Journal: Frontiers in bioscience (Landmark edition)

Article Title: Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

doi: 10.31083/j.fbl2903100

Figure Lengend Snippet: Fig. 5. ISL1 modulates neuroactive pathway in subclusters of single cell RNA-seq data and The Cancer Genome Atlas (TCGA)

Article Snippet: After cell counting, the cells were incubated with ISL LIM Homeobox 1 (ISL1) monoclonal antibody (1:200, 15661-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C in the dark for 60 min, followed by incubation for 30 min with a fluorescent secondary antibody (1:200, SA00013-2, CoraLite488-conjugated goat anti-rabbit IgG (H+L)).

Techniques: RNA Sequencing

Journal: Frontiers in bioscience (Landmark edition)

Article Title: Single-Cell Transcriptome Analysis of Small Cell Neuroendocrine Carcinoma of the Endometrium Reveals ISL1 as a Potential Biomarker for Diagnosis and Treatment.

doi: 10.31083/j.fbl2903100

Figure Lengend Snippet: Fig. 6. Flow cytometric sorting of neuroendocrine tumor cells to obtain ISL1 expression group, and explore the differences in cell function in vitro. (A) Flow cytometric sorting of neuroendocrine tumor cell line H446 according to the difference in the expression of

Article Snippet: After cell counting, the cells were incubated with ISL LIM Homeobox 1 (ISL1) monoclonal antibody (1:200, 15661-1-AP; Proteintech, Rosemont, IL, USA) at 4 °C in the dark for 60 min, followed by incubation for 30 min with a fluorescent secondary antibody (1:200, SA00013-2, CoraLite488-conjugated goat anti-rabbit IgG (H+L)).

Techniques: Expressing, Cell Function Assay, In Vitro

Journal: Nature communications

Article Title: High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1.

doi: 10.1038/ncomms4449

Figure Lengend Snippet: Figure 5 | ISL1 is a critical regulator of human motoneuron development. (a) Immunofluorescence of ISL1 (red) in cells after 15-day differentiation from H1 hESCs. Cell nuclei were stained with DAPI (4’,6-diamidino-2-phenylindole; blue). RA-D1, RA-D3 and RA-D6 denote the time of initiation of RA patterning (at day 1, day 3 and day 6, respectively). (b) Percentage of ISL1-positive cells under conditions described in a. Each bar represents mean±s.d. (error bars) of six experiments. (c) Fluorescence images of ISL1 (left), ISL1/DAPI (middle) and ISL1/ TUJ1 (right) staining in differentiated cells replated at a low density for 3 days after 15-day differentiation. (d) Fluorescence images of differentiated hESCs with NT shRNA (‘Control’, left) or with ISL1 shRNA-1 (‘ISL shRNA-1’, right) stained with antibodies against ISL1 (red) and TUJ1 (green) after 15-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). (e) Fluorescence images of differentiated hESCs with NTshRNA (‘Control’, left), ISL1 shRNA-2 (‘ISL shRNA-2’, middle) and ISL1 shRNA-2 with ectopic ISL1 expression (‘shRNA-2 þ ISL1’, right) stained with antibodies against ISL1 (red) and TUJ1 (green) after 15-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). (f) Fluorescence images of differentiated hESCs with NTshRNA (left) or with ISL1 shRNA (right) stained with antibodies against HB9 (red) and ChAT (green) after 20-day differentiation. Cell nuclei were stained with DAPI (blue). (g) Fluorescence images of differentiated hESCs with NT shRNA (left), ISL1 shRNA-2 (middle) and ISL1 shRNA-2, with ectopic ISL1 expression (right) stained with antibodies against HB9 (red) and ChAT (green) after 20-day differentiation (with patterning initiated at day 3). Cell nuclei were stained with DAPI (blue). All scale bars, 50 mm.

Article Snippet: For ISL1 rescue experiments, ISL1 expression vector was generated by inserting human ISL1 cDNA (OriGene SC125257) into pSin-EF2-alpha-Puro lentivector (Addgene).

Techniques: Staining, Fluorescence, shRNA, Control, Expressing

Journal: Nature communications

Article Title: High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1.

doi: 10.1038/ncomms4449

Figure Lengend Snippet: Figure 6 | Summary of the MN differentiation model and the stages of neural differentiation. (a) hESCs undergoing neural induction begin to express high levels of NPC markers, including PAX6 and SOX1 at day 3, while markedly downregulating the pluripotency markers such as OCT-4 and NANOG (albeit retaining some degree of expression). Thus, the cells at day 3 might represent a population of primitive NPCs that can be induced to become anterior NPCs and also possesses the potential to differentiate posteriorly. With neural patterning initiated at day 3 after neural induction, the early MN marker ISL1 can be seen at day 13 after hESC differentiation, while mature MN markers including HB9 and ChAT are expressed after 17-day differentiation. (b) Our results point to a previously unidentified primitive stage of neural progenitors that is intermediate between pluripotent hESCs and anterior NPCs. This population of potential primitive NPCs can be induced to become anterior NPCs in the absence of patterning factors, and also possesses the potential to differentiate along the posterior fate in the presence of patterning factors.

Article Snippet: For ISL1 rescue experiments, ISL1 expression vector was generated by inserting human ISL1 cDNA (OriGene SC125257) into pSin-EF2-alpha-Puro lentivector (Addgene).

Techniques: Expressing, Marker

Journal: Stem cell research & therapy

Article Title: Intra-amniotic transplantation of brain-derived neurotrophic factor-modified mesenchymal stem cells treatment for rat fetuses with spina bifida aperta.

doi: 10.1186/s13287-022-03105-6

Figure Lengend Snippet: Fig. 4 Engrafted BDNF-BMSCs expressed neuron-related specific markers in the spinal cord of SBA. A–D. Double fluorescent staining of BRN3A, ISLET1, SYT, and SYN with GFP in the sections of defective spinal cords with BDNF-BMSCs engraftment. Typical double-positive cells are labeled with arrows. E–H. The relative mRNA expression of BRN3A, ISLET1, SYN, and SYT in the spinal cords of BDNF-BMSC-, BMSC-, and PBS-injected groups was quantitatively analyzed by RT-qPCR (n = 12/group). I. Simple western system detection of BRN3A, ISLET1, SYN, and SYT protein in the spinal cords from SBA fetuses after intra-amniotic injection of PBS, BMSCs, and BDNF-BMSCs. Gray–white stripes were detected by the HRP channel, and red stripes were detected the by NIR channel. J–M. Quantification of relative protein levels determined from the special peak area of BRN3A, ISLET1, SYN, and SYT shown in I. N. Representative images of GFP and BRN3A double staining in defective spinal cords with BDNF-BMSCs and pure BMSC engraftment. The images in the small white box are enlarged in the lower right corner. SC: spinal cord. O. The BRN3A+ cells around the engrafted BMSCs-BDNF or BMSCs in defective spinal cords were counted in 40 × field (n = 6, p < 0.05). *Significant difference compared to the PBS-injected group, †Significant difference compared to the BMSC-injected group, p < 0.05

Article Snippet: The target protein antibodies were BDNF (1:25; NB10098,682, NOVUS), BCL2 (1:25; SC-7382, NOVUS), BAX (1:50; D2E11, Cell Signaling), CASP3 (1:25; NB10023,708, NOVUS), BRN3A (1:50; MAB1585, Millipore), ISLET1 (1:50; NBP2-14,999, NOVUS), SYNAPSIN1 (1:25; AB1543P, Millipore), SYNAPTOTAGMIN (1:25; MAB5200, Millipore), and GAPDH (1:100; 60,004-1-Ig, Table 1 Sequences of primers used in this study Gene Accession number Primer sequences (5’‐3’)/exon location (nt) Annealing Tm (°C) PCR product (bp) BDNF NM_001270630.1 Sense: ATG GTT ATT TCA TAC TTC GGT TGC /391-414 Antisense: CTC AAA AGT GTC AGC CAG GGA/567-547 60 177 BCL2 NM_016993.1 Sense: ACG AGT GGG ATA CTG GAG ATG AAG ACT/317-343 Antisense:ACG TCC TGG CAG CCG TGT CT/442-423 61 126 BAX NM_017059.2 Sense: TGG AAG AAG ATG GGC TGA GGC/651-671 Antisense: CAT TCC CAC CCC TCC CAA TAAT/789-768 60 139 CASP3 NM_012922.2 Sense: GGA ACG AAC GGA CCT GTG G/441-459 Antisense: CGG GTG CGG TAG AGT AAG C/660-642 60 220 BRN3A XM_001076964 Sense: ATC GCG GTG TCC CAG GGC AAGA/196-218 Antisense: CGA GAT GTG GTC CAG CAG GTCA/363-341 60 168 ISLET1 NM_017339.3 Sense:CAT CGA ATG TTT CCG CTG TG/308-327 Antisense:GGT CTT CTC GGG CTG TTT GT/548-529 60 241 SYN NM_006950.3 Sense:TGG GCA AGG TCA AGG TAG A/959-977 Antisense:TGG ACA CGC ACA TCG TAT TTA/1079-1059 60 121 SYT NM_005639.2 Sense: CGC TGA GAA AGA AGA GCA AGA/1452-1472 Antisense:ATA AGC CAC CCA CAT CCA TC/1582-1563 60 131 GAPDH NM_017008.4 Sense: TGC CGC CTG GAG AAA CCT GC/808-827 Antisense: AGC AAT GCC AGC CCC AGC AT/975-956 60 168 Proteintech).

Techniques: Staining, Labeling, Expressing, Injection, Quantitative RT-PCR, Simple Western, Double Staining