Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2017 Aug 7;214(8):2453–2470. doi: 10.1084/jem.20161595

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2017 Khalaj et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 1. — miR-99 is highly expressed in hematopoietic stem and progenitors and suppresses myeloid differentiation in vitro. (A–C) Normalized expression levels of miR-99b, miR-99a, and miR-100 as determined by quantitative RT-PCR using miRNA TaqMan probes in mouse hematopoietic cell populations: hematopoietic stem cell (HSC), multipotent progenitor (MPP) Flk⁻, MPP Flk⁺, common lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-macrophage progenitor (GMP), and megakaryocyte-erythroid progenitor (MEP) cells. Expression was normalized against mmu-mir-16. Error bars denote SEM. Representative data from five independent experiments are shown. (D) miR-99 is down-regulated 48 h post-transduction of HSCs with the lentiviral anti–miR-99 vector as shown by quantitative RT-PCR. Expression was normalized against U6 (Student’s t test; n = 3). Representative data from two independent experiments are shown. (E) Comparable number of colonies form after miR-99 KD in first plating, with an increase in the number of CFU macrophage (CFU-M) colonies. 100 GFP⁺ HSC cells were cultured in methylcellulose. The colonies were scored after 7 d. Data represent mean percentage ± SEM (Student’s t test; n = 3) and are representative of three independent experiments. (F) Smaller colonies were observed after second plating of GFP⁺ cells derived from miR-99 KD HSCs. Representative data of three independent experiments are shown. (G) Colony-forming capacity of HSCs is reduced after miR-99 KD in a second plating. 15,000 GFP⁺ cells were replated 7 d after the first plating. Colony types were scored after 7 to 10 d. Data represent mean count ± SEM (Student’s t test; n = 3) and are representative of three independent experiments. (H) miR-99 KD in HSCs induces granulocytic differentiation in methylcellulose colony assays. 7 d after plating, colonies were analyzed for expression of myeloid differentiation markers by flow cytometry. Mean percentage ± SEM (Student’s t test; n = 2). Representative data of three independent experiments are shown. (I) Flow cytometry analysis of LSK cells transduced with anti–miR-99 or Scr vectors and maintained in liquid culture for 8 d reveals a decrease in the absolute number of GFP⁺Lin⁻Sca-1⁺c-Kit⁺CD150⁺ HSCs. FSCw denotes forward scatter-width. The data shown are gated on LSK cells. Data represent mean count ± SEM (Student’s t test; n = 3) and are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.