EMBO/SourceData huggingface dataset:
Fig 7A) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours. B) The mean ±standard deviation of LC3-II intensity represents three different experiments and the values were analyzed using Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.C) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. D) LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.
Fig 8A) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (E. coli LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour.B) Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&amp;amp;2 ligands (E. coli LPS and Pam3csk4)C) The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** p<0.001).
Fig 9A) Scanning electron microscopy (SEM) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1+Pg (upper panel 10000x and lower panel 25000x). B) SEM for MoDCs interacting with Pg381 (green stains for bacteria are computer generated).C) Transmission electron microscopy (TEM) of autophagosome like structures within MoDCs infected with Pg381, Mfa1+Pg and FimA+Pg for 12 hours (upper, middle and lower panels, respectively). The right and left sections show the different magnifications of randomly selected sections. Pg381 and FimA+Pg strains are mostly enclosed in the characteristics double-membrane intracellular vesicles (Orange arrows). Contrary, Mfa1+Pg escaped these autophagic (double-membrane) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm (Green arrows).D) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage. Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection. Each strain was counted in three randomly selected grids for each sample. The analysis of the bacterial counts used Kruskal-Wallis test of different groups and Dunn's test for multiple comparisons (*p<0.01).
Fig 10A) Epifluorescence microscopy images of MoDCs infected with pre-labeled bacteria (green-CFSE). LAMP1 detected by transduction of red fluorescent protein (RFP) chimera using baculovirus transgenes to MoDCs.B) The mean of fluorescent intensities ±standard deviation of LAMP1 were quantified in three different experiments (* p<0.001).C) The upper scattered plot shows the co-localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains. The lower plot shows the average of Pearson's correlation measures of three randomly selected region of interest (ROI) within each field within 3 different experiments (* p<0.0001) (Table 4 and 5). All analysis of fluorescence intensity used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons.
Figure 1A Representative Gli1ZF/DNA structure extrapolated from MD trajectories. Gli1ZF is shown as blue cartoon, residues involved in binding to DNA (based on single point mutation study) are shown as magenta sticks, and Zn ions as gray spheres.B Effect of Gli1ZF mutants on the binding affinity to DNA as predicted by in silico alanine scanning. The ∆∆G was calculated along MD trajectories as the difference between the ∆G of each Gli1ZF alanine mutant and Gli1ZF‐WT. Results are shown as ∆∆G values in kcal/mol calculated by means of the MM‐PBSA methods ± SEM.C Effect of Gli1ZF mutants on Gli1‐dependent transcriptional activation. Luciferase assay was performed in HEK293T cells transfected with 12XGliBS‐Luc (GliBS, Gli binding site), pRL‐TK Renilla (normalization control), Flag‐Gli1 WT or the indicated Flag‐Gli1 mutants. Data show the mean ± SD of three independent experiments. *P 0.01; **P 0.05 versus Gli1 WT. Western blot analysis of Flag‐Gli1 WT or the indicated Flag‐Gli1 mutant expression levels (bottom panel).E Gli1/DNA binding. Double‐stranded oligonucleotide containing the canonical GliBS sequence (5′-TTGCCTACCTGGGTGGTCTCTCCACTT-3′) or mutated GliBS sequence used as control (5′-TTGCCTACCTCCCACTTCTCTCCACTT-3′) was used as probe (P) in EMSA experiments. The assay was performed using recombinant GST‐Gli1ZF‐WT (Gli1 zinc finger fragment: aa 242-424), GST‐Gli1ZF‐K350A and GST‐Gli1ZF‐K340A. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF mutants bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding (as described in ). *P 0.05 versus Gli1 WT.
Figure 2A Inhibition of Gli1‐induced transcription in transfected HEK293T cells. HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated with increasing concentrations of GlaB or GANT61. Treatment time was 24 h, and control cells were treated with DMSO only.B GlaB chemical structure with numbering scheme for NMR analysis.C Inhibition of Gli1‐induced transcription in transfected Smo−/−MEF cells. Smo−/−MEF cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty) or Gli1 vector and treated for 24 h with increasing concentrations of GlaB or DMSO only as control.D The graphs show the Hh target gene expression levels in Ptch1−/−MEFs treated for 48 h with GlaB and DMSO as a control. mRNA levels were determined by quantitative real‐time PCR (qRT-PCR) normalized to endogenous control (β2‐microglobulin and HPRT). Pfkfb3 gene was used as a negative control.F SuFu−/−MEFs were treated for 48 h with GlaB and DMSO as a control. Gli1 and Ptch1mRNA levels were determined by qRT-PCR normalized to β2‐microglobulin and HPRT expression. Pfkfb3 gene was used as a negative control.Promoter occupancy of Gli1 is prevented by GlaB treatment. MEF WT cells were transfected with Flag‐tagged Gli1 or empty vectors, and chromatin immunoprecipitation (ChIP) was carried out. qRT-PCR was performed using primers encompassing the Gli‐BS of mouse Ptch1 promoter (right, schematic representation). Results are indicated as fold difference, relative to empty (pcDNA3) control.H Ptch1 mRNA expression levels (left panel) were determined by qRT-PCR in Daoy cells transfected with siRNA specific for Gli1 and Gli2 (siGli1/2) or a non‐specific control siRNA (siCtr) and treated for 24 h with GlaB or DMSO as a control. (right panel) The graph shows Gli1 and Gli2mRNA expression levels determined by qRT-PCR in Daoy cells transfected with siGli1/2 or siCtr. Results are expressed as fold repression relative to control, and data were normalized to GAPDH and HPRT expression.
Figure 3A The predicted binding mode of GlaB (green sticks) to Gli1ZF (blue cartoons). Residues highlighted by the single‐point mutation study to impact on Gli1 binding to DNA are shown as blue sticks; K340 and K350 of the GlaB binding site are colored magenta. GlaB protons highlighted by the NMR study are shown as orange spheres.B Detail of GlaB binding to Gli1ZF‐WT, Gli1ZF‐K340A, GLI1ZF‐K350A, GLI1ZF‐K340A/K350A mutants and respective theoretical affinity values. Gli1 is shown as blue transparent surface; K340 and K350 are colored magenta. GlaB is shown as green sticks.C Inhibition of Gli1/DNA binding by GlaB. EMSA using recombinant GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A in the presence of different concentrations of GlaB or with DMSO only. The shifted complex is competed with a 50× excess of cold probe. The graph on the right indicates ratio (mean arbitrary units ± SD from three independent experiments) of GST‐Gli1ZF‐WT or GST‐Gli1ZF‐K340A bound to the labeled GliBS probe/GliBS‐free probe normalized to the amount of GST‐Gli1ZF‐WT/DNA binding in absence of GlaB. *P 0.05 versus DMSO; **P 0.05 K340A + GlaB versus Gli1 WT + GlaB.D HEK293T cells were transfected with 12XGliBS‐Luc and pRL‐TK Renilla (normalization control) plus control (empty vector) or Gli1 or Gli1K340A mutant and treated with increasing concentrations of GlaB. Treatment time was 24 h, and control cells were treated with DMSO only. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO; **P 0.05 K340A versus Gli1 WT.
Figure 4A qRT-PCR shows Hh target mRNA expression levels determined in 6‐day‐old mouse cerebellar progenitors after s.c. injections of GlaB.B Representative EGLHE staining of 6‐day‐old mousecerebellum after s.c. injections of GlaB (upper panel); immunohistochemistry shows Gli1 (middle panel) and Ki67 stainings (lower panel). In all images, the scale bar represents 50 μm.C The graph shows the percentage of EGL thickness reduction in GlaB‐treated mouse cerebella in comparison with vehicle mouse cerebella. Data show the mean ± SD of cerebella (n = 10) for each treatment. *P 0.05 versus Ctr.D BrdU assay in GCPs. Cerebellargranule cell progenitors (GCPs) isolated from 4‐day‐old mice were treated with Shh (recombinant mouseSonic Hedgehog, amino‐terminal peptide, ShhN, 3 μg/ml) and with different concentrations of GlaB, as indicated, for 48 h. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample. Shown is the mean of three independent experiments. Error bars indicate SD. *P 0.05 Shh versus DMSO; **P 0.05 Shh + GlaB versus Shh.E qRT-PCR and show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB. In all qRT-PCR experiments, the results were normalized to endogenous control (β2‐microglobulin and HPRT). *P 0.05 versus Ctr.F Western blot analysis show Hh and proliferation target mRNA and protein expression levels determined in ex vivo GCP culture derived from 6‐day‐old mousecerebella after s.c. injections of GlaB.
Figure 5A, B Ex vivo cell cultures from Ptch1+/−mice MBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, a trypan blue count was performed (A) to determine the growth rate of viable cells.A, B Ex vivo cell cultures from Ptch1+/−miceMBs were treated with GlaB (5 μM), GANT61 (10 μM) or DMSO only. After the indicated times, Gli1 mRNA expression levels were determined by qRT-PCR (B) normalized to endogenous control (β2‐microglobulin and HPRT).C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/−mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right).C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT).C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. Western blot analysis show Hh, proliferation and stemness target protein expression levels.C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample.G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.
Figure 6A Change of tumor volume during GlaB or vehicle treatment period (18 days).B Representative flank allografts average volumes. Error bars indicate SD. *P 0.05.C Representative Masson's trichromestaining of tumors; immunohistochemistry of Gli1, Ki67 and TUNEL stainings. Scale bars represent 35 μm for Masson's trichromestaining, 25 μm for Gli1 and TUNEL, and 10 μm for Ki67 stainings.D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (C). Error bars indicate SD. *P 0.05.E qRT-PCR analysis show Hh and proliferation target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.F Western blot analysis show Hh and proliferation target protein expression levels. Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.
Figure 7A Representative images (low and high magnification) of HE (upper panel) and Ki67immunohistochemistry staining (lower panel) of a human Daoy MB cell‐derived orthotopic tumor in NOD/SCIDmicecerebella after i.p. injections of GlaB. Scale bars represent 500 or 200 μm for HE staining, and 150 μm or 50 μm for Ki67 staining.B Representative tumor average volumes at the end of treatment. Error bars indicate SD. *P 0.05.C Quantification of Ki67 staining from immunohistochemistry shown in (A). Error bars indicate SD. *P 0.05.
Figure 8A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only. After the indicated times, a trypan blue count was performed to determine (A) the growth rate and (B) the percentage of cell death. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.C Gli1 mRNA expression levels were determined by qRT-PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (D) Change of tumor volume during GlaB or vehicle treatment period (18 days).D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (E) Representative flank allografts average volumes (upper panel).D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (F) HE and immunohistochemistry staining of Gli1, Ki67 and TUNEL of allograft tumor samples. Scale bars represent 50 μm for HE, 25 μm for Gli1, 25 μm for Ki67 and 30 μm for TUNEL stainings. (G) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (F)D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (H) qRT-PCR of Hh and proliferation target mRNA expression levels. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Shown is the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.
Figure 1Adult mouse back skin sections immunostained for LYVE1 (red), αSMA (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. LV, lymphatic vessels; apm, arrector pili muscle.Back skin sections of P55 mouse skin immunostained for LYVE1 (red), Tenascin-C (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. TNC, Tenascin-C.Adult mouse back skin sections from different postnatal (P) days immunostained for LYVE1 (red), Lhx2 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.Histogram of the quantification of the LV distance to HFSC positive for Lhx2. ). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.Back skin sections of P49 mouse skin immunostained for LYVE1 (red), CD34 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. White arrowheads denote the proximity of lymphatic capillaries to HFSC.Histogram of the quantification of the LV distance to HFSC positive for CD34. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. C, Catagen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen; A, Anagen.Back skin sections of K15CrePR+/T; Wls∆/∆ and Control K15CrePR+/+; Wlsflox/flox mice treated with mifepristone from P7 during 12 weeks (P90), immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.Histogram of the quantification of the LV angle relative to the epidermis. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.
Figure 3LV associated with individual HF further organize associating HF triads, which connect with other HF triads across the skin. P70, 70 d old mice. n= 3 - 4 mice. White arrowheads denote capillaries stemming from HF triads. Scale bar, 100 μm.3D reconstructions of whole skin mounts showing a triad of HF connected by LV. n= 3 - 4 mice. Scale bar, 50 μm. White arrowhead denotes an extending lymphatic capillary from a HF triad.Images of intravital microscopy analyses showing aligned HF rows in the back skin interconnected by LV in the Prox1CreERT2; Rosa-LSL-eYPF mice. Dotted boxes denote HF triads; White arrowheads denote capillaries stemming from HF triads interconnecting to other HF triads in adjacent rows. n= 3 - 4 mice. Scale bar, 50 μm.
Figure 4Adult back skin sections from different postnatal (P) days immunostained for LYVE1 (red) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 10 μm. White arrowheads denote LV membrane protrusion and fenestrated areas. LV, lymphatic vessels; HF, hair follicle.Histogram of the caliber (μm) of LV at different postnatal days. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. A, Anagen; C, Catagen; T, Telogen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen.H&E staining of back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. epi, epidermis; der, dermis.LYVE1 immunofluorescence (red) counterstained with DAPI (blue) (D) in back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.Histogram of the caliber (μm) of LV (E) in back skin sections from mice treated intradermally with Control or Clodronate liposomes.Back skin sections of Controls and K14ΔNβ-cateninER+/T mice immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.Histogram of the LV caliber in skin sections from Controls and K14ΔNβ-cateninER+/T mice. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.Histogram of the percentage of HF in Telogen and Anagen present in the skin of control mice or mice treated with Cilostazol. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.
Figure 5Gene set enrichment plots (GSEA) of LV using public annotations showing the enrichment of selected gene signatures involved in different pathways in late Telogen (P69, Tl) compared with mid Telogen (P55, Tm). NES: normalized enrichment score. The false discovery rate (FDR; q-value) is indicated.Heat map diagram of DEGs in LV during late Telogen compared to mid Telogen, relative to biological processes, analyzed using the Enrichr platform (Chen et al., 2013; Kuleshov et al., 2016). Data shown represent the log2 (FPKM+1).Histograms of RNA ISH analyses by RNAscope to quantify the level of expression of ITGA5 (D), DCN (E), PKD1 (F) and PLXND1 (G), assessed by the number of mRNA spots present in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= average of 30 LYVE1+ cells / sample, n= 3 mice.Histograms represent the mean fluorescence intensity for Jup (H) and Emilin1 (I) in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.
Figure 6H&E staining (A) and histogram of the HF length (B) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal Diphtheria Toxin (CSA+DT) starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.Ki67 immunostaining (C) and histogram of the number of Ki67+ cells per HF (D) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle; and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.LYVE1 immunostaining (E) and histogram of the LV caliber (F) analyzed in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). Scale bar, 10 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.H&E staining (G) and histogram of the HF length (H) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. Data represent the mean value ± SEM.Cleaved caspase 3 immunostaining (I) and histogram of the number of cleaved caspase 3+ cells per HF (J) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). Scale bar, 50 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.
Figure 1A, SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 (SB) or LGK974 (LGK) for 16 h. Total cell extracts were analyzed by WB. Protein quantification was analyzed by densitometry of three different experiments and represented B, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs from three biological replicates are presented in the right panel. Values correspond to fold variation in shRor2 versus shCtl cells (mean ± SD).C, RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT-PCR. Values correspond to fold variation in shRor2 versus shCtl cells. Results are the mean ± SD of at least three biological replicates performed in triplicate.D, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs are presented in Appendix Fig S3D as in B.
Figure 2E, HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a-producing cells for 16 h and expression of the indicated genes assessed by qRT-PCR (E) (mean ± SD of three biological replicates performed in triplicate in E).G, WB analysis of HT29 M6 and SW480 overexpressing Snail1. Quantification is presented in Appendix Fig S5I.I, SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by qRT-PCR (I, mean ± SD of three biological replicates performed in triplicate).