Fig 1: ITGA11 is a potential target of FEZF1-AS1 in NSCLC cells. (A) FEZF1-AS1 was detected in Ago2 immunoprecipitates in the RNA-binding protein immunoprecipitation assay. ***P<0.001 vs. IgG. (B) Following FEZF1-AS1-knockdown, ITGA11 was the most downregulated mRNA of the 12 selected mRNAs, as determined using RT-qPCR. **P<0.01 vs. si-NC. (C) RT-qPCR analysis of ITGA11 expression in BEAS-2B and NSCLC cell lines. *P<0.05 and ***P<0.001 vs. BEAS-2B. (D) Western blot analysis verified that ITGA11 expression was regulated by FEZF1-AS1. (E) Efficacy of FEZF1-AS1 overexpression. FEZF1-AS1 overexpression upregulated ITGA11 expression, as determined using (F) RT-qPCR and (G) western blot analysis. Data are presented as the mean ± SEM. *P<0.05; **P<0.01; ***P<0.001. ITGA11, integrin subunit all; NSCLC, non-small cell lung cancer; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering; FEZF1-AS1, FEZ family zinc finger 1 antisense RNA 1; Ago2, argonaute 2; FC, fold-change.
Fig 2: Schematic illustration of acquired features of the phenotype of cancer‐associated fibroblasts. TGF‐β1‐stimulated fibronectin and collagen type I production induce ITGA11 expression in fibroblasts, resulting in accelerated CAF‐mediated migration toward fibronectin and collagen type I via ERK1/2 signal without changing integrin α5 β1 expressions. The released fibronectin induced COL11A1 production, which was accompanied by ITGA11 expression via ERK1/2 signal in cancer stroma.
Fig 3: Effect of ITGA11 genetic modification on fibroblast features. (A) ITGA11 was knocked down using siRNA in HFL-1 and proteins were extracted and subjected to western blot analysis to detect the expression levels of each target (see Section 2.6). Vertical axis: (B) ITGA11, (C) COL11A1, (D) integrin a5, (E) integrin ß1, (F) fibronectin, and (G) a-SMA protein expression normalized to ß-actin, (H) p-ERK1/2 protein expression normalized to t-ERK1/2 and (I) p-SMAD3 protein expression normalized to t-SMAD3. (J) Migration toward fibronectin (20 µg·mL-1) was assessed after silencing ITGA11. Vertical axis: number of migrated cells per 5HPF. (K) Migration toward collagen type I (1 µg·mL-1) was assessed after silencing ITGA11. (B-K; n = 3, one-way ANOVA) Vertical axis: number of migrated cells per 5HPF. (L) NIH 3T3 cells were transfected with ITGA11-overexpression vector or GFP and subjected to western blot analysis to detect the ITGA11 and a-SMA expression levels (see Section 2.7). (M) Migration toward fibronectin (20 µg·mL-1) was assessed with ITGA11-overexpressing NIH 3T3 cells. (M; n = 8, Mann–Whitney) Vertical axis: number of migrated cells per 5HPF. The values represent the mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001.
Fig 4: Osteolectin contains conserved integrin binding motifs and binds with high affinity to integrin a11ß1.(A, B) The human (A) and mouse (B) Osteolectin proteins contain RGD and LDT sequences. (C) Alignment of Osteolectin amino acid sequences shows that the RGD and LDT domains are evolutionarily conserved among bony vertebrates. (D, E) RNA-seq analysis of integrin a (D) and ß (E) subunits in PDGFRa+CD45-Ter119-CD31- bone marrow stromal cells from enzymatically dissociated adult bone marrow (n = 2 independent samples). These cells are uniformly positive for LepR expression (Zhou et al., 2014). (F) RNA-seq analysis of Itga1, Itga6, Itga11, and Itgav in PDGFRa+CD45-Ter119-CD31- bone marrow stromal cells, VE-Cadherin+ bone marrow endothelial cells, and whole bone marrow cells (n = 2 independent samples per cell population). (G) Itga11 expression in cell populations from mouse bone marrow by qRT-PCR (n = 3 independent samples per cell population). The markers used for the isolation of each cell population are shown in Supplementary file 1. (H) In MC3T3-E1 preosteoblast cells expressing Flag-tagged Osteolectin, anti-Flag antibody co-immunoprecipitated endogenous integrin ß1 and integrin a11 with Flag-tagged Osteolectin (results are representative of two independent experiments). (I) Recombinant human Osteolectin (rhOln) selectively bound to recombinant human integrin a11ß1 and a10ß1, but not to other integrins (n = 3 independent experiments). (J) Integrin a11ß1 bound Osteolectin and recombinant human Pro-Collagen 1a (rhCol1A) with similar affinities, but not bovine serum albumin (BSA) (n = 3 independent experiments). (K) Osteolectin, but not Pro-Collagen 1a, promoted osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells (n = 3 independent experiments). (L) 200 nM RGDS peptide inhibited the binding of integrin a11ß1 to recombinant human Osteolectin. (M) 100 µM RGDS peptide inhibited osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells in response to 30 ng/ml of recombinant human Osteolectin. All numerical data reflect mean ±standard deviation. Statistical significance was determined with one-way (G) or two-way ANOVAs with Dunnett’s multiple comparisons tests (K) or Tukey’s multiple comparisons tests (M).10.7554/eLife.42274.004Figure 1—source data 1.Data for Figure 1.
Fig 5: Conditional Itga11 deletion from LepR+cells reduces cortical bone formation in adult mice.(A) Representative microCT images of cortical bone in the mid-femur diaphysis of male Lepr-Cre; Itga11fl/fl mice and littermate controls at 2, 6, and 12 months of age. (B–F) microCT analysis of the total area (B), cortical area (C), cortical thickness (D), cortical area/total area (E), and cortical bone mineral density (F) in the mid-femur diaphysis of Lepr-Cre; Itga11fl/fl mice and sex-matched littermate controls at 2, 6, and 12 months of age (n = 4–7 mice per genotype per sex per time point from at least three independent experiments). (G) Cortical bone mineral apposition rate based on calcein double labelling in the mid-femur diaphysis (n = 3–4 mice per genotype per sex per time point from three independent experiments). All numerical data reflect mean ±standard deviation. The statistical significance of differences was determined with two-way ANOVAs with Sidak’s multiple comparisons tests.10.7554/eLife.42274.016Figure 6—source data 1.Data for Figure 6.
Supplier Page from Abcam for Anti-ITGA11 antibody