Journal: bioRxiv

Article Title: E2f coordinates the cell cycle and cell fate of hematopoietic progenitors to drive stress myelopoiesis

doi: 10.1101/2025.06.16.659412

Figure Lengend Snippet: a Unfractioned BM cells from CT / TKO mice (5 per group) were isolated two weeks after Tamoxifen treatment, dyed for multiplexing purposes, merged, stained with antibodies against lineage markers (B220, Mac1, Gr1, Ter119, Cd3, Cd4, Cd8), Kit and Sca1 (HSPC) and screened for the expression of 256 surface markers (Biolegend antibody array® complemented by individual analysis for CD131 since it is not included in the array design). Background signal intensity was assessed by isotype staining. Each dot represents a surface marker expression in CT (x-axis) and TKO (y-axis) HSPCs. The dotted line represents identical positivity % in CT and TKO HSPCs. Markers of monocyte lineage (CD14 and CD11c) and MPP4 (Flt3) are highlighted for technical validation. Gene names of interest are color-coded for better visualization when expression difference between CT and TKO HSPCs is at least 10 points. b Upper panel: analysis of mouse and human Csf2rb regulatory region identifies a conserved E2f binding site in the first untranslated exon. Lower panel: ChIP analysis performed on unfractioned BM cells show that E2f1 and E2f3 bind to a region of the Csf2rb promoter that harbors the putative E2f binding site in primary BM cells (n=3). c CT and TKO HSPCs were isolated two weeks after Tamoxifen treatment, serum starved and stimulated with Il3 or Gm-Csf (10ng/ml) for 10 min. p-Erk was used as a readout for βcytokine signaling activity (n=3). d CT MPP4 were isolated by flow cytometry, serum starved for 2 hours and treated with 20ng/ml of Il3 for 1 hour. Cells were collected and RNA was extracted prior to qPCR to detect Cebpb and Zbtb7b expression. e 100 CT and TKO MPP4 cells were plated in cytokine-free methylcellulose, supplemented with either a cocktail of Il3, Il6 and Scf, or Il3, Scf and Il6 only. Colonies were counted after 9 days in culture (n=3). f 100 CT and TKO MPP3 cells were plated in cytokine-free media, supplemented with Il3. Colonies were counted after 9 days in culture (n=3). g CT and TKO MPP4 cells were infected with p-Sicor- RFP lentiviral vectors expressing either a scramble hairpin or two hairpins ( a and b ) targeted against Csf2rb . 500 RFP + cells were plated for methylcellulose culture and colonies were quantified after 9 days in culture (n=3). h CT and TKO mice were treated with Tamoxifen for four consecutive days. On day five, eight and eleven, TKO mice were injected with either isotype antibody (control, black bar), antibody against Tnfα (250ug, yellow bar) or a combination of antibodies against Gm-Csf and Il3 (250ug each, red bar). On day 14, HSPC subpopulations were analyzed for frequency per million BM cells (n=6 per groups). i DSS 2% was added to the drinking water of wild-type mice for 12 days. Mice were injected with various combinations of blocking antibodies (isotype, anti-GM-Csf, anti-Il3, combination of anti-GM-Csf and anti-Il3) on day 1, 4, 7 and 10 of colitis-inducing DSS treatment. Untreated wild-type mice were used as control. j Frequency of Mac1 + cells, Ly6g + granulocytes and Ly6c + monocytes in the BM. k Wild-type and Csf2rb deficient mice were exposed to three cycles of DSS 2% treatment (1 cycle: 12 days with DSS 2% containing drinking water followed by 12 days with normal drinking water). l Mice were weighted at the end of the treatment. m Representative H&E staining of intestine sections from wild-type and Csf2rb-/- mice shows more severe alteration of villae structure in wild-type mice compared to Csf2rb deficient mice (n=5).

Article Snippet: Antibodies against E2f1 and E2f3 were purchased from Santa Cruz.

Techniques: Isolation, Multiplexing, Staining, Expressing, Ab Array, Marker, Biomarker Discovery, Binding Assay, Activity Assay, Flow Cytometry, Infection, Injection, Control, Blocking Assay

Journal: Molecular medicine reports

Article Title: MicroRNA‑141 inhibits the differentiation of bone marrow‑derived mesenchymal stem cells in steroid‑induced osteonecrosis via E2F3.

doi: 10.3892/mmr.2022.12750

Figure Lengend Snippet: Figure 3. Luciferase assays. Relative luciferase activity was assessed in bone marrow‑derived mesenchymal stem cells following co‑transfection with miR‑141 and E2F3 WT or mut plasmids. **P<0.01 vs. control. miR, microRNA; E2F3, E2F transcription factor 3, NC negative control; WT, wild‑type; mut, mutant.

Article Snippet: Western blot analysis of E2F3 protein expression. at day 3, 7 and 14, after discarding the RASMX‐90021 medium (Cyagen; cat. no. RASMX‐90021) and α‐MEM (HyClone; Cytiva; SH30265) the cells were lysed in RIPA lysis buffer at 4 ̊C overnight.

Techniques: Luciferase, Activity Assay, Control, Negative Control, Mutagenesis

Journal: Molecular medicine reports

Article Title: MicroRNA‑141 inhibits the differentiation of bone marrow‑derived mesenchymal stem cells in steroid‑induced osteonecrosis via E2F3.

doi: 10.3892/mmr.2022.12750

Figure Lengend Snippet: Figure 4. MRNA expression of miR‑141 and E2F3 (A) Expression levels of miR‑141 in group of control and ONFH at 3d, 7d and 14d. (B) MRNA expression levels of E2F3 in group of NC, miR‑141, ONFH+ inhibitor NC and ONFH+ miR‑141 inhibitor (left part). mRNA expression levels of E2F3 in group of control and ONFH at 3d, 7d and 14d (right part). *P<0.05, **P<0.01 vs. control. miR, microRNA; E2F3, E2F transcription factor 3, NC negative control; ONFH, osteonecrosis of the femoral head.

Article Snippet: Western blot analysis of E2F3 protein expression. at day 3, 7 and 14, after discarding the RASMX‐90021 medium (Cyagen; cat. no. RASMX‐90021) and α‐MEM (HyClone; Cytiva; SH30265) the cells were lysed in RIPA lysis buffer at 4 ̊C overnight.

Techniques: Expressing, Control, Negative Control

Journal: Molecular medicine reports

Article Title: MicroRNA‑141 inhibits the differentiation of bone marrow‑derived mesenchymal stem cells in steroid‑induced osteonecrosis via E2F3.

doi: 10.3892/mmr.2022.12750

Figure Lengend Snippet: Figure 5. Protein expression levels of E2F3. (A) Protein expression of E2F3 in BMSCs from the control or ONFH rat at day 3, 7 and 14. (B) Protein expression of E2F3 in BMSCs from the normal rat transduced with NC or miR‑141 mimic lentivirus and BMSCs from the ONFH rat transduced with the inhibitor NC or miR‑141 inhibitor lentivirus. *P<0.05, **P<0.01 vs. control. miR, microRNA; E2F3, E2F transcription factor 3, NC negative control; ONFH, osteonecrosis of the femoral head; BMSCs, bone marrow‑derived mesenchymal stem cells.

Article Snippet: Western blot analysis of E2F3 protein expression. at day 3, 7 and 14, after discarding the RASMX‐90021 medium (Cyagen; cat. no. RASMX‐90021) and α‐MEM (HyClone; Cytiva; SH30265) the cells were lysed in RIPA lysis buffer at 4 ̊C overnight.

Techniques: Expressing, Control, Transduction, Negative Control