Journal: Journal of Tissue Engineering

Article Title: Development of an iPSC-derived immunocompetent skin model for identification of skin sensitizing substances

doi: 10.1177/20417314251336296

Figure Lengend Snippet: Functional and molecular characterization of iPSC-derived dendritic cells (iPSC-DC). (a and b) Phase-contrast microscopy images demonstrate distinct morphology. iPSC-DC display characteristic dendritic projections, while iPSC exhibit tightly packed colonies with a high nucleus-to-cytoplasm ratio and well-defined colony borders (magnification 20×). (c and d) iPSC-DC and iPSC were stained with fluorochrome-conjugated antibodies against HLA-DR, CD209, CD11c, CD86, and CD83. Unstained controls are shown in red histograms. (e) Relative IL-8 secretion of iPSC and iPSC-DC treated with a positive control composed of 50 ng/mL tumor necrosis factor-alpha (TNF-α) and 50 ng/mL interleukin-1 beta (IL-1β) in comparison to their untreated controls. (f) Western blot analysis of iPSC and iPSC-DC for the pluripotency marker Lin28a and the DC-markers CD11c and CD209, β-Actin was used as a loading control. (g) Mixed Lymphocyte Reaction Assay to assess antigen-presenting function of mature iPSC-DC: Mature iPSC-DC were co-cultured with 1 × 10 CFSE-labeled allogeneic CD4+ T cells at a DC:T cell ratio of 1:25. After 5 days of co-culture, proliferation of CD4+ CFSE-labeled T cells was analyzed by flow cytometry ( N = 3–6). T cells cultured without DC, either with or without CD3/CD28 T cell activator, served as negative and positive controls, respectively. Mature DC treated with DNCB (positive control) significantly induced the proliferation of allogeneic T cells, demonstrating their functional antigen-presenting capability. Statistical analysis was performed using one-way ANOVA with Dunnett’s correction. ** p -value ⩽ 0.01, ns: not significant. Data are presented as mean ± SD. ( N = 3–6).

Article Snippet: Following primary antibody incubation, membranes were washed with a TBS-T buffer (tris-buffered saline with Tween) and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-mouse (Cell Signaling Technology, Frankfurt/Main, Germany) for Lin28a and CD209, and anti-rabbit (Cell Signaling Technology, Frankfurt/Main, Germany) for CD11c.

Techniques: Functional Assay, Derivative Assay, Microscopy, Staining, Positive Control, Comparison, Western Blot, Marker, Control, Cell Culture, Labeling, Co-Culture Assay, Flow Cytometry

Journal: bioRxiv

Article Title: YY1 protein is essential for the promotion of Muller glia reprogramming and retina regeneration

doi: 10.1101/2025.01.31.635905

Figure Lengend Snippet: (A) An experimental timeline that describes injury, Lissamine-tagged Morpholino (MO) delivery, electroporation at 0dpi, BrdU pulse for 5hrs at 4dpi, followed by harvesting. (B and C) IF microscopy images of retinal cross-sections show a decrease in the number of BrdU + retinal progenitors with yy1a MO and yy1b MO alone and in combination, compared to control MO injected retina at 4dpi (B), which is quantified (C). (D) Western Blot analyses of various RAGs in retinal extracts prepared from retinae electroporated with MOs against yy1a and yy1b at 2dpi. (E) The qPCR analyses of let-7a mi-RNA in the yy1a and yy1b knockdown condition at 2dpi; *p < 0.05; n=6 biological replicates. (F and G) The schematic showing Yy1-binding sites (BS) on ascl1a promoter (F) and the retinal ChIP assays confirm the physical binding of Yy1 on ascl1a promoter, in 2dpi retina (G). N.S marks the negative control. (H and I) Luciferase assay done in zebrafish embryos at 24hpf, injected with ascl1a: GFP-luciferase construct shows upregulation in the ascl1a promoter activity in yy1a and yy1b overexpression (H), while downregulation in their combined knockdown (I). ( J and K) The schematic showing Yy1-binding sites (BS) on lin28a promoter (J) and the retinal ChIP assays confirm the physical binding of Yy1 on lin28a promoter, in 2dpi retina (K). (L and M) Luciferase assay done in zebrafish embryos at 24hpf, injected with lin28a: GFP-luciferase construct shows upregulation in the lin28a promoter activity in yy1a and yy1b overexpression (L), while opposite in their combined knockdown (M). (N and O) The schematic showing Yy1-binding sites (BS) on yy1a intron (N) and the retinal ChIP assays confirm the loss of Yy1 binding onto its intron, in 2dpi retina (O). (P) The qPCR analysis shows the downregulation of yy1a and yy1b in their own combined knockdown. (Q and R) The schematic showing Yy1-binding sites (BS) on zic2b promoter (Q) and the retinal ChIP assays confirm the physical binding of Yy1 on zic2b promoter, in 2dpi retina (R). (S) Luciferase assay done in zebrafish embryos at 24hpf, injected with zic2b: GFP-luciferase construct shows upregulation in the zic2b promoter activity in yy1a and yy1b knockdown. (T and U) The schematic showing Yy1-binding sites (BS) on oct4 promoter (T) and the retinal ChIP assays confirm the physical binding of Yy1 on oct4 promoter, in 2dpi retina (U). (V) Luciferase assay done in zebrafish embryos at 24hpf, injected with oct4: GFP-luciferase construct shows upregulation in the oct4 promoter activity in yy1a and yy1b knockdown. Error bars represent SD. *p < 0.001 in (H, L, M, P, S, V); *p < 0.05 (F). n = 6 biological replicates (C, E and P).

Article Snippet: Primary antibodies used in the study are Rb Anti-YY1, rabbit polyclonal antibody against human ASCL1/MASH1 (Abcam, cat. no. ab74065), Rabbit polyclonal antibody against LIN28a (Cell Signalling Technologies, cat. no. 3978), mouse monoclonal against Oct3/4 (sc5279; Santa Cruz Biotechnology), Mouse polyclonal antibody against Zic2b (raised in house in mice against full length zebrafish Zic2b protein as antigen), rabbit polyclonal antibody against Sox2 (cat. no. ab59776; Abcam), Rabbit polyclonal antibody against pSmad3 (Abcam, ab52903), Rabbit monoclonal antibody against Tgfbi (Abcam, ab170874).

Techniques: Electroporation, Microscopy, Control, Injection, Western Blot, Knockdown, Binding Assay, Negative Control, Luciferase, Construct, Activity Assay, Over Expression

Journal: bioRxiv

Article Title: YY1 protein is essential for the promotion of Muller glia reprogramming and retina regeneration

doi: 10.1101/2025.01.31.635905

Figure Lengend Snippet: (A) An experimental timeline that describes injury, mRNA transfection and electroporation at 0dpi, BrdU pulse for 5hrs at 4dpi, followed by harvesting. (B and C) IF microscopy images of retinal cross-sections show a significant increase in the number of BrdU + and PCNA + retinal progenitors in yy1a overexpression, while no significant increase in the case of yy1b overexpression as compared to control gfp mRNA transfected retina at 4dpi (B), which is quantified (C). (D and E) IF microscopy images of retinal cross-sections show a significant increase in the number of BrdU + and PCNA + retinal progenitors in yy1a and yy1b combined overexpression, as compared to control gfp mRNA transfected retina at 4dpi (D), which is quantified (E). (F) qPCR analysis shows an upregulation in the levels of ascl1a, lin28a, sox2 and hdac1 in the combined overexpression of yy1a and yy1b. (G) Western blot analysis shows an upregulation in the levels of Ascl1a, Lin28a, Sox2 and Hdac1, while downregulation of Oct4 and Zic2b in the combined overexpression of yy1a and yy1b. (H) qPCR analysis shows a downregulation in the levels of oct4 and zic2b in the combined overexpression of yy1a and yy1b. Scale bars represent 10μm in (B and D); asterisk marks the injury site and GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer in (B and D); dpi, days post injury. Error bars represent SD. *p < 0.005 in (E, F); *p < 0.0005 (C, H); n.s is non-significant. n = 6 biological replicates.

Article Snippet: Primary antibodies used in the study are Rb Anti-YY1, rabbit polyclonal antibody against human ASCL1/MASH1 (Abcam, cat. no. ab74065), Rabbit polyclonal antibody against LIN28a (Cell Signalling Technologies, cat. no. 3978), mouse monoclonal against Oct3/4 (sc5279; Santa Cruz Biotechnology), Mouse polyclonal antibody against Zic2b (raised in house in mice against full length zebrafish Zic2b protein as antigen), rabbit polyclonal antibody against Sox2 (cat. no. ab59776; Abcam), Rabbit polyclonal antibody against pSmad3 (Abcam, ab52903), Rabbit monoclonal antibody against Tgfbi (Abcam, ab170874).

Techniques: Transfection, Electroporation, Microscopy, Over Expression, Control, Western Blot

Journal: bioRxiv

Article Title: YY1 protein is essential for the promotion of Muller glia reprogramming and retina regeneration

doi: 10.1101/2025.01.31.635905

Figure Lengend Snippet: (A) An experimental timeline that describes injury, mRNA delivery, electroporation at 0dpi, dipping in the PFI3 drug and BrdU pulse for 5hrs at 4dpi, followed by harvesting. (B and C) IF microscopy images of retinal cross-sections at 4dpi, show the effect of combined treatment of PFI3 along with yy1a and yy1b overexpression on the number of proliferating cells, which are marked by BrdU + (B), and quantified in (C). (D) qPCR analysis of few RAGs in the experimental conditions explained in B and C. (E) Western blot analysis shows regulation of Ascl1a, Lin28a, Hdac1 and Oct4 in the experimental conditions explained in B and C. (F) An experimental timeline that describes injury, mRNA delivery, electroporation at 0dpi, dipping in the PFI3 drug until 4dpi and BrdU pulse for 5hrs at 4dpi, followed by harvesting at 20dpi. (G and H) IF microscopy images of retinal cross-sections at 20dpi, shows the decrease in number of surviving BrdU positive cells, which were labelled at 4dpi in the combined treatment of PFI3 along with overexpression of yy1a and yy1b mRNA (G), which is quantified in (H). Scale bars represent 10μm in (B and G); asterisk marks the injury site and GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer in (B and G); dpi, days post injury. Error bars represent SD. *p < 0.001 in (C, D, H), n.s is non-significant. n = 6 biological replicates.

Article Snippet: Primary antibodies used in the study are Rb Anti-YY1, rabbit polyclonal antibody against human ASCL1/MASH1 (Abcam, cat. no. ab74065), Rabbit polyclonal antibody against LIN28a (Cell Signalling Technologies, cat. no. 3978), mouse monoclonal against Oct3/4 (sc5279; Santa Cruz Biotechnology), Mouse polyclonal antibody against Zic2b (raised in house in mice against full length zebrafish Zic2b protein as antigen), rabbit polyclonal antibody against Sox2 (cat. no. ab59776; Abcam), Rabbit polyclonal antibody against pSmad3 (Abcam, ab52903), Rabbit monoclonal antibody against Tgfbi (Abcam, ab170874).

Techniques: Electroporation, Microscopy, Over Expression, Western Blot

Journal: Oncology Letters

Article Title: Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells

doi: 10.3892/ol.2024.14710

Figure Lengend Snippet: Sequences of primers used for reserve transcription-quantitative PCR.

Article Snippet: Polyclonal anti-phosphorylated-Lin28A (pLin28) (Ser200) antibodies (cat. no. PA5-105696) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

Techniques: Sequencing

Journal: Oncology Letters

Article Title: Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells

doi: 10.3892/ol.2024.14710

Figure Lengend Snippet: RSV decreases expression of Lin28A mRNA in NCCIT cells in a time-concentration manner. **P<0.01; ***P<0.001; ****P<0.0001 vs. Ctl. RSV, resveratrol; Ctl, control.

Article Snippet: Polyclonal anti-phosphorylated-Lin28A (pLin28) (Ser200) antibodies (cat. no. PA5-105696) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Concentration Assay, Control

Journal: Oncology Letters

Article Title: Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells

doi: 10.3892/ol.2024.14710

Figure Lengend Snippet: Lin28A protein is more stable than mRNA and is affected only by the highest concentrations of RSV. NCCIT cells were treated with different concentrations of RSV (25, 50, 100 and 150 µM) at different time periods. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 vs. Ctl. RSV, resveratrol; Ctl, control.

Article Snippet: Polyclonal anti-phosphorylated-Lin28A (pLin28) (Ser200) antibodies (cat. no. PA5-105696) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

Techniques: Control

Journal: Oncology Letters

Article Title: Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells

doi: 10.3892/ol.2024.14710

Figure Lengend Snippet: RSV induces Lin28A proteasomal degradation. NCCIT cells were treated with 5 µM of MG132 and 150 µM of RSV for different periods of time (24, 48 and 96 h) and then western blotting was performed. (A) Representative western blot of Lin28A protein expression. (B) Representative graph of western blotting analysis. *P<0.05; **P<0.01; ****P<0.0001 vs. Ctl. +++ P<0.001; ++++ P<0.0001 vs. R group. RSV/R, resveratrol; M, MG132; Ctl, control.

Article Snippet: Polyclonal anti-phosphorylated-Lin28A (pLin28) (Ser200) antibodies (cat. no. PA5-105696) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

Techniques: Western Blot, Expressing, Control

Journal: Oncology Letters

Article Title: Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells

doi: 10.3892/ol.2024.14710

Figure Lengend Snippet: Inhibition of USP28 with specific siRNAs is not sufficient to totally silence Lin28A protein expression. (A) Expression of USP28 mRNA in cells transfected with siUSP28, quantified using RT-qPCR. The controls were transfected NCCIT cells and the transfection of a nonspecific siRNA that did not affect the gene of interest (USP28). (B) Representative western blot of USP28 protein expression in cells transfected with siUSP28 at different times (24, 48, 72 and 96 h). (C) Expression of Lin28A mRNA in cells transfected with siUSP28, quantified by stem-loop RT-qPCR. (D) Representative western blot of the expression of Lin28A protein and pLin28A by MAPK/ERK (serine 200) in cells transfected with siUSP28 at different times (24, 48, 72 and 96 h). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 vs. Ctl. + P<0.05; +++ P<0.001; ++++ P<0.0001 vs. (−). DV, densitometry value; USP28, ubiquitin-specific protease 28; si, small-interfering; RT-qPCR, reverse transcription-quantitative PCR; Ctl, control; (−), nonspecific siRNA; pLin28A, phosphorylated Lin28A.

Article Snippet: Polyclonal anti-phosphorylated-Lin28A (pLin28) (Ser200) antibodies (cat. no. PA5-105696) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

Techniques: Inhibition, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Ubiquitin Proteomics, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Oncology Letters

Article Title: Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells

doi: 10.3892/ol.2024.14710

Figure Lengend Snippet: RSV decreases the expression of pLin28A in serine 200 caused by MAPK/ERK. Western blot and representative graph of western blot analysis of NCCIT cells treated with (A) different concentrations of RSV for 96 h and (B) 150 µM of RSV at different times (24, 48, 72 and 96 h). ****P<0.0001 vs. Ctl. DV, densitometry value; RSV, resveratrol; pLin28A, phosphorylated Lin28A; Ctl, control.

Article Snippet: Polyclonal anti-phosphorylated-Lin28A (pLin28) (Ser200) antibodies (cat. no. PA5-105696) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Letters

Article Title: Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells

doi: 10.3892/ol.2024.14710

Figure Lengend Snippet: MAPK/ERK inhibitor decreased the expression of pLin28A (serine 200), and the total protein expression of Lin28A, similar to treatment with resveratrol. Representative western blot of the NCCIT cells treatment with the MAPK/ERK inhibitor (5 or 7.5 µM) at different times (24, 48, 72 and 96 h). DV, densitometry value; pLin28A, phosphorylated Lin28A; USP28, ubiquitin-specific protease 28; Ctl, control.

Article Snippet: Polyclonal anti-phosphorylated-Lin28A (pLin28) (Ser200) antibodies (cat. no. PA5-105696) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Western Blot, Ubiquitin Proteomics, Control

Journal: Oncology Letters

Article Title: Resveratrol inhibits Lin28A expression and induces its degradation via the proteasomal pathway in NCCIT cells

doi: 10.3892/ol.2024.14710

Figure Lengend Snippet: RSV inhibits Lin28A mRNA expression in NCCIT cells, potentially through inhibiting Lin28A mRNA synthesis via the decrease in the transcription factors SP1 and STAT3; however, this has not been completely proven in the model. Conversely, it was revealed that RSV inhibits the expression of the USP28 deubiquitinase and the MAPK/ERK pathway, which is responsible for phosphorylation at serine 200, which allows its stability. This causes the Lin28A protein to lose its stability mechanisms and begin its degradation through the proteasomal pathway. Image produced in Biorender. RSV, resveratrol; USP28, ubiquitin-specific protease 28; P, phosphorylation at serine 200; Ub, ubiquitin marks. Created with BioRender.com.

Article Snippet: Polyclonal anti-phosphorylated-Lin28A (pLin28) (Ser200) antibodies (cat. no. PA5-105696) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Phospho-proteomics, Produced, Ubiquitin Proteomics