Journal: eLife

Article Title: Pathway-specific effects of ADSL deficiency on neurodevelopment

doi: 10.7554/eLife.70518

Figure Lengend Snippet: ( A ) Reduced mRNA levels of ADSL confirmed by qRT-PCR experiments. hTERT-RPE-1 were silenced with smart pool RNAi for 96 hr before harvesting. Two independent experiments in triplicate are shown in the panel (n = 2, two-tailed t -test, ****p<0.0001) ( B ) Western blot of RPE-1 cell extracts treated as in ( A ). One experiment is shown as representative of four independent experiments. Actin was used as a loading control. Quantifications of ADSL intensity in four different experiments were performed by ImageJ software and normalized to actin first and then to the relative controls (n = 4, two-tailed t -test, ****p<0.0001). Ponceau is shown as an additional loading and transfer control. ( C ) S-Ado levels in RPE-1 cells silenced with a single control or ADSL siRNA (n = 4, two-tailed t -test, *p<0.05). ( D ) Cell proliferation rates of RPE-1 cells quantified every 3 days after treatment with a single control or ADSL siRNA in medium with serum (n = 3, two-tailed t -test ****p<0.0001). ΔPDL represent the difference in population doubling levels quantified through the formula described in Materials and methods. ( E ) Ki67-positive cells (indicated by arrows) upon 96 hr of silencing with control or ADSL smart pool siRNAs. Scale bar 10 μm (n = 4, scored 767 cells for siC, 694 cells for siADSL conditions, ****p<0.0001). ( F ) The percentage of p53-positive cells (indicated by arrows) following treatment with control or ADSL smart pool siRNAs was quantified in three independent experiments (n = 3, scored 835 cells for siC and 1126 cells for siADSL, *p<0.05). ( G ) Cell proliferation rates in RPE-1 wt and p53 knockout KO cells as in ( D ) were counted for 6 days (n = 3, two-tailed t -test, **p<0.01, *p<0.05). ( H ) RPE-1 cells stably expressing EGFP or ADSL*-EGFP were transfected with a single control or ADSL siRNAs for 96 hr and immunostained with anti-Ki67 antibody. Scale bar = 20 μm. Quantification of Ki67-positive cells (n = 3, scored 278 cells for EGFP siC, 264 for EGFP siADSL, 266 for ADSL* siC, 232 cells for ADSL* siADSL conditions, ns not significant, ****p<0.0001). ( I ) Quantification of RPE-1 transfected with a single control or ADSL siRNA for 96 hr in the presence or absence of 60 μM nucleosides. Cells were fixed and immunostained with anti-Ki67 antibody (n = 3, at least 100 cells per conditions were counted in each experiment, ns not significant, ****p<0.0001). ( J ) Quantification of RPE-1 in the same conditions of ( I ) and immunostained with anti-p53 antibody (n = 3, at least 100 cells were counted for each condition per experiment, ns not significant, ****p<0.0001). ( K ) Quantification of p53-positive cells in ADSL -depleted cells in the presence or absence of the phosphoribosylaminoimidazole carboxylase (PAICS) inhibitor MRT00252040 (n = 3, scored 223 cells for siC, 248 cells for siADSL, 336 cells for siC+ MRT00252040, 365 cells for siADSL + MRT00252040, ns not significant, ****p<0.001, *p<0.05). ( L ) Quantification of Ki67-positive cells in ADSL-depleted cells in the presence or absence of MRT00252040 (n = 5, more than 60 cells were counted in each condition for each experiment, ns not significant, ***p<0.001). Positive cells are indicated with arrows in siADSL panels. All graphs depict means ± SD with individual values shown in circles. Figure 1—source data 1. Related to . GraphPad Prism file. Figure 1—source data 2. Related to . jpg file.

Article Snippet: Staining of human cells was performed with the following primary antibodies: α-ADSL (MilliporeSigma, rabbit, 1:100 IF, 1:1000 western), α-ARL13B (Santa Cruz Biotechnology, mouse monoclonal C5, 1:100), PCNT (Novus Biologicals, rabbit, 1:400), α-p53 (Cell Signaling, mouse monoclonal 1C12, 1:100), α−RPA2 (Calbiochem, mouse monoclonal Ab-3, 1:100), a-53BP1 (Novus Biologicals, rabbit, 1:400), α-pSer139-H2A.X (Santa Cruz Biotechnology, rabbit, 1:100), α-AKT (Cell Signaling, rabbit polyclonal, 1:2000), α-pAKT-S473 (Cell Signaling, rabbit polyclonal, 1:2000), α-vinculin (Cell Signaling, rabbit polyclonal, 1:2000), α-actin (MilliporeSigma, mouse monoclonal AC-40, 1:1500), α-vimentin (Abcam, rabbit, 1:100), α-CK20 (DaKo, mouse, 1:200), α-Ki67 (Novocastra, mouse, 1:500), α-centrobin (a kind gift from Ciaran Morrison, mouse, 1:500 [ ]), α-centrin (EMD Millipore, mouse, 1:1000), α-CP110 (a kind gift from Andrew Holland, rabbit, 1:1000), α-mouse-Alexa Fluor 594 (Molecular Probes, goat, 1:400), and α-rabbit-Alexa Fluor 488 (Molecular Probes, goat, 1:400).

Techniques: Quantitative RT-PCR, Two Tailed Test, Western Blot, Control, Software, Knock-Out, Stable Transfection, Expressing, Transfection

Journal: eLife

Article Title: Pathway-specific effects of ADSL deficiency on neurodevelopment

doi: 10.7554/eLife.70518

Figure Lengend Snippet: ( A ) Analysis of AMP and GMP levels following ADSL depletion in RPE-1 cells (see also ). ( B ) Analysis of S-Ado and SAICAr levels following ADSL depletion in RPE-1 cells treated or not with 4 μM MRT00252040 (phosphoribosylaminoimidazole carboxylase [PAICS] inhibitor). Box–Cox transformation was applied using lambda = 0 for SAICAr and lambda = −0.5 for S-Ado values. ( C ) Analysis of S-Ado and SAICAr levels following ADSL depletion in HeLa cells treated or not with 4 μM MRT00252040 (PAICS inhibitor). Box–Cox transformation was applied using lambda = 0 for SAICAr and lambda = −0.5 for S-Ado values. ( D ) qRT-PCR confirmed ADSL depletion with a single siRNA against ADSL (siRNA#2; n = 4 in triplicate, two-tailed t -test, ****p<0.0001). ( E ) Flow cytometry analysis of cell cycle using propidium iodide staining in RPE-1 cells treated with control (Ci) siRNA or siRNA against ADSL. Representative data is shown (left) and data from n = 3 independent cultures is graphed (right). Student’s t -test was applied (n = 3 scored more than 10,000 cells for each condition in each experiment, *p<0.05). ( F ) Quantification of cell viability by Trypan Blue in RPE-1 cells transfected with single control or ADSL siRNAs (n = 5, scored 807 cells for siC, 682 cells for siADSL, ns not significant). ( G ) β-Galactosidase assay in RPE-1 cells upon ADSL depletion treated as in panel ( F ). Doxorubicin (doxo) was used as a positive control to induce senescence. ( H ) Flow cytometry analysis of cell cycle using propidium iodide staining in RPE-1 p53 KO cells treated with control (Ci) siRNA or siRNA against ADSL. Representative data is shown (left) and data from n = 3 independent cultures is graphed (right). Student’s t -test was applied (n = 3 scored more than 10,000 cells for each condition in each experiment, ns not significant). ( I ) Ki67 staining in RPE-1 p53 KO upon silencing with a single control or ADSL siRNA (siRNA#2; n = 2, at least 200 cells for each condition per experiment were counted, ns not significant). Scale bar = 20 μm. All graphs depict means ± SD with individual values shown in circles. ( J ) Control of ADSL depletion and siRNA-resistant mutant expression 96 hr post-ADSL depletion. Scale bar = 20 μm. ( K ) Cells were transfected with a single control or ADSL siRNA (siADSL#2) for 96 hr, fixed, and stained against vimentin and cytokeratin-20 (CK20). Serum starvation (SS) for 144 hr was used as positive control for differentiation and CK20 staining. Quantification of the percentage of cells positive for vimentin is shown. No CK20-positive cells were observed in ADSL-depleted cells. Scale bar = 20 μm. Figure 1—figure supplement 2—source data 1. Related to . GraphPad Prism file.

Article Snippet: Staining of human cells was performed with the following primary antibodies: α-ADSL (MilliporeSigma, rabbit, 1:100 IF, 1:1000 western), α-ARL13B (Santa Cruz Biotechnology, mouse monoclonal C5, 1:100), PCNT (Novus Biologicals, rabbit, 1:400), α-p53 (Cell Signaling, mouse monoclonal 1C12, 1:100), α−RPA2 (Calbiochem, mouse monoclonal Ab-3, 1:100), a-53BP1 (Novus Biologicals, rabbit, 1:400), α-pSer139-H2A.X (Santa Cruz Biotechnology, rabbit, 1:100), α-AKT (Cell Signaling, rabbit polyclonal, 1:2000), α-pAKT-S473 (Cell Signaling, rabbit polyclonal, 1:2000), α-vinculin (Cell Signaling, rabbit polyclonal, 1:2000), α-actin (MilliporeSigma, mouse monoclonal AC-40, 1:1500), α-vimentin (Abcam, rabbit, 1:100), α-CK20 (DaKo, mouse, 1:200), α-Ki67 (Novocastra, mouse, 1:500), α-centrobin (a kind gift from Ciaran Morrison, mouse, 1:500 [ ]), α-centrin (EMD Millipore, mouse, 1:1000), α-CP110 (a kind gift from Andrew Holland, rabbit, 1:1000), α-mouse-Alexa Fluor 594 (Molecular Probes, goat, 1:400), and α-rabbit-Alexa Fluor 488 (Molecular Probes, goat, 1:400).

Techniques: Transformation Assay, Quantitative RT-PCR, Two Tailed Test, Flow Cytometry, Staining, Control, Transfection, Positive Control, Mutagenesis, Expressing

Journal: eLife

Article Title: Pathway-specific effects of ADSL deficiency on neurodevelopment

doi: 10.7554/eLife.70518

Figure Lengend Snippet:

Article Snippet: Staining of human cells was performed with the following primary antibodies: α-ADSL (MilliporeSigma, rabbit, 1:100 IF, 1:1000 western), α-ARL13B (Santa Cruz Biotechnology, mouse monoclonal C5, 1:100), PCNT (Novus Biologicals, rabbit, 1:400), α-p53 (Cell Signaling, mouse monoclonal 1C12, 1:100), α−RPA2 (Calbiochem, mouse monoclonal Ab-3, 1:100), a-53BP1 (Novus Biologicals, rabbit, 1:400), α-pSer139-H2A.X (Santa Cruz Biotechnology, rabbit, 1:100), α-AKT (Cell Signaling, rabbit polyclonal, 1:2000), α-pAKT-S473 (Cell Signaling, rabbit polyclonal, 1:2000), α-vinculin (Cell Signaling, rabbit polyclonal, 1:2000), α-actin (MilliporeSigma, mouse monoclonal AC-40, 1:1500), α-vimentin (Abcam, rabbit, 1:100), α-CK20 (DaKo, mouse, 1:200), α-Ki67 (Novocastra, mouse, 1:500), α-centrobin (a kind gift from Ciaran Morrison, mouse, 1:500 [ ]), α-centrin (EMD Millipore, mouse, 1:1000), α-CP110 (a kind gift from Andrew Holland, rabbit, 1:1000), α-mouse-Alexa Fluor 594 (Molecular Probes, goat, 1:400), and α-rabbit-Alexa Fluor 488 (Molecular Probes, goat, 1:400).

Techniques: Western Blot, Transfection, Construct, Plasmid Preparation, Recombinant, In Situ, Sequencing, Luciferase, Control, Imaging, TA Cloning, Mutagenesis, DC Protein Assay, Staining, Ligation, Lysis, Saline, Concentration Assay, Cell Culture, Software

Journal: eLife

Article Title: Pathway-specific effects of ADSL deficiency on neurodevelopment

doi: 10.7554/eLife.70518

Figure Lengend Snippet:

Article Snippet: Staining of human cells was performed with the following primary antibodies: α-ADSL (MilliporeSigma, rabbit, 1:100 IF, 1:1000 western), α-ARL13B (Santa Cruz Biotechnology, mouse monoclonal C5, 1:100), PCNT (Novus Biologicals, rabbit, 1:400), α-p53 (Cell Signaling, mouse monoclonal 1C12, 1:100), α−RPA2 (Calbiochem, mouse monoclonal Ab-3, 1:100), a-53BP1 (Novus Biologicals, rabbit, 1:400), α-pSer139-H2A.X (Santa Cruz Biotechnology, rabbit, 1:100), α-AKT (Cell Signaling, rabbit polyclonal, 1:2000), α-pAKT-S473 (Cell Signaling, rabbit polyclonal, 1:2000), α-vinculin (Cell Signaling, rabbit polyclonal, 1:2000), α-actin (MilliporeSigma, mouse monoclonal AC-40, 1:1500), α-vimentin (Abcam, rabbit, 1:100), α-CK20 (DaKo, mouse, 1:200), α-Ki67 (Novocastra, mouse, 1:500), α-centrobin (a kind gift from Ciaran Morrison, mouse, 1:500 [ ]), α-centrin (EMD Millipore, mouse, 1:1000), α-CP110 (a kind gift from Andrew Holland, rabbit, 1:1000), α-mouse-Alexa Fluor 594 (Molecular Probes, goat, 1:400), and α-rabbit-Alexa Fluor 488 (Molecular Probes, goat, 1:400).

Techniques: In Vitro, Expressing, Flow Cytometry, Inhibition, In Vivo, Fluorescence In Situ Hybridization