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. 2021 Aug 17;10:e64846. doi: 10.7554/eLife.64846

Figure 7. FOSL1 contributes to mesenchymal (MES) genes activation, cell growth, and stemness in MES brain tumor stem cells (BTSCs).

(A) Western blot analysis using the specified antibodies of human BTSC lines, characterized as non-MES (left) and MES (right). (B) Western blot detection of FRA-1 in MES BTSC 380 upon transduction with inducible shRNAs targeting GFP (control) and FOSL1, analyzed after 3 and 7 days of doxycycline (Dox) treatment; vinculin was used as loading control. (C) Cell growth of BTSC 380 shGFP and shFOSL1, in the absence or presence of Dox, measured by MTT assay; absorbance values were normalized to day 1. Data from a representative of three independent experiments are presented as mean ± SD (n = 15, technical replicates). Two-way ANOVA, –Dox vs. +Dox: ***p≤0.001. (D) BrdU of BTSC 380 shGFP and shFOSL1, in the absence or presence of Dox, analyzed by flow cytometry. Data from a representative of two independent experiments are presented as mean ± SD (n = 3, technical replicates). Student’s t test, relative to the respective control (–Dox): *p≤0.05. (E) Representative limiting dilution analysis on BTSC380 for shGFP and shFOSL1, in the presence or absence of Dox, calculated with extreme limiting dilution assay (ELDA) analysis; bar plot inlets show the estimated stem cell frequency with the confidence interval; chi-square p-values are indicated. (F) Principal component analysis of H3K27Ac signal over FOSL1/FRA-1 binding sites, calculated using MACS on ENCODE samples (see Materials and methods), in non-MES (n = 10) and MES BTSC (n = 10) (from Mack et al., 2019). (G) Volcano plot illustrating the log2 fold-change differences in H3K27Ac signal between non-MES and MES BTSCs against the p-value for that difference. Blue and red probes represent statistically significant differences (FDR < 0.005) in H3K27Ac signal between non-MES and MES BTSCs. (H) Heatmap of ChIP-seq enrichment of FOSL1/FRA-1 or OLIG2 binding sites for the indicated profiles. (I) View of the PLAU, CD44, and OLIG2 loci of selected profiles. (J) Representative ChIP experiment in BTSC 349 cells. The panel shows FRA-1 binding to the promoter of a subset of MES targets (n = 3, technical replicates) expressed as a percentage of the initial DNA amount in the immune-precipitated fraction. NANOG gene was used as a negative control. Student’s t test, relative to IgG: ns = not significant, **p≤0.01, ***p≤0.001.

Figure 7—source data 1. Source data of Figure 7A.
Figure 7—source data 2. Source data of Figure 7B.
Figure 7—source data 3. Source data of Figure 7C–G, J.

Figure 7.

Figure 7—figure supplement 1. Further characterization of FOSL1 role in human brain tumor stem cells (BTSCs).

Figure 7—figure supplement 1.

(A) Western blot detection of FRA-1 in BTSC 349 upon transduction with inducible shGFP (control) or shFOSL1_3, analyzed after 3 and 7 days of doxycycline (Dox) treatment; vinculin was used as loading control. (B) Cell growth of BTSC 349 shGFP and shFOSL1_3 cells, in the absence or presence of Dox, measured by MTT assay; absorbance values were normalized to day 1. Data from a representative of three independent experiments are presented as mean ± SD (n = 15, technical replicates). Student’s t test on day 7, relative to shFOSL1_3 –Dox: ***p≤0.001. (C) BrdU incorporation of BTSC 349 shGFP and shFOSL1_3, in the absence or presence of Dox, analyzed by flow cytometry. Data from a representative of two independent experiments are presented as mean ± SD (n = 3). Student’s t test, relative to the respective control (–Dox): ns = not significant, **p≤0.01. (D) Representative limiting dilution analysis on BTSC 349 shFOSL1_3 in the presence or absence of Dox, calculated with extreme limiting dilution assay (ELDA) analysis; p<0.0001. (E) mRNA expression of FOSL1, MES, and PN genes in BTSC 349 shFOSL1_3 cells in the absence or presence of Dox for 3 days. Data from a representative of three experiments are presented as mean ± SD (n = 3, technical replicates), normalized to GAPDH expression. Student’s t test, relative to –Dox: ns = not significant, *p≤0.05, **p≤0.01, ***p≤0.001. (F) Representative images of BTSC 380 grown in either neurosphere medium (NS) or in differentiation conditions (NS + 0.5% FBS + TNFalpha 5 ng/mL) for 5 days. Scale bar = 250 μm. (G) Cell growth of BTSC 380 grown in differentiation conditions, in the absence or presence of Dox, measured by MTT assay; absorbance values were normalized to day 1. Data from a representative of two independent experiments are presented as mean ± SD (n = 10, technical replicates). (H) Western blot detection of FRA-1 in h543 and h676 upon transduction with pBabe (control) or pBabe-FOSL1; vinculin was used as loading control. (I) Cell growth of cells as in (H), measured by MTT assay; absorbance values were normalized to day 1. Data from a representative of three independent experiments are presented as mean ± SD. (J) Western blot detection of FRA-1 in h543 and h676 upon transduction with the indicated shRNA. To note that FRA-1 is barely detectable in h676 cells (see also panel H and Figure 1A). (K) Cell growth of cells as in (J), measured by MTT assay; absorbance values were normalized to day 1. Data from a representative of two independent experiments are presented as mean ± SD (n = 10, technical replicates).
Figure 7—figure supplement 1—source data 1. Source data of Figure 7—figure supplement 1A.
Figure 7—figure supplement 1—source data 2. Source data of Figure 7—figure supplement 1H.
Figure 7—figure supplement 1—source data 3. Source data of Figure 7—figure supplement 1J.
Figure 7—figure supplement 1—source data 4. Source data of Figure 7—figure supplement 1B–E, G, I, K.