no expression of Procr. b Quantification of the percentage of endothelial cells that express Procr within the lesions; n = 10 lesions from 3 animals. c Procr CreERT2-IRES-tdTomato/+ / Ccm3 f/f mice received tamoxifen injection at P1 and were analysed at P30. Representative images of the cerebellum stained for PECAM1 and whole-mount retina stained for Isolectin B4. d Fluorescence-activated cell sorting analysis on endothelial cells isolated from wild-type mice at P2. The plot shows the percentage of Procr + cells among the Cd45 − /Cd31 + endothelial cells. Data are means ± SE; n = 3 animals. e RNAseq analysis on endothelial cells isolated from the brain of wild-type mice at different ages, showing the reduction of Procr expression during development. Data are means ± SE from 3 animals each group. Source data are provided as a Source Data file. f Procr CreERT2-IRES-tdTomato/+ / Ccm3 f/f /R26-EYFP mice received tamoxifen injection at P1 and were analysed at P30. Representative images of whole-mount retina stained for Isolectin B4 and EYFP showing endothelial cells lining the lesion that underwent recombination. g – j Representative confocal images of brain sections double stained for PECAM1 (red) and the mesenchymal markers (green) KLF4 ( g ), SCA1 ( h ), FN1 ( i ) and αSMA ( j ). White arrows, cells lining the cavernoma; yellow arrows, cells in normal vessels. Scale bars, 500 μm ( c and f main panels); 100 μm ( a main panel, c and f magnifications), 50 μm ( g – j main panels); 20 μm ( a , g – j magnifications)

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques: In Situ Hybridization, RNAscope, Staining, Expressing, Injection, Fluorescence, FACS, Isolation

Journal: Nature Communications

Article Title: Endothelial cell clonal expansion in the development of cerebral cavernous malformations

doi: 10.1038/s41467-019-10707-x

Figure Lengend Snippet: Model for the onset and growth of cavernomas. Cavernoma-initiating cells undergo clonal expansion in response to Ccm3 loss and start to form cavernomas. In the further step, recombined cells recruit normal cells, which in turn, contribute to cavernoma expansion

Article Snippet: To generate Ccm 3 −/− cells re-expressing mGFP-tagged CCM3, Ccm3 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc).

Techniques:

Journal: BMC biochemistry

Article Title: Protein phosphatase 2a (PP2A) binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3.

doi: 10.1186/1471-2091-12-54

Figure Lengend Snippet: Figure 6 CCM3 binding to striatin is affected by the same deletions that reduce Mst3 binding. (A) Three days after transfection with HA-tagged wild-type and mutant striatins, HEK293 cells stably expressing FLAG-CCM3 were lysed and FLAG-CCM3 immune complexes were isolated, separated by SDS-PAGE, and immunoblotted. Lysate levels of the transfected HA-tagged striatins are shown for comparison to the amount of HA-striatins that coimmunoprecipitated with FLAG-CCM3. (B) Relative binding of wild-type and mutant striatins to FLAG-CCM3 was measured as described in the legend to Figure 2B, except that in this experiment the values were normalized to expression of the HA-constructs in the cell lysates. The error bars represent the standard deviation of three independent experiments. **, p ≤0.01 relative to wild-type.

Article Snippet: The cDNA of human CCM3 (Catalog No. SC320246) was obtained from OriGene Technologies, Inc. and the hCCM3 ORF was PCR-amplified and inserted into a modified pLenti6/V5-D-Topo plasmid with an N-terminal FLAG epitope tag.

Techniques: Binding Assay, Transfection, Mutagenesis, Stable Transfection, Expressing, Isolation, SDS Page, Comparison, Construct, Standard Deviation

Journal: BMC biochemistry

Article Title: Protein phosphatase 2a (PP2A) binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3.

doi: 10.1186/1471-2091-12-54

Figure Lengend Snippet: Figure 10 Model of human wild-type striatin complex, based on results from previous studies and the current study. Schematic of wild-type human striatin, drawn to scale, showing the locations of its previously published protein-interaction motifs/domains and residues or domains determined in this study to be important for binding of the striatin-associated proteins. Previously published domains: Caveolin (Cav)- binding motif (aa53-63); C-C, coiled-coil region (aa70-116); Calmodulin (CaM)-binding domain (aa149-166); WD-repeat domain (aa419-780). The Cav-binding motif and CaM-binding domain are indicated by arrows with corresponding proteins below. Estrogen receptor alpha (not shown) has been found to bind to striatin amino acids 1-203 [23]. Upper labels (from this study): Names are as indicated. Residues important for the binding of these proteins are indicated by arrows or by brackets. Mst4 binding was not affected as dramatically as Mst3 by Δ(227-309) so it may have an overlapping, but not identical, binding site. Note: The amino acid stretch in striatin important for Mst3/CCM3 association contains a small region of amino acid homology with PP2A B’ B-type subunits that we previously identified [8]. Whether these residues play a role in binding Mst3/CCM3 remains to be tested.

Article Snippet: The cDNA of human CCM3 (Catalog No. SC320246) was obtained from OriGene Technologies, Inc. and the hCCM3 ORF was PCR-amplified and inserted into a modified pLenti6/V5-D-Topo plasmid with an N-terminal FLAG epitope tag.

Techniques: Binding Assay

Journal: Genes

Article Title: Transcriptome Analysis Reveals Altered Expression of Genes Involved in Hypoxia, Inflammation and Immune Regulation in Pdcd10 -Depleted Mouse Endothelial Cells

doi: 10.3390/genes13060961

Figure Lengend Snippet: RNA-seq data. ( a ) Total lysates were obtained from si Pdcd10 -ECs compared to siCNT-EC, separated on 10% SDS-gel and subjected to immunoblotting with indicated antibodies. ( b ) Relative levels of protein intensity related to Pdcd10/β-Actin was quantified by densitometry using Image J analysis software, and the mean of each quantification was reported in the graph. Graphs show averages calculated on three different biological experiments represented by three points (green, yellow and orange). Scale bars represent standard errors. Values are expressed as mean ± SEM ( * p < 0.05, n = 3). ( c ) Heatmap of gene ontology enrichment analysis of functional differences between si Pdcd10 -EC and siCNT-EC lines. The statistical significance in the heatmap was calculated and presented based on the -log10 false discovery rate (FDR) corrected p -values (blue indicates significant upregulated genes; red indicates significant downregulated genes). The colored scale bar below shows the color scaling with FDR values. The horizontal or vertical bars (violet, blue, orange and green) represented the different clusters of genes coming from a gene ontology analysis generated by Rosalind analysis. On the right-hand side of the Heatmap, a list of DEGs was reported. ( d ) Volcano plot showing the differentially expressed genes (violet points represent downregulated genes, green points represent upregulated genes, and the adjusted p -value threshold plotted on the Y-axis is 1.3). ( e ) Treemap representing over-represented biological functions, grouped into processes. Sizes of rectangles are proportional to the number of genes involved in a specific biological process. On the right of the Treemap, the more representative biological function for each cluster is indicated.

Article Snippet: In brief, to generate Pdcd10 −/− cells re-expressing mGFP-tagged PDCD10, Pdcd10 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc., Rockville, MD, USA).

Techniques: RNA Sequencing Assay, SDS-Gel, Western Blot, Software, Functional Assay, Generated

Journal: Genes

Article Title: Transcriptome Analysis Reveals Altered Expression of Genes Involved in Hypoxia, Inflammation and Immune Regulation in Pdcd10 -Depleted Mouse Endothelial Cells

doi: 10.3390/genes13060961

Figure Lengend Snippet: Differential expressed genes (Log Fold Change < 0.05).

Article Snippet: In brief, to generate Pdcd10 −/− cells re-expressing mGFP-tagged PDCD10, Pdcd10 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc., Rockville, MD, USA).

Techniques: Sequencing, Binding Assay

Journal: Genes

Article Title: Transcriptome Analysis Reveals Altered Expression of Genes Involved in Hypoxia, Inflammation and Immune Regulation in Pdcd10 -Depleted Mouse Endothelial Cells

doi: 10.3390/genes13060961

Figure Lengend Snippet: RNA-seq validation analysis. ( a ) Q-PCR results of a set of DEGs in siPdcd10-ECs and siCNT-EC. The fold change value relates to the mean expression levels of siCNT-EC, which were set as value 1. The mean expression levels of siCNT-EC derived from three biological replicates; each of these was run in three technical replicates. Graphs show averages calculated on three different biological experiments represented by three points (green, yellow and orange); each point characterized the mean of three technical replicates. Error bars represent standard errors. Values are expressed as mean +/− SEM (** p < 0.01, n = 3). ( b ) Q-PCR of Pdcd10 expression level of mouse (left) and human (right) gene results came from Pdcd10iEC-KO, EC-Ctrl, and Pdcd10iEC-KO +Pdcd10 cell lines. ( c ) Q-PCR of some DEGs came from Pdcd10iEC-KO, EC-Ctrl and Pdcd10iEC-KO +Pdcd10 lines were reported. Graphs show averages calculated on three different biological experiments represented by three points (green, yellow and orange); each point characterized the mean of three technical replicates. The fold change value relates to the mean expression levels of EC-Ctrl, which were set as value 1. The mean expression levels of EC-Ctrl derived from three biological replicates; each of these was run in three technical replicates. Values are expressed as mean +/− SEM (** p < 0.01, * p < 0.05, n = 3).

Article Snippet: In brief, to generate Pdcd10 −/− cells re-expressing mGFP-tagged PDCD10, Pdcd10 −/− were transduced with the recombinant lentivirus Lenti ORF clone mGFP-tagged PDCD10 (OriGene Technologies Inc., Rockville, MD, USA).

Techniques: RNA Sequencing Assay, Expressing, Derivative Assay

Journal: Journal of cellular and molecular medicine

Article Title: MicroRNA-425-5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10.

doi: 10.1111/jcmm.12742

Figure Lengend Snippet: Fig. 4 miR-425-5p directly targets the 30UTR of PDCD10 in colon cancer cells. (A) Western Blot analysis of PDCD10 protein level in HCT116 and HCT116-R cells. b-actin was used as loading control. (B) Western Blot analysis of PDCD10 protein level in HCT116-R cells transfected with control or miR-425-5p inhibitor. b-actin was used as loading control. (C) Western Blot analysis of PDCD10 protein level in HCT116 cells transfected with control or miR-425-5p mimic. b-actin was used as loading control. (D) The wild-type 30-UTR of mammalian PDCD10 mRNA contains a putative miR-425-5p-binding site. The mutant form was shown below. (E) Luciferase reporter assays. Cells were transfected with reporters containing the wild-type or mutant form after transfection with miR-425-5p mimics or control miRNA. Data represent the mean S.D., n = 3. **P < 0.01.

Article Snippet: Transfection of miR-425-5p inhibitor, miR-425-5p mimic and its nonspecific control (Invitrogen, Hampton, VA, USA) were performed according to the manual provided with the siPORTM NeoFXTM Transfection Agent (Ambion, Grand Island, NE, USA). pLenti-C-Myc-DDK PDCD10 cDNA (RC221815L1) and two individual siRNAs (SR307707) targeting PDCD10 were obtained from Origene. siRNAs were transfected with Lipofectamine RNAi Max reagent (Invitrogen, Grand Island, NE, USA) as per manufacturer’s protocol. cDNA transfections were performed with Lipofectamine LTX reagent (Invitrogen) as per manufacturer’s protocol.

Techniques: Western Blot, Control, Transfection, Binding Assay, Mutagenesis, Luciferase

Journal: Journal of cellular and molecular medicine

Article Title: MicroRNA-425-5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10.

doi: 10.1111/jcmm.12742

Figure Lengend Snippet: Fig. 5 miR-425-5p regulates chemoresistance through PDCD10. (A) Western blot analysis of PDCD10 protein level in HCT116-R cells cotransfected with miR-425-5p inhibitor and scramble or PDCD10 siRNAs. b-actin was used as loading control. (B) Western Blot analysis of PDCD10 protein level in HCT116-R cells transfected with PDCD10 cDNA. b-actin was used as loading control. (C) Transient knockdown of PDCD10 reversed the chemosensitizing effects of miR-425-5p inhibitor in HCT116-R cells with 5-FU (5 lM) or OX (10 lM) treatments. Data represent the mean S.D., n = 2. *P < 0.05. 425 inh: miR-425-5p inhibitor; Scr: scramble miRNA; (D) Overexpression of PDCD10 phenocopied the effects of miR-425-5p inhi- bitor to sensitize HCT116-R to 5-FU (5 lM) or OX (10 lM). Data represent mean S.D., n = 3. *P < 0.05. (E) Caspase activity in control and PDCD10 overexpression cells treated with or without 5-FU and OX. Data represent mean S.D., n = 3. **P < 0.01.

Article Snippet: Transfection of miR-425-5p inhibitor, miR-425-5p mimic and its nonspecific control (Invitrogen, Hampton, VA, USA) were performed according to the manual provided with the siPORTM NeoFXTM Transfection Agent (Ambion, Grand Island, NE, USA). pLenti-C-Myc-DDK PDCD10 cDNA (RC221815L1) and two individual siRNAs (SR307707) targeting PDCD10 were obtained from Origene. siRNAs were transfected with Lipofectamine RNAi Max reagent (Invitrogen, Grand Island, NE, USA) as per manufacturer’s protocol. cDNA transfections were performed with Lipofectamine LTX reagent (Invitrogen) as per manufacturer’s protocol.

Techniques: Western Blot, Control, Transfection, Knockdown, Over Expression, Activity Assay

Journal: Journal of cellular and molecular medicine

Article Title: MicroRNA-425-5p regulates chemoresistance in colorectal cancer cells via regulation of Programmed Cell Death 10.

doi: 10.1111/jcmm.12742

Figure Lengend Snippet: Fig. 6 miR-425-5p regulates chemoresistance of colon tumours in vivo. (A and B) Co-administration of miR-425-5p inhibitor enhanced tumour growth inhibitory effects of 5-FU and OX in vivo. HCT116 cells were subcutaneously implanted into nude mice and received i.p. injection of either miR-425-5p inhibitor, 5-FU (25 mg/kg) or OX (25 mg/kg) lone or combined. Tumour volumes (mean S.D.) were obtained from five mice for each group and plotted. Data represent mean S.D. *P < 0.05, **P < 0.01 versus the vector control. (C and D) Tumour weights of the mice treated as described above. Data represent mean S.D. **P < 0.01. (E) Representative IHC staining of PDCD10 in control and miR-425-5p inhibitor- transfected xenografts. The scale bar indicates 50 lm.

Article Snippet: Transfection of miR-425-5p inhibitor, miR-425-5p mimic and its nonspecific control (Invitrogen, Hampton, VA, USA) were performed according to the manual provided with the siPORTM NeoFXTM Transfection Agent (Ambion, Grand Island, NE, USA). pLenti-C-Myc-DDK PDCD10 cDNA (RC221815L1) and two individual siRNAs (SR307707) targeting PDCD10 were obtained from Origene. siRNAs were transfected with Lipofectamine RNAi Max reagent (Invitrogen, Grand Island, NE, USA) as per manufacturer’s protocol. cDNA transfections were performed with Lipofectamine LTX reagent (Invitrogen) as per manufacturer’s protocol.

Techniques: In Vivo, Injection, Plasmid Preparation, Control, Immunohistochemistry, Transfection