Journal: bioRxiv

Article Title: FGF9 treatment reduces off-target chondrocytes from iPSC-derived kidney organoids

doi: 10.1101/2024.08.23.609318

Figure Lengend Snippet: (A–D) Alcian blue staining revealed the presence of cartilage at days 7+18 and 7+25 in the whole organoid (left images, scale bars represent 1000 µm) and cryosections (right images; scale bars represent 50 µm). (E–I, N=5) Progressively and significantly increased expression of five different markers of chondrogenesis was detected by qPCR at day 7+18 and 7+25. * p < 0.05; ** p < 0,01; *** p < 0.001 based on -fold change relative to day 7+18. (J) Western blotting of SOX9 and COL2A1 protein levels in kidney organoids from day 7+18 to 7+25. GAPDH levels are shown as loading controls. (K–L) Quantification of protein levels using ImageJ normalized to GAPDH showed a significant increase (*** p < 0.001; N=5) of COL2A1 (L) from day 7+18 to 7+25. SOX9 levels (K; N=3) did not show significant differences.

Article Snippet: Primary antibodies used were the following: anti-SOX-9 (1/1000, Cell Signaling Technology, 82630), anti-GAPDH (1/10000, Cell Signaling Technology, 2118), anti-vimentin (1/500, Thermo Fisher Scientific, MA5-16409), anti-α-SMA (1/1000, Cell Signaling, 19245S) and anti-COL2A1 (1/1000; Abcam, ab34712).

Techniques: Staining, Expressing, Western Blot

Journal: bioRxiv

Article Title: FGF9 treatment reduces off-target chondrocytes from iPSC-derived kidney organoids

doi: 10.1101/2024.08.23.609318

Figure Lengend Snippet: (A–E) FGF9 treatment significantly decreased five markers of chondrogenesis in kidney organoids at day 7+25 compared to control organoids. Gene expression was assessed by qPCR and shown as -fold change compared to expression at day 7+18. ** p < 0.01; *** p < 0.001; N=5. (F) Western blotting of SOX9 and COL2A1 protein levels in kidney organoids at day 7+25 showed decreased expression in FGF9-treated organoids compared to untreated controls. GADPH levels are shown as loading controls. (G–H) Quantification of protein levels confirmed significantly decreased expression of SOX9 (G) and COL2A1 (H) at day 7+25 with FGF9 treatment (+) compared to nontreated (–) organoids. ** p < 0.01 from 3–4 samples. Relative expression was assessed as the -fold change compared to day 7+18.

Article Snippet: Primary antibodies used were the following: anti-SOX-9 (1/1000, Cell Signaling Technology, 82630), anti-GAPDH (1/10000, Cell Signaling Technology, 2118), anti-vimentin (1/500, Thermo Fisher Scientific, MA5-16409), anti-α-SMA (1/1000, Cell Signaling, 19245S) and anti-COL2A1 (1/1000; Abcam, ab34712).

Techniques: Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: FGF9 treatment reduces off-target chondrocytes from iPSC-derived kidney organoids

doi: 10.1101/2024.08.23.609318

Figure Lengend Snippet: (A-D) Cartilage (asterisks) stained with Alcian blue in whole organoids (left images, scale bars represent 1000 μm) and on cryosectionos (rights images, scale bars represent 50 μm) was less abundant with FGF9 treatment but the appearances of small islands of cartilage were visible (bottom row). (E-I) FGF9 treatment (+) significantly decreased four of five markers of chondrongenesis in kidney organoids at day 7+32 compared to control (−) organoids. Gene expression assessed by qPCR and show as -fold change compared to expresssin in untreated organoids at day 7+18. ***p < 0.001 from 4 samples. (J) Western blotting COL2A1 protein in control (−) kidney organoids showed increased expression over time which was abrogated with FGG9 treatment (+). GADPH levels shown as loading controls.

Article Snippet: Primary antibodies used were the following: anti-SOX-9 (1/1000, Cell Signaling Technology, 82630), anti-GAPDH (1/10000, Cell Signaling Technology, 2118), anti-vimentin (1/500, Thermo Fisher Scientific, MA5-16409), anti-α-SMA (1/1000, Cell Signaling, 19245S) and anti-COL2A1 (1/1000; Abcam, ab34712).

Techniques: Staining, Control, Expressing, Western Blot

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Schematics of the experimental strategy to measure senescence markers (p21, p16 and SA-βGal activity) and SASP cytokines levels in mice lungs at 10 and 20 days p.i. Day 1 CFU= 55. (b) %p21+p16+ and (c) %SA-βGal+ (CellEvent Senescence green+) cells out of all live lung (uninfected and Mtb- infected mice) and Spleen ( Mtb- infected mice) cells at 10 days p.i. as determined by multicolor flow cytometry. (d) %p21+p16+ and (e) %SA-βGal+ cells out of different lung-cell types at 10 days p.i., as determined by multicolor flow cytometry. Gating for p16⁺, p21⁺, and SA-βGal⁺ events for each cell type were established using reference cells from Mtb -infected WT B6 mice (set at 1 %). (AMs: Alveolar macrophages, IMs: Interstitial macrophages) (f) Normalized concentration of SASP-cytokines in lung homogenates at 20 dpi and 4 wpi as measured by a LEGENDplex™ Mouse Inflammation Panel (13-plex). The data are means ± SEM. Each data point represents a mouse (n=11-12). (two-way ANOVA with Tukey’s (b-e) or Dunnett’s (f) multiple comparisons test).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Activity Assay, Infection, Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Representative H&E-stained images of Mtb -infected mice lungs treated with Vehicle or drugs, and respective ImageJ-based quantification. Yellow arrow indicates necrotic granuloma in Vehicle treated B6.Sst1S mice at 5 wpi. The data are means ± SEM. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. Square represents necrotic granuloma formation in the lung. (b) Representative γH2A.X-Immunohistochemistry images of mice after lungs vehicle/ drugs treatment, and respective Violin plots to show ImageJ quantification of γH2A.X-stained area. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. (c) %p21+βGal+ and %p16+βGal+ subpopulation observed in all live lung cells of Mtb -infected mice (n= 5-8). The data are means ± SEM. Each data point represents a mouse. Statistical analysis was calculated by two-way ANOVA with Dunnett’s multiple comparisons test. Bubble plot to show %p21+βGal+ and %p16+βGal+ subpopulation in different lung cell types in Mtb -infected (d) B6.Sst1S mice (n= 8) and (e) WT B6 old mice (n= 5) at indicated time point and treatment groups. The size of the bubble indicates %subpopulation out of all of particular cell types and color is adjusted p values relative to Veh group. Statistics are calculated by one-way ANOVA with Tukey’s multiple comparisons test ( p > 0.15: ns).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Staining, Infection, Two Tailed Test, Immunohistochemistry