Journal: eLife

Article Title: Paraxial mesoderm organoids model development of human somites

doi: 10.7554/eLife.68925

Figure Lengend Snippet:

Article Snippet: For generation of PM organoids, 500 dissociated iPSCs resuspended in mTeSR1 media containing 10 μM Y-27362 dihydrochloride (ROCKi; Tocris Bioscience, Cat# 1254) and 0.05% PVA were dispensed into 96-well U-bottom non-adherent suspension culture plates (Greiner Bio-One, 650185) and allowed to aggregate for 24 hr.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A) Schematic visualizing how hydroxychloroquine (HQ) and verteporfin (VP) interfere with autophagy. (B) Percentage of CD41 + /CD42d + -positive cells after treatment of HSPCs with DMSO, 5 µM HQ or 5 µM VP for 72h was assessed using flow cytometry. Values were normalized to the DMSO control. n=3-4 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (C-E) Analysis of TGFβ1 (C) , IL1β (D) and PF4 levels (E) in cell culture supernatant after treatment of HSPCs with DMSO, 5 µM HQ or 5 µM VP for 72h. Supernatant derived from cells cultured in the absence of TPO were included as a control. Values were normalized to DMSO values. n=4-6 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (F) Visualization of LC3B, LAP/TGFβ1 and CD42b in enriched bone marrow-derived MKs treated with DMSO, HQ or VP for 72h. Nuclei were counterstained using DAPI. Scale bars: 20 µm. (G-I) MK area (G) , LAP/TGFβ1 (H) and LC3B MFI (I) were analyzed in cultured MKs. At least 70 MKs were analyzed per condition. n=3. One-way ANOVA with Dunnett’s test for multiple comparisons. (J, K) Immunoblot (J) and densitometric analysis (K) of enriched bone marrow-derived MKs treated with DMSO or HQ for 72h. n=6. Unpaired, two-tailed Student’s t-test. (L) Schematic on the inhibitory function of CCG1423 (CCG) and Y27632 (Y) on RhoA and Rho kinases (ROCKs). (M) Percentage of CD41 + /CD42d + -positive cells after treatment of HSPCs with DMSO, 5 µM CCG or 500 nM Y for 72h was assessed using flow cytometry. Values were normalized to the DMSO control. n=3 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (N-P) Analysis of TGFβ1 (N) , IL1β (O) and PF4 levels (P) in cell culture supernatant after treatment of HSPCs with DMSO, CCG or Y for 72h. Supernatant derived from cells cultured in the absence of TPO were included as a control. Values were normalized to DMSO values. n=3-6 mice. One-way ANOVA with Dunnett’s test for multiple comparisons. (Q, R) MK area was assessed (Q) and LC3B, LAP/TGFβ1 and CD42b were visualized (R) in enriched bone marrow-derived MKs treated with DMSO, CCG or Y for 72h. Nuclei were counterstained using DAPI. Scale bars: 20 µm. (S, T) , LAP/TGFβ1 (S) and LC3B MFI (T) were analyzed in cultured MKs. At least 70 MKs were analyzed per condition. n=3. One-way ANOVA with Dunnett’s test for multiple comparisons. Schematics were generated using Biorender.com.

Article Snippet: For inhibitor studies, MKs were treated with 5 µM CCG1423 (HY-13991), 500 nM Y27632 (HY-10583), 50 µM endosidin2 (HY-120821), 5 µM Verteporfin (HY-B0146) or 5 µM Hydroxychloroquine (HY-B1370, all MedChem Express).

Techniques: Flow Cytometry, Control, Cell Culture, Derivative Assay, Western Blot, Two Tailed Test, Generated

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A, B) Percentage of EGFP + CD45 + cells (A) and platelets (B) of PBS- or Y27632 (Y)-treated mice transplanted with MPL W515L -transduced cells three weeks after transplantation. n=5 mice. Two-way ANOVA with Sidak’s correction for multiple comparisons and unpaired, two-tailed Student’s t-test. (C) Spleen to body weight of transplanted PBS- and Y-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. (D, E) White blood cell (WBC) (D) and red blood cell (RBC) counts (E) of transplanted PBS- and Y-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. (F-H) Platelet count (F), mean platelet volume (MPV) (G) and immature platelet fraction (IPF) (H) in transplanted PBS- and HQ-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD. (I-K) Visualization (I) and quantification of intracellular LAP/TGFβ1 (J) and collagen IV (K) in transplanted PBS- and Y-treated mice. n=5. Unpaired, two-tailed Student’s t-test. Scale bars: 50 µm. (L) Quantification of MK numbers in femoral cryosections in transplanted PBS- and Y-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. (M-O) Analysis of TGFβ1 (M) , IL1β (N) and PF4 levels (O ) in bone marrow fluid of transplanted PBS- and Y-treated mice. n=5 mice. Unpaired, two-tailed Student’s t-test. All data are presented as mean ± SD. Schematic was generated using Biorender.com.

Article Snippet: For inhibitor studies, MKs were treated with 5 µM CCG1423 (HY-13991), 500 nM Y27632 (HY-10583), 50 µM endosidin2 (HY-120821), 5 µM Verteporfin (HY-B0146) or 5 µM Hydroxychloroquine (HY-B1370, all MedChem Express).

Techniques: Transplantation Assay, Two Tailed Test, Generated

Journal: bioRxiv

Article Title: Inhibition of RhoA-mediated secretory autophagy in megakaryocytes mitigates myelofibrosis in mice

doi: 10.1101/2024.12.04.626665

Figure Lengend Snippet: (A, B) Percentage of EGFP + CD45 + cells (A) and platelets (B) of vehicle (Veh; carboxymethylcellulose), ruxolitinib (R)-, ruxolitinib and hydroxychloroquine (HQ) (R+HQ)- and ruxolitinib and Y27632 (R+Y)-treated mice transplanted with MPL W515L -transduced cells three weeks after transplantation. n=5 mice. Two-way ANOVA with Sidak’s correction for multiple comparisons and one-way ANOVA with Sidak’s correction for multiple comparisons. (C) Spleen to body weight of transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (D, E) White blood cell (WBC) (D) and red blood cell (RBC) counts (E) of transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (F, G) Platelet count (F) and immature platelet fraction (IPF) (G) in transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. (H-J) Visualization (H) and quantification of MK numbers (I) and collagen IV deposition (J) in transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. Scale bars: 50 µm. (K-M) Analysis of TGFβ1 (K) , IL1β (L) and PF4 levels (M ) in bone marrow fluid of transplanted Veh-, R-, R+HQ- and R+Y-treated mice. n=5 mice. One-way ANOVA with Sidak’s correction for multiple comparisons. All data are presented as mean ± SD.

Article Snippet: For inhibitor studies, MKs were treated with 5 µM CCG1423 (HY-13991), 500 nM Y27632 (HY-10583), 50 µM endosidin2 (HY-120821), 5 µM Verteporfin (HY-B0146) or 5 µM Hydroxychloroquine (HY-B1370, all MedChem Express).

Techniques: Transplantation Assay