Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: a, After gene editing, LMNB1-edited clones #5 and #8 were established as cell lines, expanded, and their normal karyotype confirmed. b-c, Western blot and immunocytochemical analysis of gene-edited and clone cell line demonstrating expression of typical markers for pluripotent (OCT4) and differentiated cells (Brachyury for mesoderm; SOX17 for endoderm; PAX6 for ectoderm) after directed differentiation in adherent cultures. Scale bar in c : 50 μm.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Clone Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: a, EB formation in the presence of DMSO, Y-27632 and CEPT. Human ESCs (WA09) cells were dissociated with Accutase and plated into 6-well ULA plates in E6 medium. Representative phase-contrast images were taken at 24 h post-plating. Scale bar, 50 μm. b, To generate single EBs, hESCs were dissociated with Accutase and plating into AggreWell plates (5,000 cells/well). Images were taken 24 h post-plating. In the presence of DMSO the vast majority cells underwent cell death and EB formation was not observed. Treatment with Y-27632 supported EB formation but significant numbers of dead cells were detected surrounding the EB. Note that CEPT enables EB formation without apparent cell death. Scale bar, 100 μm. c, Quantification of the diameter of single EBs (24 h post-plating). Data are mean ± s.d. (n = 20 EBs for Y-27632 and n = 22 for CEPT), **** P < 0.0001, two-tailed Student’s t -test. d, Single EB formation in 96-well ULA plates. Dissociated hESCs were plated into 96-well ULA plates at 2,000 cells/well. Live and dead cells were stained with calcein green AM and propidium iodide (PI) 24 h after cell seeding. Scale bars, 100 μm. e, Quantification of cell survival in single EBs at day 1 and day 7 by using the CTG 3D assay. Note the significant difference between Y-27632 and CEPT treatment at both timepoints. Data represent mean ± s.d. (n = 24 EBs for each group), **** P < 0.0001, one-way ANOVA. f, CEPT supports differentiation of single EBs into the three germ layers. Individual EBs were cultured in E6 medium to allow for spontaneous differentiation and analyzed on day 7 for the expression of PAX6, SOX17, and Brachyury using an optimized quantitative RT-PCR protocol that enabled detection of low transcript levels in single EBs (see Materials and Methods section for details). Data represent mean ± s.d. (n = 3 experiments and in each experiment 24 EBs were analyzed for each group), * P = 0.0327, two-tailed Student’s t -test. g, h, Cerebral organoids were generated by using Y-27632 and CEPT for the first 24 h. At day 30, organoids were fixed, sectioned, processed for histology (hematoxylin and eosin stain) and immunohistochemistry for FOXG1. Representative images show that CEPT treatment resulted in larger organoids and more abundant FOXG1-expressing cells. Scale bars, 400 μm.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Two Tailed Test, Staining, Cell Culture, Expressing, Quantitative RT-PCR, Generated, H&E Stain, Immunohistochemistry

Journal: bioRxiv

Article Title: A Versatile Polypharmacology Platform Promotes Cytoprotection and Viability of Human Pluripotent and Differentiated Cells

doi: 10.1101/815761

Figure Lengend Snippet: Basal expression levels of Caspase-3 in hESCs (WA09) in comparison to their lineage-restricted precursors after directed differentiation into ectoderm (PAX6), mesoderm (Brachyury), and endoderm (SOX17). Note the strong Caspase-3 expression at the pluripotent state and downregulation upon differentiation.

Article Snippet: On day 7, mRNA from individual EBs was extracted using TurboCapture 96 mRNA kit (Qiagen), reverse-transcribed into cDNA using Sensiscript RT kit (Qiagen), and pre-amplified using TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and Taqman probes for PAX6 (Hs01088114 ) , Brachyury (Hs00610080) and SOX17 (Hs00751752), followed by qPCR quantification of PAX6, Brachyury, and SOX17.

Techniques: Expressing, Comparison

Journal: eLife

Article Title: An antisense oligonucleotide-based strategy to ameliorate cognitive dysfunction in the 22q11.2 Deletion Syndrome

doi: 10.7554/eLife.103328

Figure Lengend Snippet: ( A ) Schematic diagram depicting the human chromosome 22q11.2 region. Bright grey and red horizontal bars indicate the two most common hemizygous genomic deletions found in the 22q11.2 Deletion Syndrome. The location of the coding genes and non-coding RNAs (miRNAs, underlined) are shown for chromosome 22q11.2. The microprocessor DGCR8 (DiGeorge Syndrome Critical Region Gene 8) and MIR185 are shown in bold. ( B ) Cortical marker TBR1 and pan-neuronal marker TUJ1 expression in cortical neurons as detected by immunocytochemistry at day 13 of differentiation. TBR1 (red), TUJ1 (green) and DAPI (blue) expression are shown. Scale bar: 100 μm. ( C ) Volcano plot showing differentially expressed human mature miRNAs (DEmiRs) in cortical neurons at day 8 of differentiation. Significant DEmiRs (p-value <5%) are shown above red line; Q5 (Ctrl) n=3, Q6 (22q11.2) n=3. 153/133 miRNAs were significantly up- and downregulated in Q6 (22q11.2) hiPSC-derived cortical neurons, respectively. 22q11.2 deletion region residing miRNAs miR-185, miR-1286 and miR-1306 are highlighted. ( D–F ) Consistent upregulation of EMC10 mRNA in Q6 (22q11.2) line derived cortical neurons as assayed by qRT-PCR at ( D ) day 8 (p=0.031; Q5: n=3, Q6: n=3), ( E ) day 20 (p=0.0478; Q5: n=4, Q6: n=4) and ( F ) day 34 (p=0.0358; Q5: n=3, Q6: n=3) of differentiation. ( G ) Western blot analysis showing upregulated EMC10 protein levels in Q6 (22q11.2) line derived cortical neurons at day 8 of differentiation. Tubulin was probed as a loading control. ( H ) Immunofluorescence images of NGN2 generated cells. Representative images of NGN2-iNs at DIV21 from Q5 (Ctrl) and Q6 (22q11.2) hiPSC lines identified via EGFP fluorescence and immunostained for neuronal dendrite marker MAP2 and the nuclear marker DAPI. Scale bar = 100 µm. ( I–K ) qRT-PCR assay of EMC10 mRNA expression level in NGN2-iNs at DIV21. ( I ) Upregulation of EMC10 mRNA in Q6 (22q11.2) line derived neurons compared to the healthy control line Q5 (p=0.0222). Q5 (Ctrl) n=4, Q6 (22q11.2) n=4. ( J ) Upregulation of EMC10 mRNA in Q1 (22q11.2) patient line compared to healthy control line Q2 (p0.0441). Q2 (Ctrl) n=5 and Q1 (22q11.2) n=7. ( K ) Upregulation of EMC10 mRNA in QR27 (22q11.2) patient line compared to healthy control line QR20 (p=0.0414). QR20 (Ctrl) n=5 and QR27 (22q11.2) n=5. Data are presented as mean ± SEM, unpaired two-tailed t-test, *p<0.05, **p<0.01. Figure 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 1—source data 2. Original files for western blot analysis shown in .

Article Snippet: Sequence-based reagent , TaqMan Hs00232429 ( TBR1 ) , Thermo Fisher , cat#4331182 , Human TBR1 qRT-PCR.

Techniques: Marker, Expressing, Immunocytochemistry, Derivative Assay, Quantitative RT-PCR, Western Blot, Control, Immunofluorescence, Generated, Fluorescence, Two Tailed Test

Journal: eLife

Article Title: An antisense oligonucleotide-based strategy to ameliorate cognitive dysfunction in the 22q11.2 Deletion Syndrome

doi: 10.7554/eLife.103328

Figure Lengend Snippet: ( A ) Characterization of Q5 (Ctrl) and Q6 (22q11.2) hiPS cells demonstrate normal karyotype distribution in both lines. ( B–D ) Characterization of the Q5 and Q6 hiPSC lines. Multiplex Ligation-dependent Probe Amplification (MLPA) assay of gene copies within and around 22q11.2 locus for Q5 (Ctrl) ( B ) and Q6 (22q11.2) ( C ) line shows that copy number of genes in the 22q11.2 locus (highlighted in light blue) are reduced by half in the Q6 (22q11.2) hiPSC line. ( D ) qRT-PCR assays of embryonic stem cell marker OCT4/POU5F1 and NANOG shows that they are highly expressed in both hiPSC lines (Q5 Ctrl and Q6 22q11.2). ( E ) TBR1 and TUJ1 expression of cortical neurons by immunocytochemistry at day 20 of differentiation indicates the efficiency of hiPSC differentiation into cortical neurons. TBR1 (red), TUJ1 (green) and DAPI (blue). Scale bar: 100 μm. ( F–G ) Time-course qRT-PCR analysis at day 0 and day 20 of differentiation of human pluripotency marker ( F ) OCT4/POU5F1 (p=0.0012) and ( G ) TBR1 (p=0.0273), a preplate, subplate and cortical Layer VI neuron marker in Q5 (Ctrl) line. Day 0: n=4, Day 20 n=4. Data are presented as mean ± SEM, unpaired two-tailed t-test, *p<0.05, **p<0.01. Figure 1—figure supplement 1—source data 1. PDF file containing original gel for . Figure 1—figure supplement 1—source data 2. Original file for hiPSC validation shown in .

Article Snippet: Sequence-based reagent , TaqMan Hs00232429 ( TBR1 ) , Thermo Fisher , cat#4331182 , Human TBR1 qRT-PCR.

Techniques: Multiplex Assay, Ligation, Amplification, Quantitative RT-PCR, Marker, Expressing, Immunocytochemistry, Two Tailed Test, Biomarker Discovery

Journal: BMC Oral Health

Article Title: Systemic inhibition of 5-lipoxygenase by MK-886 exacerbates apical periodontitis bone loss in a mouse model

doi: 10.1186/s12903-023-02712-w

Figure Lengend Snippet: Detection of genes related to bone metabolism after treatment with MK-886. Relative expression of Acp5 , Mmp9, Ctsk and Calcr genes that encode the tartrate-resistant acid phosphatase enzyme (TRAP), matrix-9 metalloproteinase, cathepsin K and the calcitonin receptor, after LPS inoculation into root canals, and at 7 and 14 days after treatment with MK-886 (median and interquartile range). * p < 0.05 compared with healthy periodontal ligament; # p < 0.05 compared with inoculation of LPS into the root canals; & p < 0.05 comparison between 7 and 14 days of the same treatment

Article Snippet: Aliquots of 2 μL of the total cDNA were amplified by qRT-PCR using primers for Tnfrsf11a (RANK; Mm00437135), Tnfsf11 (RANKL; Mm00441906), Tnfrsf11b (OPG; Mm01205928), Acp5 (Mm00475698), Mmp9 (Mm00442991), Ctsk (Mm00484039) and Calcr (Mm01197736) (TaqMan ® Gene Expression Assay, Applied Biosystems) in an StepOne Plus equipment (Applied Biosystems).

Techniques: Expressing, Comparison