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. 2020 Dec 15;22(1):e50535. doi: 10.15252/embr.202050535

Figure EV3. Short PLB treatment exhibits minor cytotoxic effects.

Figure EV3

  1. Expression of LAS1L in day 2‐differentiated APLNR+ cells and day 5‐differentiated CD31+CD34+ cells by RNA‐Seq. The bottom panel showing the inclusion/exclusion of LAS1L exon 9 during hematopoietic differentiation by RT–PCR.
  2. The inclusion/exclusion of exon 9 of ATP5F1C and HDAC7 detecting by RT–PCR during hematopoietic differentiation.
  3. The top panel is a representative RT–PCR electropherogram showing the inclusion or exclusion of exon 9 in ATP5F1C (left) and HDAC7 (right) in cells at days 2 and 5 without or with PLB treatment at indicated concentrations, respectively. The quantification of PSI is presented in the bottom bar graph. P‐values were calculated by one‐way followed by Dunnett’s test.
  4. The cellular morphology on days 3, 4, and 5 during hematopoietic differentiation after treatment with 1.25 or 2.5 nM PLB from day 2.5 to 5. Scale bar = 40 μm.
  5. Cellular apoptosis assessed using Annexin V and 7‐AAD at days 3 and 4 of differentiation by flow cytometry, with or without PLB treatment from day 2.5 to 5.
  6. The proportion of G0/G1, S, and G2/M cells at days 3 and 4 of differentiation, with or without PLB treatment from day 2.5 to 5, respectively. The cell cycle was determined using propidium iodide staining by flow cytometry.
  7. Immunofluorescent staining depicting low‐density lipoprotein (AcLDL) uptake from FACS‐sorted CD31+ cells without (DMSO) or with PLB (1.25 nM) treatment. CD144, LDL, and DAPI were stained (upper) by red, green, and blue, respectively. Scale bar = 40 μm.
  8. The bar graph showing the quantification of the LDL fluorescent intensity by the Volocity 3D image analysis software.
  9. The schematic illustrating the DOX‐inducible knockdown system with expression of SF3B1 shRNAs. The knockdown of SF3B1 was confirmed by RT–qPCR (left) and Western blotting (right) after inducing with DOX.
  10. The representative FACS plots of CD31+CD34+ EPCs after SF3B1 depletion at day 5 of differentiation.
  11. The percentage of CD31+CD34+ EPCs after SF3B1 depletion at day 5 of differentiation.

Data information: Results given are mean ± SD. P‐values were determined by Student’s t‐test in (A), (E), (F), (H), (I), and (K). ns represents no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were conducted for at least 3 biological replicates.

Source data are available online for this figure.