doxycycline inducible h2begfp expression  (Addgene inc)

Journal: bioRxiv

Article Title: Androgen Deprivation-Induced TET2 Activation Fuels Prostate Cancer Progression via Epigenetic Priming and Slow-Cycling Cancer Cells

doi: 10.1101/2025.03.26.645495

Figure Lengend Snippet: ( A ) Schematic of the construct used for generating C4-2B SCC cells. The H2B-eGFP fusion gene expression is regulated by the reverse tetracycline transactivator (rtTA2) and activated by doxycycline (DOX). After DOX withdrawal, cells with high GFP intensity were considered as SCCs, while cells with low GFP intensity were non-SCCs Figure adapted from Addgene. ( B ) Experimental workflow for in vitro and in vivo SCC studies. In vitro , cells were treated with DOX for 4 days, followed by 10 days culturing with or without ADT. In vivo , xenografted tumors were labeled with DOX for 7 days. 8 days after DOX withdrawal, mice were either left untreated (Intact) or subjected to castration and ENZ (10 mg/kg) treatment (ADT) for 7 days. Workflow created using BioRender. ( C ) Quantification of GFP-positive cells under androgen-deprivation conditions compared to controls over a 10-day culture period, n=6. Student’s t-test, p < 0.01. ( D ) Live-cell imaging of GFP expression and confluency masks in control (Ctrl) and ADT groups at day 0, day 5, and day 10. Arrows indicate SCCs. ( E ) IHC staining of GFP in xenograft tumors. Upper panels show stitched images (20× magnification), and lower panels provide magnified views. Arrows highlight SCCs. Scale bars are indicated under the images. ( F ) Quantification of IHC results using QuPath. Cancer cells were categorized by GFP intensity into four groups: GFP-, GFP 1+ (0-0.2 raw pixel intensity, yellow color), GFP 2+ (0.2-0.4 raw pixel intensity, orange color), and GFP 3+ (0.4-0.8 raw pixel intensity, red color). Similar trends were observed in repeated experiments (n=3),two-way ANOVA, p < 0 . 05 .

Article Snippet: The SCCs reporter plasmid, which carries Doxycycline-inducible H2BeGFP expression (pSIN-TRE-H2BeGFP-rtTA2, #165494, Addgene), or TET2 knockdown construct (p-LV[shRNA]-mCherry:T2A:Puro-U6>hTET2 [shRNA#1], #VB900137-4927mqb, VectorBuilder) was used for transfection.

Techniques: Construct, Gene Expression, In Vitro, In Vivo, Labeling, Live Cell Imaging, Expressing, Control, Immunohistochemistry

Journal: Pulmonary Circulation

Article Title: PPARγ/ETV2 axis regulates endothelial‐to‐mesenchymal transition in pulmonary hypertension

doi: 10.1002/pul2.12448

Figure Lengend Snippet: PPARγ increases the expression of ETV2 in HPAECs and PPARγ gain‐of‐function increases ETV2 and endothelial makers and attenuates the expression of EndoMT markers. (a, b) HPAECs treated with AdPPARγ (25 MOI) or green fluorescent protein (GFP) constructs were subjected to qRT‐PCR analysis. n = 3–5/group, * p < 0.05 vs GFP. (c) PPARγ expressing plasmid (PPARγ) or control plasmid (Mock) was co‐transfected with pGL3‐basic‐ETV2 promoter and treated with dimethyl sulfoxide (RSG/−) or rosiglitazone (RSG/+) (10 µM), then incubated for 72 h. Firefly luciferase activity was normalized by Renilla luciferase activity. n = 3–5/group, * p < 0.05 vs Mock/RSG(−). + p < 0.05 vs PPARγ/RSG(−). (d) HPAECs were treated with AdPPARγ + RSG for 6 h, then incubated with fresh medium for an additional 72 h under normoxic (NOR) or hypoxic (HYP) condition. RSG was treated for last 24 h. (e) HPAECs treated with AdPPARγ or green fluorescent protein (GFP) constructs with RSG (10 µM) were cultured under hypoxic condition and subjected to qRT‐PCR analysis. Each bar represents mean ± SE PPARγ, ETV2, EndoMT or EC markers level relative to GAPDH expressed as fold‐change vs cells treated with GFP. n = 5–6/group, * p < 0.05 vs HYP/GFP. (f–h) whole lungs were collected from littermate control (FulCon) or endothelial‐targeted PPARγ overexpression ( ePPARγOX ) mice. Levels of lung PPARγ (f) or ETV2 (g) or EndoMT and EC markers (h) were measured with qRT‐PCR and expressed relative to GAPDH mRNA * p < 0.05 vs FulCon, n = 5–8/group.

Article Snippet: HEK/293 T cells (1.3 × 10 5 /well of a 24‐well plate) were transfected with 2 µg PPARγ expression plasmid (pcDNA3.1‐FLAG‐PPARγ), 200 ng pGL3‐basic‐ETV2 promoter, and 30 ng pRL‐null using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Construct, Quantitative RT-PCR, Plasmid Preparation, Control, Transfection, Incubation, Luciferase, Activity Assay, Cell Culture, Over Expression

Journal: Pulmonary Circulation

Article Title: PPARγ/ETV2 axis regulates endothelial‐to‐mesenchymal transition in pulmonary hypertension

doi: 10.1002/pul2.12448

Figure Lengend Snippet: Endothelial depletion of PPARγ reduces ETV2, PECAM1, and VE‐Cad expression and increases levels of EndoMT markers in mouse lungs. (a, b) HPAECs were treated with scrambled (SCR) or PPARγ (20 nM) siRNAs (a, b) or ETV2 (20 nM) siRNAs (c) for 6 h, washed with fresh medium, then incubated for an additional 72 h. Each bar represents mean ± SE PPARγ or ETV2 level relative to GAPDH expressed as fold‐change vs cells treated with SCR. n = 3/group, * p < 0.05 vs SCR. (d–f) Whole lungs were collected from littermate control (LitCon) or endothelial‐targeted PPARγ knockout ( ePPARγKO ) mice. Lung levels of PPARγ (d), ETV2 (e), or EndoMT and EC markers (f) mRNA levels were measured using qRT‐PCR in littermate control (LitCon) or endothelial‐targeted PPARγ knockout ( ePPARγKO ) mice and expressed relative to GAPDH mRNA * p < 0.05 vs LitCon, n = 4/group.

Article Snippet: HEK/293 T cells (1.3 × 10 5 /well of a 24‐well plate) were transfected with 2 µg PPARγ expression plasmid (pcDNA3.1‐FLAG‐PPARγ), 200 ng pGL3‐basic‐ETV2 promoter, and 30 ng pRL‐null using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Incubation, Control, Knock-Out, Quantitative RT-PCR

Journal: Pulmonary Circulation

Article Title: PPARγ/ETV2 axis regulates endothelial‐to‐mesenchymal transition in pulmonary hypertension

doi: 10.1002/pul2.12448

Figure Lengend Snippet: Establishment of EndoMT ( i ‐EndoMT) model. (a–f) i ‐EndoMT cells were induced by addition of 0.1 ng/mL interleukin‐1 beta (IL‐1β), 10 ng/mL tumor necrosis factor alpha (TNF‐α), and 10 ng/mL transforming growth factor beta (TGFβ) to HPAECs up to 72 h. Morphology change (a), mean HPAEC PPARγ (b), ETV2 (c), EndoMT or EC marker (d) levels were measured with qRT‐PCR. All bars represent the mean PPARγ, ETV2 , EndoMT or EC marker mRNA levels relative to GAPDH ± SE expressed as fold‐change versus CON. * p < 0.05 versus CON, n = 3/group. (e) Cell contraction assay. CON or i ‐EndoMT cells were harvested at 72 h after the treatment and resuspended with a total of 5 × 10 5 cells in a 1:4 ratio of cell suspension and collagen mixture provided in the cell contraction assay. The gels were imaged at 0 h and 72 h post‐incubation and analyzed by ImageJ software. n = 3/group. Red arrow (dashed line in photomicrograph) indicates the cell contraction. (f) Wound healing assay. CON or i ‐EndoMT cells harvested at 72 h posttreatment were harvested and replated in a wound healing chamber. Images were taken 5 h later.

Article Snippet: HEK/293 T cells (1.3 × 10 5 /well of a 24‐well plate) were transfected with 2 µg PPARγ expression plasmid (pcDNA3.1‐FLAG‐PPARγ), 200 ng pGL3‐basic‐ETV2 promoter, and 30 ng pRL‐null using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Marker, Quantitative RT-PCR, Contraction Assay, Suspension, Incubation, Software, Wound Healing Assay

Journal: Pulmonary Circulation

Article Title: PPARγ/ETV2 axis regulates endothelial‐to‐mesenchymal transition in pulmonary hypertension

doi: 10.1002/pul2.12448

Figure Lengend Snippet: Overexpression of ETV2 induces endothelial cell phenotype. (a, b) HPAECs infected lentiviral particles of doxycycline‐inducible ETV2 (lenti‐ETV2) were incubated with 0.1 ng/mL IL‐1β, 10 ng/mL TNF‐α, and 10 ng/mL TGFβ for 72 h. The resulting cells were then treated ± Dox up to 6 days. A representative image of the resulting cells was shown in (a) (left panels). The cells were randomly selected and counted, the ratio of cobblestone morphology to elongated‐spindle‐shaped phenotype (a, right panel) was determined, and immunocytochemistry was performed (b). (c, d) HPAECs transfected with ETV2 plasmid constructs (oxETV2, 1 µg) or vector (VEC) constructs (c) or infected with AdPPARγ (25 MOI) (d) or green fluorescent protein (GFP) constructs were incubated with 0.1 ng/mL IL‐1β, 10 ng/mL TNF‐α, and 10 ng/mL TGFβ to HPAECs for 72 h. RNAs from the resulting cells were subjected to qRT‐PCR analysis. Twenty‐4 h before cell harvest (d), the cells were treated with rosiglitazone (RSG, 10 μM). Each bar represents the mean ± SE EndoMT or EC marker level relative to GAPDH as indicated. * p < 0.05 versus CON/VEC or i ‐EndoMT/GFP. n = 3–4/group. (e) Lung fibroblasts of IPAH patients were infected with AdETV2 and 3 days later, the resulting cells were subjected to qRT‐PCR analysis. The results are presented as fold‐change versus CON. * p < 0.05 vs NOR, n = 5/group. (f) A hypothetical schema defining the role of PPARγ/ETV2 on EndoMT in PH pathogenesis.

Article Snippet: HEK/293 T cells (1.3 × 10 5 /well of a 24‐well plate) were transfected with 2 µg PPARγ expression plasmid (pcDNA3.1‐FLAG‐PPARγ), 200 ng pGL3‐basic‐ETV2 promoter, and 30 ng pRL‐null using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Over Expression, Infection, Incubation, Immunocytochemistry, Transfection, Plasmid Preparation, Construct, Quantitative RT-PCR, Marker

Journal: Research

Article Title: Brain Short-Chain Fatty Acids Induce ACSS2 to Ameliorate Depressive-Like Behavior via PPARγ–TPH2 Axis

doi: 10.34133/research.0400

Figure Lengend Snippet: PPARγ binds directly ACSS2 to allow Tph2 histone acetylation. (A) Representative BiFC fluorescent images of HEK293T cells transfected with 2 μg of plasmid encoding ACSS2 and PPARγ alone or together fused to the fluorescent protein fragments indicated in each panel. DAPI stain demonstrated nuclear locus. The intensity yellow fluorescent protein (YFP) signal indicates the amounts and localization of BiFC complex (ACSS2–PPARγ). (B) Co-IP of ACSS2 and PPARγ in HEK293T cells or hippocampus of mice. IB, immunoblot. (C) Pull-down assay of purified recombination proteins His-ACSS2 and GST-PPARγ from BL21 (DE3). (D) Pull-down assay of DNA probes and hippocampal ACSS2 was performed. The equal (10 mg) hippocampus lysate from 9 mice of individual groups were incubated with excess biotin-labelled DNA probes (20 nM) containing predicated PPARγ binding site within human TPH2 promoter for 6 h. Streptavidin agarose (20 μl) was added for another 6 h. The precipitations were subject to immunoblot with anti-PPARγ and anti-ACSS2 antibody. GAPDH signals was presented as NC. (E) ChIP analyses using an anti-ACSS2 and anti-PPARγ antibodies were performed in hippocampus of mice. The histogram shows the amount of immunoprecipitated DNA expressed as a percentage of the total input DNA. The data are presented as the means ± SEM of triplicate samples. Data are shown as means ± SEM ( n = 3 per group) and were analyzed using unpaired 2-tailed Student’s t test, ACSS2 ( t = 8.121, df = 4, P = 0.0013) and PPARγ ( t = 16.69, df = 4, P < 0.0001). ** P < 0.01 and **** P < 0.0001. (F) SH-SY5Y cells were transfected with PPARγ expression plasmid for 24 h. ChIP analyses using an anti-H3K9Ac, anti-H3K27Ac, anti-CTD, and anti-Sin3A antibodies were performed. The histogram shows the amount of immunoprecipitated DNA expressed as a percentage of the total input DNA. Data are shown as means ± SEM ( n = 3 per group) and were analyzed using unpaired 2-tailed Student’s t test, H3K9Ac ( t = 4.933, df = 4, P = 0.0079), H3K27Ac ( t = 3.478, df = 4, P = 0.0254), p-Ser 2/5 polymerase II (pol II) CTD ( t = 2.928, df = 4, P = 0.0429), and Sin3a ( t = 5.746, df = 4, P = 0.0045) * P < 0.05 and ** P < 0.01.

Article Snippet: BiFC expression plasmids for ACSS2 and PPARγ were constructed by inserting the PCR fragment containing full-length ACSS2, PPARγ, or their derivatives (primers in Table ) into pBiFC-VN173 and pBiFC-VC155 (Addgene, Cambridge, MA, USA) with ClonExpress II One Step Cloning Kit.

Techniques: Transfection, Plasmid Preparation, Staining, Co-Immunoprecipitation Assay, Western Blot, Pull Down Assay, Purification, Incubation, Binding Assay, Immunoprecipitation, Expressing

Journal: Research

Article Title: Brain Short-Chain Fatty Acids Induce ACSS2 to Ameliorate Depressive-Like Behavior via PPARγ–TPH2 Axis

doi: 10.34133/research.0400

Figure Lengend Snippet: SCFAs trigger PPARγ to mediate the antidepressant responses in CRS-exposure mice via TPH2. (A) The mRNA levels of PPARγ in SH-SY5Y cells with or without 10 μM NaAC, 1 μM NaPPA, and 0.1 μM NaBA alone or together for 12 h. Data were normalized with GAPDH mRNA levels. Scale bars represent means values, and error bars represent SEM of triplicate samples. Data are shown as means ± SEM ( n = 4 per group) and were analyzed using one-way ANOVA, F 4,15 = 11.7, P = 0.0002, and Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01, and **** P < 0.0001. (B) The lysates of SH-SY5Y cells with or without 10 μM NaAC, 1 μM NaPPA, and 0.1 μM NaBA alone or together for 24 h were subjected to Western blot with indicated antibodies. The levels of TPH2, PPARα, PPARβ, and PPARγ were quantitatively analyzed ( n = 3 biological replicates). Data are shown as means ± SEM ( n = 3 per group) and were analyzed using one-way ANOVA, TPH2 ( F 4,10 = 8.281, P = 0.0032), PPARα ( F 4,10 = 0.1347, P = 0.9658), PPARβ ( F 4,10 = 0.1479, P = 0.9597), and PPARγ ( F 4,10 = 13.1, P = 0.0005), and Tukey’s multiple comparison test, ** P < 0.01 and *** P < 0.001. (C) SH-SY5Y cells were incubated with or without 10 μM NaAC, 1 μM NaPPA, and 0.1 μM NaBA alone or together for 24 h. ChIP analyses using an anti-ACSS2, anti-CTD, and anti-Sin3A antibodies were performed. The histogram shows the amount of immunoprecipitated DNA expressed as a percentage of the total input DNA. The data are presented as the means ± SEM of quadruplicate samples. Data are shown as means ± SEM ( n = 4 per group) and were analyzed using one-way ANOVA, PPARγ ( F 4,15 = 29.04, P < 0.0001), p-Ser 2/5 pol II CTD ( F 4,15 = 33.26, P < 0.0001), and Sin3a ( F 4,15 = 23.71, P < 0.0001), and Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. (D) Representative BiFC fluorescent images of SH-SY5Y cells transfected with 2 μg of plasmid encoding ACSS2 or PPARγ fused to the fluorescent protein fragments indicated in each panel in response to 10 μM NaAC, 1 μM NaPPA, and 0.1 μM NaBA alone or together for 24 h. DAPI stain demonstrated nuclear locus. The intensity YFP signal indicates the amounts and localization of BiFC complex (ACSS2–PPARγ). (E) Two weeks after injecting the shPPARγ virus into the hippocampus of mice using stereotaxic injection techniques, the CRS modeling experiment was initiated for 4 weeks, during which mice had free access to drinking SCFAs or water. Behavioral tests were then conducted for 1 week ( n = 10 per group). (F) Immobility time in the TST in mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups were detected. Data are shown as means ± SEM ( n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, t = 2.946, df = 18, P = 0.0086. ** P < 0.01. (G) Immobility time in the FST in mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups were detected. Data are shown as means ± SEM ( n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, t = 2.522, df = 18, P = 0.0213. * P < 0.05. (H) SPTs in mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups were detected. Data are shown as means ± SEM ( n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, t = 2.258, df = 18, P = 0.0366. * P < 0.05. (I) Raw traces of mice in the OFT were shown. Total distance traveled in the OFT and time spent exploring the center area in the OFT from mice in individual animals from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups. Data are shown as means ± SEM ( n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, time ( t = 3.185, df = 18, P = 0.0051) and locomotion ( t = 1.329, df = 18, P = 0.2004). ** P < 0.01. (J) Raw traces of mice in the EPM were shown. Time spent in the open arms and probability of entering open arms in the EPM test from mice in individual animals from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups. Data are shown as means ± SEM ( n = 10 per group) and were analyzed using unpaired 2-tailed Student’s t test, time ( t = 2.923, df = 17, P = 0.0095) and entries ( t = 4.064, df = 17, P = 0.0008). ** P < 0.01 and *** P < 0.001. (K) Analysis of 5-HT content of hippocampus by ELISA in male mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups. Data are shown as means ± SEM ( n = 6 per group) and were analyzed using unpaired 2-tailed Student’s t test, ** P < 0.01. (L) RT-PCR analysis of PPARγ and TPH2 expression levels in hippocampus of male mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups ( n = 6 per group). Data were normalized with GAPDH mRNA levels and presented as fold changes compared with control group. Scale bars represent means values, and error bars represent SEM. Data are shown as means ± SEM ( n = 6 per group) and were analyzed using unpaired 2-tailed Student’s t test, * P < 0.05 and **** P < 0.0001. (M) Representative immunoblots and quantification of PPARγ and TPH2 protein levels normalized to loading controls in hippocampal male mice from CRS + SCFAs + shNC and CRS + SCFAs + shPPARγ groups. Data are shown as means ± SEM ( n = 3 per group) and were analyzed using unpaired 2-tailed Student’s t test, PPARγ ( t = 6.951, df = 4, P = 0.0023) and TPH2 ( t = 7.074, df = 4, P = 0.0021). ** P < 0.01.

Article Snippet: BiFC expression plasmids for ACSS2 and PPARγ were constructed by inserting the PCR fragment containing full-length ACSS2, PPARγ, or their derivatives (primers in Table ) into pBiFC-VN173 and pBiFC-VC155 (Addgene, Cambridge, MA, USA) with ClonExpress II One Step Cloning Kit.

Techniques: Comparison, Western Blot, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation, Staining, Virus, Injection, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Control

Journal: Research

Article Title: Brain Short-Chain Fatty Acids Induce ACSS2 to Ameliorate Depressive-Like Behavior via PPARγ–TPH2 Axis

doi: 10.34133/research.0400

Figure Lengend Snippet: d -Mannose significantly elevates brain SCFAs by altering the gut microbiota of the CRS-exposure mice. (A) RT-PCR analysis of ACSS2 , TPH2 and PPARγ expression levels in hippocampus of male mice from control, CRS, mannose, and CRS + mannose groups. Data were normalized with GAPDH mRNA levels and presented as fold changes compared with control group. Data are shown as means ± SEM ( n = 6 to 10 per group) and were analyzed using 2-way ANOVA and Tukey’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. (B) Representative immunoblots and quantification of ACSS2, TPH2, and PPARγ protein levels normalized to loading controls in hippocampal male mice from control, CRS, mannose, and CRS + mannose groups. Data are shown as means ± SEM ( n = 3 per group) and were analyzed using 2-way ANOVA and Tukey’s multiple comparison test, * P < 0.05 and ** P < 0.01. (C) Pull-down assay of DNA probes and hippocampal ACSS2 was performed. The equal (10 mg) hippocampus lysates from 9 mice of individual groups were incubated with excess biotin-labeled DNA probes (20 nM) containing predicated PPARγ binding site within human TPH2 promoter for 6 h. Twenty microliters of streptavidin agarose was added for another 6 h. The precipitations were subject to immunoblot with anti-PPARγ and anti-ACSS2 antibody. GAPDH signals was presented as NC. (D) ChIP analyses using an anti-PPARγ antibody were performed in hippocampus of mice from control, CRS, mannose, and CRS + mannose groups ( n = 5 per group). The histogram shows the amount of immunoprecipitated DNA expressed as a percentage of the total input DNA. Data are shown as means ± SEM ( n = 5 to 6 per group) and were analyzed using 2-way ANOVA and Tukey’s multiple comparison test, * P < 0.05 and *** P < 0.001.

Article Snippet: BiFC expression plasmids for ACSS2 and PPARγ were constructed by inserting the PCR fragment containing full-length ACSS2, PPARγ, or their derivatives (primers in Table ) into pBiFC-VN173 and pBiFC-VC155 (Addgene, Cambridge, MA, USA) with ClonExpress II One Step Cloning Kit.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Comparison, Western Blot, Pull Down Assay, Incubation, Labeling, Binding Assay, Immunoprecipitation

Journal: Research

Article Title: Brain Short-Chain Fatty Acids Induce ACSS2 to Ameliorate Depressive-Like Behavior via PPARγ–TPH2 Axis

doi: 10.34133/research.0400

Figure Lengend Snippet:

Article Snippet: BiFC expression plasmids for ACSS2 and PPARγ were constructed by inserting the PCR fragment containing full-length ACSS2, PPARγ, or their derivatives (primers in Table ) into pBiFC-VN173 and pBiFC-VC155 (Addgene, Cambridge, MA, USA) with ClonExpress II One Step Cloning Kit.

Techniques: High Performance Liquid Chromatography, Magnetic Beads, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Sonication, Fractionation, Luciferase, Bacteria, Mutagenesis, Real-time Polymerase Chain Reaction, Recombinant, Software

Journal: Cell reports

Article Title: DPPA3-HIF1α axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype.

doi: 10.1016/j.celrep.2023.112927

Figure Lengend Snippet: Figure 1. DPPA3 gene is overexpressed in slow-cycling cancer cells (SCCCs) (A) Pulse-chase experimental design to evaluate SCCCs. After a doxycycline (DOX) treatment, the accumulated H2BeGFP signal in cells was diluted (dil) upon cell divisions revealing label-retaining cells (SCCCs). (B) Gene set enrichment analysis (GSEA) plot showing enrichment of a custom GERM_CELL gene set in SCCC versus RCCC expression profiles from two CRC models grown in 3D (ArrayExpress: E-MTAB-4004). (C and D) qRT-PCR analysis showing DPPA3 expression in SCCCs (C and D), RCCCs (C and D), and super-rapid cycling cancer cells (sRCCCs) (D) obtained from a DOX pulse-chase sorting experiment of each indicated CRC model. Mean ± SD of triplicates. (E and F) Experimental design (E) and colonies formation capacity evaluation (F) of control (shCTRL) and DPPA3 knockdown (shDPPA3) RCCCs and SCCCs sorted from CRC SW1222-H2BeGFP pool organoids. Dots indicate the percentage of organoids grown embedded in each single Matrigel. (G) Representative pictures (left) and percentage (right) of colonies (<400 mm) and megacolonies (R400 mm) generated from SCCCs in the self-renewal assay shown in (F). Scale bar, 100 mm. (H) Schematic representation of paired CRC primary tumor (pT) and liver metastasis (Met) biopsies collection. (I) Representative pictures (left) and immunohistochemical staining quantification (right) of DPPA3 in paired pTs and liver metastases (Met) of CRC patients. Patients were categorized according to the percentage of nuclear DPPA3-positive cells into DPPA3-High (>20%, n = 41) and DPPA3-Low (%20%, n = 24). Scale bar, 250 mm; high-magnification scale bar, 25 mm. (J) Percentage of nuclear DPPA3-positive cells in metachronous metastases analyzed in (I) according to the early (%2 years) or late (>2 years) relapse time of CRC patients. Mean ± SEM. (C, D, F, G, I, and J) *p % 0.05, ***p % 0.001,****p % 0.0001, unpaired t test (C and J), one-way ANOVA (D), two-way ANOVA (F), chi-square exact test (G), and paired t test (I). NES, normalized enrichment score; p, one-way ANOVA p value; RCCCs, rapid-cycling cancer cells. See also Figure S1 and Table S1.

Article Snippet: To obtain SW1222 Histone2B fused to the eGFP (H2BeGFP) clone, cells were transduced with lentiviruses expressing H2BeGFP protein8 (pSIN-TRE-H2BeGFP-rtTA2; Addgene, plasmid ID: 165494).

Techniques: Pulse Chase, Expressing, Quantitative RT-PCR, Control, Knockdown, Generated, Immunohistochemical staining, Staining

Journal: Cell reports

Article Title: DPPA3-HIF1α axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype.

doi: 10.1016/j.celrep.2023.112927

Figure Lengend Snippet: Figure 2. DPPA3 overexpression identifies CRC patients with high risk of disease progression after treatment (A) Pulse-chase experimental design to evaluate the chemoresistance of SCCCs in CRC cells growing in 3D cultures. After a DOX treatment, the accumulated H2BeGFP signal in cells was diluted upon cell divisions revealing label-retaining cells (SCCCs). Vehicle (VH) or 5-fluorouracil (5FU) was added, after which annexin-V (anx-V) staining was performed and subsequently analyzed by flow cytometry (FC). (B) Percentage of apoptotic (left) SW1222 RCCCs and SCCCs and proportion of SCCCs (right) upon 5FU exposure. Mean ± SD of triplicates. (C) Percentage of apoptotic DPPA3 knockdown (shDPPA3) and control (shCTRL) RCCCs and SCCCs upon 5FU exposure. Mean ± SD of triplicates. (D) Experimental design (top) and colonies formation capacity evaluation (bottom) of control (shCTRL) and DPPA3 knockdown (shDPPA3) RCCCs and SCCCs sorted from CRC SW1222-H2BeGFP organoids after 5FU exposure. Dots indicate the percentage of organoids grown embedded in each single Matrigel. Mean ± SD. (E) Schematic representation of samples collection workflow. (F) Representative pictures of indicated samples immunostained with DPPA3. Scale bar, 20 mm. (G and H) Bar plots showing percentage distributions of no responder (No resp.), intermediate responder (Interm. R.) and responder (Resp.) (G), and dead and alive (H) LARC patients according to the percentage of nuclear DPPA3-positive cells in naive biopsies assessed in (F).

Article Snippet: To obtain SW1222 Histone2B fused to the eGFP (H2BeGFP) clone, cells were transduced with lentiviruses expressing H2BeGFP protein8 (pSIN-TRE-H2BeGFP-rtTA2; Addgene, plasmid ID: 165494).

Techniques: Over Expression, Biomarker Discovery, Pulse Chase, Staining, Cytometry, Knockdown, Control