yu ss c  (ATCC)

Journal: bioRxiv

Article Title: Midline Assembloids Reveal Regulators of Human Axon Guidance

doi: 10.1101/2024.06.26.600229

Figure Lengend Snippet: (A) Schematic illustrating a developing human neural tube (purple) and location of the FP (green). (B) Schematic illustrating the generation of human FP organoids (hFpO, green) and human spinal organoids (hSpO, purple). (C) Representative immunocytochemistry images of FOXA2 expression and RedDot2 (nuclear stain) at day 8 of differentiation in cleared hFpO and hSpO. Scale bar, 100 μm. (D) Quantification of FOXA2 positive nuclei from immunocytochemistry images of individual hFpO and hSpO (n = 27 hFpO and 11 hSpO in 1-3 differentiation experiments from 3 hiPS cell lines; two-tailed t-test: ****P < 0.0001). Left bar shows individual organoids and the right bar indicates different hiPS cell line. Data is presented as mean ± SEM. (E) Gene expression analysis (by RT-qPCR) of FOXA2, NKX2-2, PAX6, PAX3, SHH , and NTN1 in hFpO and hSpO at day 8 of in vitro differentiation. Two-tailed Mann-Whitney test was used. For FOXA2 : n = 11 hFpO and 8 hSpO from 8 and 6 hiPS cell lines, respectively; ****P < 0.0001. For NKX2-2 : n = 7 hFpO and 7 hSpO from 5 hiPS cell lines; ***P = 0.0006. For PAX6 : n = 7 hFpO and 7 hSpO from 5 hiPS cell lines; **P = 0.0023. For PAX3 : n = 7 hFpO and 7 hSpO from 5 hiPS cell lines; ***P = 0.0006. For SHH : n = 11 hFpO and 5 hSpO from 8 and 5 hiPS cell lines, respectively; **P = 0.0018. For NTN1 : n = 5 hFpO and 5 hSpO from 4 hiPS cell lines; **P = 0.0019. Points indicate organoid samples from different differentiation experiments. Data is presented as mean ± SEM (F) Western blot of SHH and NTN1(cell extract and conditioned media) collected from hFpO and hSpO. Immunoblotting of GAPDH was used as loading control. (G) Western blot quantification of SHH and NTN1 (cell extract and conditioned media) from hFpO and hSpO normalized to GAPDH luminescence. Data is presented as mean ± SEM. Two-tailed Mann-Whitney test was used. For NTN1 (cell extract): n = 5 hFpO and 5 hSpO from 5 hiPS cell lines; **P = 0.0079. For NTN1 (media): n = 5 hFpO and 5 hSpO from 5 hiPS cell lines; **P = 0.0079. For SHH: n = 5 hFpO and 5 hSpO from 5 hiPS cell lines; **P = 0.0079. (H) Representative z-projected confocal images of E11.5 mouse dorsal spinal explants cultured for 40hr in conditioned media (CM) collected from either hFpO or hSpO. Explants were stained for Tubb3 and phalloidin (actin stain) to visualize axons. Scale bar, 100 μm. (I) Sholl analysis of axon outgrowth from conditioned media treated E11.5 mouse dorsal spinal explants and area under the curve quantification. Two-tailed Mann-Whitney test ***P = 0.0003; n = 8 for hSpO and n = 8 hFpO CM treated explants dissected from 2-3 embryos. (J) UMAP visualization of single cell gene expression of hFpO at day 8, separately colored by individual cell lines (shades of green). (n = 11,894 cells from 3 hiPS cell lines) (K) UMAP and violin plots showing gene expression of selected FP markers across 3 hiPS cell lines (shades of green). Color scale indicates normalized gene expression level. (L) Dot plots showing the expression of rostral-caudal defining genes expressed in hFpO cells. The size of the circle represents the percent of cells expressing each gene in 3 hiPS cell lines (shades of green). (M) UMAP visualization of subsetted primary human spinal progenitor clusters (left, CS12-19) and mapped hFpO cells (day 8) onto the primary atlas (right, color scale indicates prediction score of mapped hFpO cells and light gray points indicates reference atlas cells). (N) Bar plot displaying the proportion of predicted domains of hFpO cells from mapping onto a primary spinal progenitor atlas in each of the three hiPS cell lines.

Article Snippet: The following antibodies were used for immunostaining: anti-FOXA2 antibody (1:1000 dilution, rabbit, Abcam, ab108422), anti-TUBB3 antibody (1:500 dilution, rabbit, Cell Signaling Technology, MAB1195), anti-NKX2.2 antibody (1:100 dilution, mouse, DSHB, 74.5A5), anti-NKX6.1 antibody (1:100 dilution, mouse, DSHB, F55A10), anti-hROBO3 antibody (1:100 dilution, goat, R&D Systems, AF3076).

Techniques: Immunocytochemistry, Expressing, Staining, Two Tailed Test, Quantitative RT-PCR, In Vitro, MANN-WHITNEY, Western Blot, Control, Cell Culture

Journal: bioRxiv

Article Title: Midline Assembloids Reveal Regulators of Human Axon Guidance

doi: 10.1101/2024.06.26.600229

Figure Lengend Snippet: (A) Schematic illustrating the assembly of day 8 hFpO (green) and day 22 hSpO (purple) to form hMA for modeling hFpO-dependent axon guidance. (B) UMAP visualization of single cell gene expression of cells collected from day 35 (d35) hSpO. (n = 17,574 cells from 3 hiPS cell lines). (C) Bar plots of the proportion of annotated d35 hSpO cell clusters across 3 hiPS cell lines. (D) UMAP visualization of single cell gene expression of markers enriched in dorsal commissural neuron populations, ROBO3, EBF2, TAG-1 , and NHLH2 . Color scale indicates normalized gene expression level. (E) Representative z-projected immunocyto-chemistry images of cleared hSpO showing expression of ROBO3 and TUBB3 with RedDot2 nuclear stain from day 20 to day 30 of differentiation. Scale bar, 200 μm. (F) Quantification of fold-change in ROBO3, TUBB3 and Reddot2 fluorescence relative to day 20 of differentiation measured from 22–32 individual organoids from 3 hiPS cell lines (individual samples shown as gray points, mean per line is shown as a colored points, and the dashed line shows mean and SEM across samples). Line indicates mean while shaded area represents SEM. Plotted values are Log2 transformed. One-way ANOVA followed by Dunnett’s multiple comparison test was used. For ROBO3: F 5,161 = 39.26, P <0.0001; ***P = 0.0003, ****P < 0.0001. For TUBB3: F 5,161 = 6.864, P < 0.0001; *P = 0.0164, ***P = 0.0005, ****P <0.0001. For Reddot2: F 5,161 = 17.81, P < 0.0001; **P = 0.0036, *P = 0.0171, ***P = 0.0008. Scale bar, 200 μm. (G) Representative z-projected immunocytochemistry images of ROBO3 and FOXA2 expression in cleared hMA expression at 2 days after fusion (d.a.f.). Scale bar, 200 μm; inset, 100 μm). (H) Quantification on the proportion of ROBO3+ axon coverage normalized to the area of either the assembled hFpO (green) or hSpO (purple) over time. Data points represent individual assembloids (n = 11-12 per condition from 2 hiPS cell lines) while bar plots indicate the mean at 1 d.a.f. (light gray), 2 d.a.f. (medium gray) and 3 d.a.f. (dark gray). Comparison of the effects across time points in each condition; Two-way ANOVA and Tukey’s test for multiple comparison, F 2,63 = 79.23, P < 0.0001; ****P < 0.0001. (I) Quantification of the amount of oriented ROBO3 + axons from the hSpO relative to either an assembled hSpO or hFpO at 2 d.a.f. (n = 12 per condition from 2 hiPS cell lines). Zero degrees indicates projection directly towards and +-90 degrees indicates away from the assembled hFpO/hSpO. The analyzed ROI is indicated by the dotted line on the hMA schematic. Two-tailed Mann-Whitney test comparing means at 0 degrees: ****P < 0.0001.

Article Snippet: The following antibodies were used for immunostaining: anti-FOXA2 antibody (1:1000 dilution, rabbit, Abcam, ab108422), anti-TUBB3 antibody (1:500 dilution, rabbit, Cell Signaling Technology, MAB1195), anti-NKX2.2 antibody (1:100 dilution, mouse, DSHB, 74.5A5), anti-NKX6.1 antibody (1:100 dilution, mouse, DSHB, F55A10), anti-hROBO3 antibody (1:100 dilution, goat, R&D Systems, AF3076).

Techniques: Expressing, Staining, Fluorescence, Transformation Assay, Comparison, Immunocytochemistry, Two Tailed Test, MANN-WHITNEY

Journal: Inflammopharmacology

Article Title: Resveratrol protects against Schistosoma mansoni- induced liver fibrosis by targeting the Sirt-1/NF-κB axis

doi: 10.1007/s10787-023-01382-y

Figure Lengend Snippet: Effect of resveratrol on hepatic TGF-β1, α-SMA, Sirt-1 and IκB activity assessed by western blotting. Representative immunoblots of TGF-β1, α-SMA, Sirt-1, IκB, p-IκB and β-actin proteins are shown in ( A ). The level of protein expression was normalized to that of β-actin and the relative expressions of TGF-β1 (B), α-SMA ( C ), and Sirt-1 ( D ) were represented. The ratio of phosphorylated to unphosphorylated Iβκ was calculated and presented in (E). Data are expressed as means ± SD ( n = 12) and analysed by one way ANOVA followed by Tukey’s multiple comparisons test. P < 0.05 was as set as the level of significance. * significant difference versus the normal control group, # significant difference versus untreated infected group, and €significant difference versus the Res-20-infected mice. Res, resveratrol; TGF-β1, tissue growth factor beta-1; α-SMA, alpha smooth muscle actin; Sirt-1rtuin 1, and IκB, inhibitor kappa B

Article Snippet: The membranes were incubated with the primary antibodies; SIRT-1 (2310), α-SMA (14,968), TGF-β (3711), Iκβ (9242), p-Iκβ Ser32 (9241), and β-actin (4970) (Cell signaling technology, Beverly, MA, USA) overnight at 4 °C after blocking with 5% non-fat milk in TBST.

Techniques: Activity Assay, Western Blot, Expressing, Infection