Journal: Scientific Reports

Article Title: Oct4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts

doi: 10.1038/s41598-019-52294-3

Figure Lengend Snippet: Characterisation of Oct4+ hDFs. (a) Morphology of control GFP+ hDFs and Oct4+ hDFs 6 days post transduction, as determined by light microscopy. Scale bar = 100 μm. (b) Analysis of POU5F1 expression in Oct4+ hFDs relative to GFP+ hDFs 6 days post transduction, as determined by RT-qPCR. Error bars represent ± SD. (c) Western blot analysis of mesenchymal/epithelial markers and Oct4 expression in control GFP+ hDFs and Oct4+ hDFs 6 days post transduction. α-tubulin and β-actin were used as a loading control. Uncropped western blot images are shown in Supplementary Fig. . (d) Analysis of SNAI2, COL1A, CDH1, EPCAM, CRB3 expression in Oct4+ hFDs relative to GFP+ hDFs 6 days post transduction, as determined by RT-qPCR. Error bars represent ± SD. (e) Analysis of cell migration of Oct4+ and GFP+ hDFs, as determined by scratch-wound healing assay. The graph shows cell-free area during time upon making a straight scratch on tissue culture plate. Error bars show ± SE, n = 5.

Article Snippet: Briefly, HEK293T cells were transfected with vectors for Oct4 over-expression pSin-EF2-Oct4-Pur (a gift from James Thomson), (Addgene #16579) , or GFP (for control experiments) PL-SIN-EF1α-EGFP (a gift from James Ellis) (Addgene #21320) , or pCW57-GFP-miR-302cluster (for mir-302 over-expression experiments), or pCW57-GFP-2A-MCS (for control experiments) together with 2 nd generation of lentiviral production plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) kindly provided by Didier Trono.

Techniques: Control, Transduction, Light Microscopy, Expressing, Quantitative RT-PCR, Western Blot, Migration, Wound Healing Assay

Journal: Scientific Reports

Article Title: Oct4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts

doi: 10.1038/s41598-019-52294-3

Figure Lengend Snippet: Variation of miRNA expression between Oct4+ and GFP+ hDFs. (a) Scheme illustrating experimental scenario. (b) Hierarchical clustering, (c) heatmap, and (d) PCA analysis showing differences in miRNA expression between Oct4+ and GFP+ hDFs in each replicate.

Article Snippet: Briefly, HEK293T cells were transfected with vectors for Oct4 over-expression pSin-EF2-Oct4-Pur (a gift from James Thomson), (Addgene #16579) , or GFP (for control experiments) PL-SIN-EF1α-EGFP (a gift from James Ellis) (Addgene #21320) , or pCW57-GFP-miR-302cluster (for mir-302 over-expression experiments), or pCW57-GFP-2A-MCS (for control experiments) together with 2 nd generation of lentiviral production plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) kindly provided by Didier Trono.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Oct4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts

doi: 10.1038/s41598-019-52294-3

Figure Lengend Snippet: miRNA expression associated with Oct4-induced plastic state. (a) Heatmap shows the expression of miRNAs in three biological replicates of Oct4+ and GFP+ hDFs. (b) Pairwise analysis (volcano plot) of miRNAs expression between Oct4+ and GFP+ hDFs. Purple dots represent significantly (p < 0.05, Fold change(log2) > 1.5) differentially expressed miRNAs.

Article Snippet: Briefly, HEK293T cells were transfected with vectors for Oct4 over-expression pSin-EF2-Oct4-Pur (a gift from James Thomson), (Addgene #16579) , or GFP (for control experiments) PL-SIN-EF1α-EGFP (a gift from James Ellis) (Addgene #21320) , or pCW57-GFP-miR-302cluster (for mir-302 over-expression experiments), or pCW57-GFP-2A-MCS (for control experiments) together with 2 nd generation of lentiviral production plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) kindly provided by Didier Trono.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Oct4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts

doi: 10.1038/s41598-019-52294-3

Figure Lengend Snippet: Validation of NGS using RT-qPCR. Expression levels of selected miRNAs that are differentially expressed based on NGS data, as determined by RT-qPCR. (a) miRNAs up-regulated in Oct4+ hDFs: mir-183, mir-302 cluster members, and miR-9-5p. (b) miRNAs down-regulated in Oct4+ hDFs: miR-34c-5p, miR-193a-5p, miR-424-5p, miR-503-5p, miR-143-3p and miR-145-5p. The results are shown as expression relative to corresponding control GFP+ hDFs. Error bars represent ± SD.

Article Snippet: Briefly, HEK293T cells were transfected with vectors for Oct4 over-expression pSin-EF2-Oct4-Pur (a gift from James Thomson), (Addgene #16579) , or GFP (for control experiments) PL-SIN-EF1α-EGFP (a gift from James Ellis) (Addgene #21320) , or pCW57-GFP-miR-302cluster (for mir-302 over-expression experiments), or pCW57-GFP-2A-MCS (for control experiments) together with 2 nd generation of lentiviral production plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) kindly provided by Didier Trono.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Control

Journal: Scientific Reports

Article Title: Oct4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts

doi: 10.1038/s41598-019-52294-3

Figure Lengend Snippet: miRNA clusters and families analysis. (a) Up-regulated and (b) down-regulated miRNA clusters in Oct4+ hDFs. (c) Up-regulated and (d) down-regulated miRNA families in Oct4+ hDFs. (e) The Circos plot showing genomic locations and expression of miRNA clusters in Oct4+ and GFP+ hDFs. The outer circle represents chromosome ideogram as the reference for the genomic localization of individual miRNAs and clusters. Names of miRNA clusters are represented by name of the first miRNA molecule in a particular cluster. Clusters significantly up-regulated in Oct4+ hDFs are marked with two asterisks (**), clusters significantly down-regulated in Oct4+ hDFs are marked with one asterisk (*), (p < 0.05, Fold change(log2) > 1.5). The expression level of individual miRNAs (heatmap), miRNA clusters in Oct4+, and miRNA clusters in GFP+ hDFs are shown in the external, middle, and inner circles, respectively. The lines in the centre area represent interconnections between miRNA clusters and families. Red lines represent miRNA clusters that are also families.

Article Snippet: Briefly, HEK293T cells were transfected with vectors for Oct4 over-expression pSin-EF2-Oct4-Pur (a gift from James Thomson), (Addgene #16579) , or GFP (for control experiments) PL-SIN-EF1α-EGFP (a gift from James Ellis) (Addgene #21320) , or pCW57-GFP-miR-302cluster (for mir-302 over-expression experiments), or pCW57-GFP-2A-MCS (for control experiments) together with 2 nd generation of lentiviral production plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) kindly provided by Didier Trono.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Oct4-mediated reprogramming induces embryonic-like microRNA expression signatures in human fibroblasts

doi: 10.1038/s41598-019-52294-3

Figure Lengend Snippet: ( a ) miRNA clusters found to be significantly down- or up-regulated in Oct4+ hDFs as well as in human pluripotent stem cells. (b) Venn diagram shows number of miRNAs that are up-regulated in Oct4+ hDFs and also found to be highly-expressed in human blood and/or brain tissues (according to miRmine database).

Article Snippet: Briefly, HEK293T cells were transfected with vectors for Oct4 over-expression pSin-EF2-Oct4-Pur (a gift from James Thomson), (Addgene #16579) , or GFP (for control experiments) PL-SIN-EF1α-EGFP (a gift from James Ellis) (Addgene #21320) , or pCW57-GFP-miR-302cluster (for mir-302 over-expression experiments), or pCW57-GFP-2A-MCS (for control experiments) together with 2 nd generation of lentiviral production plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) kindly provided by Didier Trono.

Techniques:

Journal: Cell Death & Disease

Article Title: CAMK2A supported tumor initiating cells of lung adenocarcinoma by upregulating SOX2 through EZH2 phosphorylation

doi: 10.1038/s41419-020-2553-6

Figure Lengend Snippet: a , b Immunohistochemical (IHC) evaluation of p-CAMK2A T286 expression in lung adenocarcinoma. a High-level expression, showing higher frequency of tumor cells staining positively for cytoplasmic and nuclear signals (arrows). b Low-level expression, showing fewer positively stained tumor nuclei (arrows) in the same unit area as shown in Fig. 1b. c COX regression analysis for recurrence-free survival (RFS) and overall survival (OS) stratified by p-CAMK2A T286 levels. Survival curves were generated by SPSS 18.0. d COX regression analysis of CAMK2A effects on RFS and OS using public expression array data ( www.kmplot.com/lung ).

Article Snippet: Human full length CAMK2A was cloned into PCDH-CMV-MCS-EF1-COPGFP vector (SBI, Mountain View, CA) for stable CAMK2A overexpression. pSin-EF2-SOX2-Pur (16577, Addgene) was used for stable SOX2 overexpression.

Techniques: Immunohistochemical staining, Expressing, Staining, Generated

Journal: Cell Death & Disease

Article Title: CAMK2A supported tumor initiating cells of lung adenocarcinoma by upregulating SOX2 through EZH2 phosphorylation

doi: 10.1038/s41419-020-2553-6

Figure Lengend Snippet: a Western blot of shRNA-mediated CAMK2A KD in HCC827 and PDCL#24 cells. b Activated CAMK2A (p-T286) relative to total CAMK2A expression was increased in spheres compared with cell monolayers of H441, HCC827 and PDCL#24. c , d Sphere formation of both cell lines with CAMK2A KD were inhibited for two generations. e Colony formation of CAMK2A KD cells were decreased in both cell lines. f Flow cytometry of HCC827 with CAMK2A KD showed reduction of ALDH-expressing cells. g , h Tumor sizes and growth curves of xenografts with CAMK2A KD from HCC827 ( g ) and PDCL#24 ( h ) were suppressed compared with controls (shNTC). i Western blot of CAMK2A OE in H1299 and A549 cells. j , k Sphere formation ( j ) and colony formation ( k ) of H1299 and A549 with CAMK2A OE were increased. l Tumor growth of H1299-derived xenografts with CAMK2A OE was facilitated. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with control. Data represented mean ± SD.

Article Snippet: Human full length CAMK2A was cloned into PCDH-CMV-MCS-EF1-COPGFP vector (SBI, Mountain View, CA) for stable CAMK2A overexpression. pSin-EF2-SOX2-Pur (16577, Addgene) was used for stable SOX2 overexpression.

Techniques: Western Blot, shRNA, Expressing, Flow Cytometry, Derivative Assay, Control

Journal: Cell Death & Disease

Article Title: CAMK2A supported tumor initiating cells of lung adenocarcinoma by upregulating SOX2 through EZH2 phosphorylation

doi: 10.1038/s41419-020-2553-6

Figure Lengend Snippet: a , b Cell viability of HCC827 with CAMK2A KD showed increased sensitivity to gefitinib ( a ) and cisplatin ( b ). c , d Cell viability of A549 ( c ) and H1299 ( d ) with CAMK2A OE showed increased resistance to cisplatin. e , f Western blot showed that short term treatment of gefitinib and cisplatin increased p-CAMK2A level in HCC827 ( e ) and H1299 cells ( f ). g , h Western blot of gefitinib-resistant HCC827 (GR) ( g ) and cisplatin-resistant A549 (CR) cells ( h ) showed increased p-CAMK2A T286 relative to total CAMK2A compared with parental control cells. i , j Cell viability of HCC827 GR cells ( i ) and A549 CR cells ( j ) under CAMK2A inhibition by KN93 showed sensitization to gefitinib and cisplatin, respectively. k Flow cytometry analysis of cisplatin treated HCC827 and PDCL#24 cells with CAMK2A KD showed increased apoptosis. * p < 0.05; ** p < 0.01, compared with control, mean ± SD was presented.

Article Snippet: Human full length CAMK2A was cloned into PCDH-CMV-MCS-EF1-COPGFP vector (SBI, Mountain View, CA) for stable CAMK2A overexpression. pSin-EF2-SOX2-Pur (16577, Addgene) was used for stable SOX2 overexpression.

Techniques: Western Blot, Control, Inhibition, Flow Cytometry

Journal: Cell Death & Disease

Article Title: CAMK2A supported tumor initiating cells of lung adenocarcinoma by upregulating SOX2 through EZH2 phosphorylation

doi: 10.1038/s41419-020-2553-6

Figure Lengend Snippet: a , b Quantitative PCR of SOX2 , NANOG and POU5F1 transcripts in HCC827 and PDCL#24 cells with CAMK2A KD ( a ), and in H1299 and A549 cells with CAMK2A OE ( b ). c Western blot showing SOX2 decrease in HCC827 and PDCL#24 cells with CAMK2A KD. d Western blot showing SOX2 changes in HCC827 and PDCL#24 cells with CAMK2A KD and SOX2 OE. e – h Overexpression of SOX2 increased sphere formation ( e , f ) and colony formation ( g , h ) in CAMK2A-downregulated HCC827 and PDCL#24 cells. i SOX2 overexpression enhanced tumorigenicity in HCC827 shCAMK2A-1-derived xenografts. n = 4. j Expression of SOX2 in CAMK2A-overexpressed H1299 and A549 cells by western blot. k Downregulation of SOX2 in CAMK2A-overexpressed H1299 and A549 cells was confirmed by western blot. l , m Downregulation of SOX2 abrogated CAMK2A’s tumor initiating properties in sphere formation ( l ) and colony formation ( m ) in H1299 and A549 CAMK2A-overexpressed cells. * p < 0.05; ** p < 0.01; *** p < 0.001, compared with control, mean ± SD was presented.

Article Snippet: Human full length CAMK2A was cloned into PCDH-CMV-MCS-EF1-COPGFP vector (SBI, Mountain View, CA) for stable CAMK2A overexpression. pSin-EF2-SOX2-Pur (16577, Addgene) was used for stable SOX2 overexpression.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Derivative Assay, Expressing, Control

Journal: Cell Death & Disease

Article Title: CAMK2A supported tumor initiating cells of lung adenocarcinoma by upregulating SOX2 through EZH2 phosphorylation

doi: 10.1038/s41419-020-2553-6

Figure Lengend Snippet: a , b Expression of EZH2 and H3K27me3 in CAMK2A-downregulated HCC827 and PDCL #24 cells ( a ) as well as CAMK2A-overexpressed H1299 and A549 cells ( b ) by western blot. c , d Chromatin immunoprecipitation (ChIP) assay coupled with qPCR (ChIP-qPCR) analysis revealed the relative enrichment of H3K27me3 ( c ) and EZH2 ( d ) on SOX2 regulatory region in CAMK2A-downregulated HCC827 cells. e , f ChIP-qPCR analysis revealed the relative decrease of H3K27me3 ( e ) and EZH2 ( f ) on SOX2 regulatory region in CAMK2A-overexpressed H1299 cells. g – j Downregulation of EZH2 rescued the inhibitory effects of CAMK2A knockdown on SOX2 expression ( g ), sphere formation ( h ), ALDH + population ( i ) and cisplatin sensitivity ( j ) in HCC827 cells. * p < 0.05; ** p < 0.01; *** p < 0.001, compared with control, ## p < 0.01 compared with shCAMK2A, mean ± SD was presented.

Article Snippet: Human full length CAMK2A was cloned into PCDH-CMV-MCS-EF1-COPGFP vector (SBI, Mountain View, CA) for stable CAMK2A overexpression. pSin-EF2-SOX2-Pur (16577, Addgene) was used for stable SOX2 overexpression.

Techniques: Expressing, Western Blot, Chromatin Immunoprecipitation, ChIP-qPCR, Knockdown, Control

Journal: Cell Death & Disease

Article Title: CAMK2A supported tumor initiating cells of lung adenocarcinoma by upregulating SOX2 through EZH2 phosphorylation

doi: 10.1038/s41419-020-2553-6

Figure Lengend Snippet: a Co-immunoprecipitation (CoIP) in HEK293T cells with dual overexpression of CAMK2A and EZH2, either using CAMK2A or EZH2 as bait. b CoIP of EZH2 in HCC827 cells using CAMK2A as bait. c CoIP of EZH2 in H1299 CAMK2A-OE cells. d CoIP of EZH2 in A549 CAMK2A-OE cells with or without EZH2 knock-down using CAMK2A anti-body. e CoIP of EZH2 in HEK293T cells with overexpression of EZH2, CAMK2A WT or CAMK2A T286A using CAMK2A as bait. f Expression of total EZH2 and p-EZH2 T487 in monolayer and spheres from PDCL#24, HCC827, and A549 cells. g Expression of total EZH2 and p-EZH2 T487 in HCC827, H1299 and A549 cells with CAMK2A knock-down or overexpression by immunoblot. h Expression of p-EZH2 T487 in HCC827 cells treated with KN92/KN93 by immunoblot. i Expression of p-EZH2 T487 in HCC827 GR cells by immunoblot. j Immunoprecipitated p-EZH2 T487 level by EZH2 in HCC827 cells with or without CAMK2A knock-down. k CoIP of EZH2 in HEK293T cells with overexpression of CAMK2A, EZH2 WT or EZH2 T487A using CAMK2A as bait. j Correlation analysis between p-CAMK2A T286 and p-EZH2 T487 expression in 169 primary resected lung AD by immunohistochemistry. k Schematic representation depicting supportive effect of CAMK2A on TIC maintenance through EZH2/H3K27me3/SOX2 pathway.

Article Snippet: Human full length CAMK2A was cloned into PCDH-CMV-MCS-EF1-COPGFP vector (SBI, Mountain View, CA) for stable CAMK2A overexpression. pSin-EF2-SOX2-Pur (16577, Addgene) was used for stable SOX2 overexpression.

Techniques: Immunoprecipitation, Over Expression, Knockdown, Expressing, Western Blot, Immunohistochemistry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein

doi: 10.3389/fbioe.2022.911614

Figure Lengend Snippet: Gene construction and protein expression of Lin28 and Lin28-30Kc19. (A) Construction of E. coli expression vectors pET-23a/ Lin28 and pET-23a/ Lin28-30Kc19 . (B) PCR gel for confirming gene cloning of both genes, Lin28 and Lin28-30Kc19 in the expression vector pET-23a. Gray arrows indicate each gene of pET-23a , Lin28, and Lin28-30Kc19 , respectively. (C) SDS-PAGE gel for the quantification of purified proteins, Lin28 and Lin28-30Kc19. (D) Western blot analysis of the recombinant proteins. Red arrows indicate the Lin28 protein and the Lin28-30Kc19 protein, respectively. M indicates the protein marker (kDa).

Article Snippet: For the Lin28 gene, obtained from pSin-EF2-LIN28-Pur (Addgene plasmid #16580), primers contained Bam HI and Eco RI on the N and C terminals, respectively.

Techniques: Expressing, Cloning, Plasmid Preparation, SDS Page, Purification, Western Blot, Recombinant, Marker

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein

doi: 10.3389/fbioe.2022.911614

Figure Lengend Snippet: In vitro properties of the Lin28-30Kc19 protein. (A) Confocal images for detecting the intracellular delivery of the Lin29-30Kc19 protein. HDFs treated with 10 and 20 μg ml −1 of the Lin28-30Kc19 protein were observed, respectively. Blue fluorescence indicates the nuclei and green fluorescence indicates the Lin28-30Kc19 protein. Scale bars indicate 30 μm. (B) Western blot analysis of the cytosolic fraction of protein-treated cells to confirm the cell penetration property. After cultivation of cells with proteins for 24 h, cells were disrupted and then cell lysates were analyzed. Red arrows indicate the intracellularly delivered Lin28-30Kc19 protein. (C) Cytotoxicity of the Lin28-30Kc19 protein, depending on the concentration of the protein. HDFs were treated with each concentration of the Lin28-30Kc19 protein for 24 h and then cell viability was measured. The red dotted line indicates 100%, which is the value of the control group (untreated cells). n = 3. (D) In vitro stability of the Lin28-30Kc19 protein by Western blotting. HDFs treated with 50 μg ml −1 of the Lin28-30Kc19 protein were analyzed after 0, 6, 12, 24, and 48 h, respectively.

Article Snippet: For the Lin28 gene, obtained from pSin-EF2-LIN28-Pur (Addgene plasmid #16580), primers contained Bam HI and Eco RI on the N and C terminals, respectively.

Techniques: In Vitro, Fluorescence, Western Blot, Concentration Assay, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein

doi: 10.3389/fbioe.2022.911614

Figure Lengend Snippet: Aggregate-like forms of the Lin28-30Kc19 protein. HDFs treated with the Lin28-30Kc19 protein were observed through a microscope, depending upon the concentration of the protein and incubation time with the protein. Based on the red dotted line, the right region showed an aggregation tendency of the protein. Scale bars indicate 200 μm.

Article Snippet: For the Lin28 gene, obtained from pSin-EF2-LIN28-Pur (Addgene plasmid #16580), primers contained Bam HI and Eco RI on the N and C terminals, respectively.

Techniques: Microscopy, Concentration Assay, Incubation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein

doi: 10.3389/fbioe.2022.911614

Figure Lengend Snippet: Optimal protein treatment interval for reprogramming fibroblasts to hiPSCs. (A) Timeline for determining the optimal interval of the protein treatment. From Day 4, the cell culture medium was switched from a fibroblast medium to an Essential 8 ™ medium, and the Lin28-30Kc19 protein was treated every 24 or 48 h, respectively. Then, the cells were analyzed by AP staining on day 14. (B) Naked eye images of the AP-stained cells treated with the Lin28-30Kc19 protein for every 24 or 48 h, respectively. Scale bars indicate 5 mm. (C) Number of AP-stained colonies in a well. The AP-positive colonies were counted and compared in 24 and 48 h groups. (D) Size of the AP-positive single colony. Each AP-stained area was quantified and compared in the 24 and 48 h groups. (E) The AP-positive area in a well. Positively detected area was measure and compared in 24 and 48 h groups. (F) Total area positively stained with AP in three wells. The area of AP-positive colonies was summed up and compared in 24 and 48 h groups. * p < 0.05 and *** p < 0.001. n = 3.

Article Snippet: For the Lin28 gene, obtained from pSin-EF2-LIN28-Pur (Addgene plasmid #16580), primers contained Bam HI and Eco RI on the N and C terminals, respectively.

Techniques: Cell Culture, Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein

doi: 10.3389/fbioe.2022.911614

Figure Lengend Snippet: Morphological comparison of hiPSCs. Morphological changes of the fibroblasts were observed in untreated HDF (left), hiPSCs generated by four factors (middle), and hiPSCs produced by four factors with the Lin28-30Kc19 protein (right), at days 3, 7, and 11, respectively. Scale bars indicate 200 μm.

Article Snippet: For the Lin28 gene, obtained from pSin-EF2-LIN28-Pur (Addgene plasmid #16580), primers contained Bam HI and Eco RI on the N and C terminals, respectively.

Techniques: Comparison, Generated, Produced

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein

doi: 10.3389/fbioe.2022.911614

Figure Lengend Snippet: Characterization of hiPSCs generated by the addition of the Lin28-30Kc19 protein. (A) Timeline for characterization of hiPSCs at an early stage (day 7). (B) Naked eye images to detect reprogramming efficiency on day 7. HDFs treated with the Lin28-30Kc19 protein on days 4 and 6 were analyzed by AP staining. Scale bars indicate 5 mm. (C) Number of AP-stained colonies in a well on day 7. The AP-positive colonies were counted and compared in the without and with protein groups. (D) Size of an AP-positive single colony on day 7. Each AP-stained area was quantified and compared in the without and with Lin28-30Kc19 protein groups. (E) The AP-positive area in a well on day 7. Positively detected area was measured and compared without and with the recombinant protein groups. (F) The total area positively stained with AP in three wells on day 7. The area of AP-positive colonies was summed up and compared in without and with the protein groups. * p < 0.05 and *** p < 0.001. n = 3. (G) Timeline for the characterization of hiPSCs after passaging (day 14). (H) Microscopic images to confirm reprogramming efficiency on day 14. HDFs treated with the Lin28-30Kc19 protein on days 4, 6, 8, 10, and 12 were analyzed by AP staining. Scale bars indicate 500 μm.

Article Snippet: For the Lin28 gene, obtained from pSin-EF2-LIN28-Pur (Addgene plasmid #16580), primers contained Bam HI and Eco RI on the N and C terminals, respectively.

Techniques: Generated, Staining, Recombinant, Passaging

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein

doi: 10.3389/fbioe.2022.911614

Figure Lengend Snippet: Overall schematic of hiPSC generation using the Lin28-30Kc19 protein. Addition of the Lin28-30Kc19 protein improved the efficiency of hiPSC generation, due to the cell-penetrating and protein-stabilizing properties of the 30Kc19 protein, enabling intracellular delivery and maintenance of the stability of transcription factor, Lin28.

Article Snippet: For the Lin28 gene, obtained from pSin-EF2-LIN28-Pur (Addgene plasmid #16580), primers contained Bam HI and Eco RI on the N and C terminals, respectively.

Techniques:

Journal: EMBO Reports

Article Title: Acidic pH can attenuate immune killing through inactivation of perforin

doi: 10.1038/s44319-024-00365-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: MSCV IRES GFP , Addgege , Cat. No. 20672 RRID: Addgene_20672.

Techniques: Recombinant, Software