EMBO/SourceData huggingface dataset:
Figure 6Calu6, H157, and H1355 TPR-Met-expressing or vector control (pBABE)-expressing cells were treated as indicated for 24 hours and apoptosis was measured using the Apo-BrdU assay.Parental and TPR-Met-expressing cell lines were treated with the indicated drugs for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. β-actin was used as a loading control.Parental and TPR-Met-expressing cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg correction for multiple comparisons are indicated.Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies. *p < 0.05. **p < 0.01.
Figure 7Cell viability measured by CellTiter-Glo in Calu6 parental and Calu6-VAR cells treated with different concentrations of volasertib for 72 hours.Micrograph shows acquisition of a mesenchymal-to-epithelial transition phenotype in VAR cells.Basal protein and mRNA expression in parental and VAR cells for the indicated proteins was measured using immunoblotting (upper) and reverse-transcriptase PCR (lower), with GAPDH expression used for normalization.Parental and VAR cells were treated as indicated for 48 hours and apoptosis was measured using the Apo-BrdU assay.Parental and VAR cells were treated with the indicated drug concentrations for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Parental and VAR cells were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ.
Figure 8Epithelial and mesenchymal non-small cell lung cancer (NSCLC) cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Epithelial and mesenchymal NSCLC cell lines after Plk1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Calu6 and H1792 mesenchymal NSCLC cell lines after VIM silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after Plk1 and INTB1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after treatment with fibronectin for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.Calu6 and H1975 NSCLC cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoprecipitated (IP) using β1-integrin, vimentin, and Plk1 antibodies, then immunoblotted for the indicated proteins.
Figure 1Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK-p25 animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte.  Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. (E) Heatmap showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S animals. (F-G) Relative abundances across all tissues for phosphotyrosine peptides: (F) SVyTEIK (Siglec-F pY561); (G) LyDFVK (Inpp5d pY868). Bars indicate mean ± 95% confidence interval (CI); n = 3-8 samples; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.
Figure 2(A) Immunofluorescence (IF) staining showing Siglec-F and Iba1 localization in hippocampus CA3 region of 3wk induced CK (top) or CK-p25 (bottom) mice. Scale bars = 50 μm. Colors are: Magenta = Iba1, Cyan = Siglec-F. Images are 60x super-resolution max z-stack projections taken from coronal slices. (B-C) Percent area coverage of (B) Iba1 and (C) Iba1-proximal Siglec-F between CK and CK-p25 mice across hippocampal regions. Bars indicate mean ± 95% CI; n = 3-4 animals; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (D-E) IF staining of Siglec-F and Iba1 in (D) 5XFAD 2 month hypothalamus (HY) and (E) 5XFAD 6 month dentate gyrus (DG). Yellow = Aβ from methoxy X04 (D) or D54D2 (E). Scale bars = 50 μm. (F) Percent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6 month WT (non-Tg) and 5XFAD mice. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (G) Percent area coverage of Siglec-F localized to Iba1 compared to total Aβ plaque volume in each field of view across all analyzed images from 5XFAD and non-Tg control mice. Linear regression R2 value is shown for images with ≥10 μm2 total amyloid volume.  (H) Cumulative distribution function (CDF) of the distance between Siglec-F+ and Iba1+ mask voxels and nearest Aβ plaques in 6 month 5XFAD mice. Each value in the CDF represents the mean distance calculated from a single z-stack of a ROI containing ≥ 1 plaque of size ≥10 μm2. n = 55 images (4 animals). (I-J) Relative RNA abundances for Siglec-8 in (H) hippocampus and (I) posterior cingulate cortex post-mortem patient tissue. Linear regression R2 values are shown on plots.(K) IF staining showing Siglec-8 and Iba1, and MHC-II in cortical white matter tissue from a patient with late-onset AD (20x wide-field; Case ID: 01-43). Scale bars = 50 μm. Images are 60x super-resolution max z-stack projections.(L-M) Percent area coverage of Siglec-8 localized to Iba1 or MHC in individuals with non-AD or AD. In (K) each point represents one WM or GM region average from each individual. In (L) values and statistics are shown for each image analyzed. Boolean mask operators indicate: ° = mask dilation, ⋂ = intersection, ⋃ = union, NOT = inverse mask. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 8-43 images (n = 1 ND, 3 early-onset AD, 4 late-onset AD); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.
Figure 3(A) Flow cytometry quantification of BV-2 Siglec-F expression levels. Cells were cotreated with 0-10 ng/mL IFNγ and 0-10 μM tofacitinib. Values are geometric mean fluorescence intensities (gMFI). Bars indicate mean ± 95% CI; n = 2-4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group. (B) IF staining of iMGLs treated with IFNγ. Siglec-8 fluorescence is only shown on non-nuclear (Hoechst 33342-) regions. Images are 60x super-resolution max z-stack projections. Colors are: Red = Iba1, Green = Siglec-8, Blue = 33342. Scale bars = 20 μm. (C) Quantification of Siglec-8 on iMGLs treated with PBS or 25 ng/mL IFNα, IFNβ, or IFNγ. Values are mean Siglec-8 intensity values from Iba1+;33342- regions. Bars indicate mean ± 95% CI; n = 8 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.(D) Quantification of Siglec-8 on iMGLs treated ± 25 ng/mL IFNγ and 5 μM tofacitinib. Legend is same as (C). Bars indicate mean ± 95% CI; n = 5-20 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.
Figure 4 (A) Confluency doubling times quantified from BV-2 cells with stable retroviral expression of Siglec-F and related human Siglec constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Cells grown in media alone or + 50 mU sialidase (+SA). Bars indicate mean ± 95% CI; n = 3-12 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F groups. (B) Flow cytometry quantification of cell live / dead markers Annexin-V and propidium iodide on BV-2 cells with stable retrovirus expression of Siglec-F constructs. % parent values are shown for early apoptotic (Annexin-V+;PI‑), late apoptotic (Annexin-V+;PI‑), and necrotic (Annexin-V-;PI+) populations. Bars indicate mean ± SD; n = 8 replicates. (C) Confluency doubling times quantified from BV-2 cells with dox-inducible expression of Siglec-F constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Bars indicate mean ± SD; n = 8 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. (D) Selected gene expression changes (top) and enriched gene sets (bottom) taken from RNA-seq analysis of BV-2 cells with stable retroviral expression of Siglec constructs, 2xY->F, and empty vector controls. Heatmaps share a colormap range of 0.25 to 4 for z-score values (top), normalized enrichment scores (NES, bottom).(F) Expression levels of C1qa, IL-1β, and β-actin by qPCR analysis in BV-2 cells with inducible Siglec-F expression after 48 hours of treatment with 500 ng/mL doxycycline. Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.(G) Western blot showing pro-IL-1β and β-tubulin in BV-2 cells induced to express Siglec-F at 0 and 48 hours.
Figure 5(B-C) Volcano plots showing phosphotyrosine peptides that were (B) increased or (C) decreased after Siglec-F expression. Plots show Siglec-F compared to 2xY->F mutant data integrated across all three runs. Protein names are shown for altered peptides. Labels are only shown for peptides with maximum directional change from each protein. Labels colored according to GO terms: magenta = signal transduction, red = endosome, cyan = cell adhesion, yellow = cytoskeleton, green = metabolism. (D) Heatmap showing phosphosites in CK-p25 which overlapped in their directional change with BV-2 Siglec-F-associated sites. Colors indicate fold change relative to 2xY->F controls.  Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were increased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D).  Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D). (G) Western blot quantification of myc, SHP-2, and SHP-1 from myc co-IP eluates. Co-IP lysates were prepared from BV-2 cells stably expressing Siglec-F and optionally treated with 1 μM SHP099.(H) Heatmap showing phosphosites that were perturbed by SHP099 treatment. Color scale is same as (D).
Figure 6(B-C) Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec-F expression for (B) Siglec-F and (C) Siglec-F 2xY->F. Siglec-expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates.Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of monomeric Aβ, Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 10,000 MW Dextran Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.  Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 1 μm FluoSpheres. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group. (G) pHrodo Dextran uptake in BV-2 cells with stable expression of Siglec-F estimated by Incucyte measurements. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (H) pHrodo dextran uptake in BV-2 cells stably expressing Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G).  (I) Confluency doubling times for BV-2 cells with stable expression of Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G). Bars indicate mean ± 95% CI; n = 4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated 2xY->F group.
Figure 1Multiple tissue (eye, brain, adrenal gland, submandibular gland, oesophagus, lung, heart, liver, kidney, spleen, pancreas, stomach, duodenum, jejunum, ileum, caecum, colon, spinal cord, dorsal root ganglia, skeletal muscle, testis, epididymis, female gonad, skull) northern blot for CAVII. In the P56 mouse, CAVII is prominently expressed in the central nervous system including brain and spinal cord. Gapdh served as the loading control.(C) Developmental profile of CAVII protein expression. Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30. The band of the appropriate size (∼30 kDa) was absent in protein lysates from Car7 KO tissue.beta Actin served as the loading control in C and D.(D) In western blots from protein lysates of cultured cells, CAVII was detected in mixed glial/neuron cultures but not in glia cell cultures. The presence of astrocytes in all cultures was confirmed by detection of GFAP. Actin served as the loading control in C and D.
Figure 2Original single-cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons (baseline pHi 7.21, 7.13 and 7.11, respectively). Superfusion with CO2/HCO3−-free HEPES solution (upper horizontal bars) evoked an intracellular alkalinization, which in P14 WT was large and suppressed by 100 μM acetazolamide (AZ, lower horizontal bar), indicating the presence of CA activity (see Supplementary Materials and methods). The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide (a poorly permeant CA inhibitor). Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT (scale bar 10 μm).Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity. Data from CA II KO and CA II/VII KO neurons were obtained only at >P35. The number of cells tested is indicated for each bar. The animal numbers for WT mice at the different age points was n=3 (P5-8), n=2 (P10-11), n=3 (P14-16), n=2 (P17-18), n=2 (P20-30) and n=2 (>P35); for CAVII KO mice the numbers were n=3 (P5-8), n=2 (P10-11), n=5 (P14-16), n=3 (P17-18), n=4 (P20-30) and n=2 (>P35); and for CA II KO and CA II/CAVII KO mice the number was n=2 (>P35).Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2/HCO3− (horizontal bar, HEPES) in slices from >P35 WT, CAVII KO and CA II/VII KO mice. The rate of rise and the amplitude of the alkaline shift are identical in WT (P39, baseline pHi 7.01) and CAVII KO neurons (P40, 7.06), whereas alkalinization develops much more slowly in the CA II/VII KO (P46, 7.04)
Figure 3Excitatory GABAergic response in WT, but not in CAVII KO neurons. Picrotoxin blocked the biphasic response (PiTX 90 μM; n=3/genotype). The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO (13.53±1.27 versus 6.39±0.96 mV/s, P<0.001, Student's t-test). The resting membrane potential of WT and CAVII KO neurons did not differ (−71.0±1.1 versus −72.3±1.4 mV, respectively; n=15 neurons for both genotypes, P=0.47, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)Microinjection of GABA (5 mM for 100 ms, sr/slm border in the presence of CGP55845/TTX, arrowheads) induced a pronounced depolarization in WT neurons (black) that was abolished in the absence of CO2/HCO3− (grey). The depolarization was smaller in CA VII KO (red). Bar diagram illustrates GABAergic mean peak depolarization in P12-16 WT and CAVII KO (13.3±0.5 versus 8.3±0.6 mV, P<0.001, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D).In the presence of AP5/CNQX/CGP55845, microinjection of GABA (5 mM, sr/slm border) triggered SR 95531-sensitive field potential spikes in WT slices already upon 8-ms injection, while even 16-ms injection evoked hardly any spikes in CAVII KO (left). Mean number of spikes was plotted against GABA puff duration in P12-16 WT and CAVII KO (P<0.01 and P<0.001, Student's t-test; right). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)After P35, depolarizations upon GABA microinjection (arrowheads) in the presence of CGP55845/TTX were indistinguishable in WT (black), CA VII KO (red) and CA II KO (blue) but smaller in CA II/VII KO (green). The depolarization in WT was abolished in the absence of CO2/HCO3− (grey, n=5). Bar diagram summarizes mean peak depolarization for each genotype (right; ANOVA, Bonferroni). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)
Figure 4Representative raw traces of epidural EEG recordings during hyperthermia (HT) from a WT and a CA VII KO mouse. Seizures were reliably induced in WT (n=9) but not in CA VII KO (n=7). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.Specimen recordings of breath rate from a WT and a CA VII KO mouse. The baseline breath rate (WT 153±11 breaths/min and CA VII KO 157±8 breaths/min) was increased during HT in both genotypes (WT 272±28 breaths/min and CA VII KO 279±12 breaths/min). The bar diagram summarizes the mean increase in breath rate in WT (177±12%) and CA VII KO mice (178±6%, P=0.98).Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.Hyperthermia caused a comparable alkalosis in blood pH in both WT and CA VII KO mice. The blood pH did not differ between the two genotypes under control conditions (P=0.37) or during HT (P=0.44). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.Electrographic seizures were not detected in WT mice when hyperthermia was induced in the continuous presence of 5% CO2 (n=5). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.
Figure 5Diazepam (DZP, 150 μg/kg) reduced the latency to seizure onset in P14 rat pups (P<0.05). There was no statistical difference between 50 μg/kg and saline-injected animals. DZP 2.5 mg/kg completely blocked the seizures. DZP was given intraperitoneally 15 min before hyperthermia (HT) onset.Diazepam given at 150 μg/kg did not affect the baseline breath rate, and HT induced an analogous increase in the breath rate in saline- and diazepam-injected rats (P>0.05). At a higher concentration (2.5 mg/kg), DZP suppressed breathing significantly (P<0.01) and HT caused only a small increase in the breath rate.In slice preparation, DZP (1 μM) potentiated the high-frequency stimulation (40 pulses at 100 Hz, horizontal bar) -evoked GABAAR-mediated depolarization. Sample trace from a slice from a P14 rat pup where stimulation to the border of stratum radiatum and stratum lacunosum-moleculare was given in the presence of AP5, CNQX and CGP 55845. The bar diagrams summarize the effects of DZP on the duration of the depolarization (time to half-maximum) and the number of action potentials associated with the depolarizing phase. P-values (*P<0.05, **P<0.01 and ***P<0.001) are based on ANOVA, Bonferroni (A, B) or Student's t-test (C), and error bars denote s.e.m. The number of animals (A, B) or cells (C) is indicated in bar diagrams.
Figure 6CA VII expression during human brain development. Line plots show the log2-transformed exon array signal intensity in the neocortex (NCX) and hippocampal formation (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.CA VII expression during human brain development. Line plots show the log. Line plots show the normalized ratio of absolute copy number of CA VII to Gapdh by droplet digital PCR (ddPCR, right) in the neocortex (NCX) and hippocampus (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.
Figure 1Normalised Wnt/STOP signalling activity by quantification of the protein expression of two Wnt/STOP target proteins, β-catenin and c-Myc, in mitotic (M) and asynchronised cells (A) after silencing 47 Wnt signalling regulators.ELISA validation of selected Wnt/STOP signalling regulators (n=3).Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n=3).Representative pictures (left panel) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n=10). Scale bars represent 10 μm.Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9.The representative xenograft tumours (top panel) and tumour growth analysis (bottom panel) of scramble and BCL9 stable knockdown cells. LF: left flank of mouse, RF: right flank. n=6 for shS1 vs. shB1, n=5 for shS1 vs. shB2.Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm.