Inhibition of GLI signaling reduces the viability of liver progenitor cell lines.

<p>LPC lines BMOL1.2 <b>(A, C, E, G)</b> and BMOL-TAT <b>(B, D, F, H)</b> were grown over 72 h in the presence of signaling inhibitors for GLI (GANT61; <b>A, B</b>), Notch (DAPT; <b>C,D</b>) c-Met (SGX523; <b>E, F</b>), Wnt (XAV939; <b>G, H</b>) signaling and matching [DMSO]. Viability was determined by ALAMAR Blue assays, and an interaction between time and dose was assessed using two-way ANOVA followed by Tukey’s multiple comparisons test (α = 0.001) (Prism, GraphPad). **** <i>p</i><0.001.</p

A very minimal population of human vimentin⁺ HSCs/myofibroblasts express a primary cilium, with none detected on CD31⁺ endothelial cells.

<p>Human ALD liver tissue was examined for the expression of primary cilia (α-acetylated tubulin, green; γ-tubulin, red) by vimentin⁺ (grey) HSCs/myofibroblasts <b>(A)</b> or CD31+ (grey) ECs <b>(C)</b>. <b>(A)</b> The majority of vimentin⁺ cells were Pc^-ve in the tissues examined. Representative image shown, displaying absence of Pc on vimentin⁺ cells. To confirm this result, ciliary protein Arl13b (green) was co-stained with vimentin (grey). Rare Arl13b ciliary structures (arrow) co-localised with vimentin⁺ cells. Final panel in A illustrates rare Pc⁺ (α-acetylated tubulin, green; γ-tubulin, red) vimentin⁺ (grey) HSCs/myofibroblasts, at the cirrhotic interface. <b>(B)</b> Number of vimentin⁺ Pc⁺ cells or vimentin⁺ Pc^neg cells per FOV (<i>n</i> = 3 ALD samples, 8 FOV/sample). <b>(C)</b> No Pc were detected on CD31⁺ cells in the tissues examined (ALD <i>n</i> = 3, 8 FOV/sample). Representative image shown. All images obtained using confocal microscopy, 63x objective. DAPI, blue. White arrows illustrate Pc. * Non-specific liver autofluorescence.</p

Changes to transcript expression in two liver progenitor cell lines following GLI inhibition.

<p>Heat map representing changes to gene transcript levels in BMOL1.2 (<i>n</i> = 3) or BMOL-TAT (<i>n</i> = 3) lines following GANT61 (10 μM) 8 h treatment. Log₂ intensity scale shown. Downregulated genes (blue) are shown above the yellow dashed line, with upregulated genes (red) below. Grey indicates no data.</p

Gene changes in BMOL1.2 cells following GANT61 treatment.

<p>Gene changes in BMOL1.2 cells following GANT61 treatment.</p

Primary cilium expression in human EpCAM⁺ ductular reaction and liver progenitor cells <i>in vivo</i>.

<p><b>(A)</b> Immunofluorescence identified primary cilia (Pc) in human ALD tissue at the injury interface. Fully assembled Pc are identified by extension of an axoneme (α-acetylated tubulin, green) from the basal body (γ-tubulin, red). Pc were identified (white arrows) on EpCAM⁺ (grey) ductular reaction/LPCs in human donor liver and in a range of cirrhotic liver tissues (ALD, NASH, PBC). <b>(B)</b> In human ALD tissue, α-acetylated tubulin (green) co-localised with Arl13b (red), confirming axoneme staining specificity. Arl13b (green) also localised with EpCAM⁺ (grey) ductular reaction/LPCs in an identical staining pattern to α-acetylated tubulin. Note the absence of Arl13b expression on adjacent EpCAM⁺ cells differentiating into hepatocytes. Confocal microscopy, 63x objective. Pc (black arrow) were also detected at the cirrhotic interface in human ALD tissue using TEM. Cells expressing Pc via TEM were not biliary cells or HSCs/myofibroblasts. <b>(C)</b> Quantitation of ductular reaction/LPC (EpCAM⁺ or pan-CK⁺) Pc⁺ cells vs. ductular reaction/LPC Pc^-ve cells per FOV (<i>n</i> = 3 ALD samples, 10 FOV/sample). Percentage of LPCs (EpCAM⁺ or pan-CK⁺) with Pc in donor or ALD liver vs. vimentin⁺ HSCs (<i>n</i> = 3 patients/condition). Two-tailed <i>t-test</i>; ***<i>p</i> = 0.0001; ****<i>p</i><0.0001.</p

Patient sample, age, sex, fibrosis staging (Scheuer score) and SHH expression.

<p>Patient sample, age, sex, fibrosis staging (Scheuer score) and SHH expression.</p

SHH expression in human donor and cirrhotic liver.

<p><b>(A)</b> Frozen (4 μm) human donor (<i>n</i> = 5), and cirrhotic liver sections [ALD (<i>n</i> = 6), NASH (<i>n</i> = 3), PBC (<i>n</i> = 1)] were screened for SHH (C-terminus) by immunofluorescence. 5x objective. Representative images shown. <b>(B)</b> qRT-PCR for <i>SHH</i> in human donor and ALD samples. Mean±S.E.M. Significant (*) difference between means (One-sided student t-test, *<i>p</i><0.05). <b>(C)</b> SHH expression (red) by EpCAM⁺ LPCs (green) in donor liver. 63x objective. <b>(D, E)</b> Comprehensive characterisation of SHH expression by liver cell populations in ALD. Images obtained using 63x objective. The majority of SHH is produced by hepatocytes. <b>(D)</b> SHH (red) is expressed by EpCAM⁺ LPCs (green). Co-localisation in merged images indicated by yellow. A subset of CD31⁺ ECs (green) at the cirrhotic interface express SHH. <b>(E)</b> SHH (red) is not expressed by CD45⁺ leukocytes (green). Minimal SHH is expressed by vimentin⁺ ECs (solid arrows), with negligible SHH expressed by vimentin⁺ myofibroblasts (dashed arrows). DAPI, blue.</p

Common gene changes in BMOL1.2 and BMOL-TAT cells following GANT61 treatment.

<p>Common gene changes in BMOL1.2 and BMOL-TAT cells following GANT61 treatment.</p

Gene changes in BMOL-TAT cells following GANT61 treatment.

<p>Gene changes in BMOL-TAT cells following GANT61 treatment.</p

Widespread GLI expression in human donor and cirrhotic liver.

<p><b>(A)</b> Frozen (4 μm) human donor (<i>n</i> = 5), and cirrhotic liver sections [ALD (<i>n</i> = 6), NASH (<i>n</i> = 3), PBC (n = 1)] were screened for GLI2 (red) expression by immunofluorescence. Representative images taken at 5x or 40x (insets) objective shown. DAPI, blue. <b>(B)</b> qRT-PCR for <i>GLI1</i> and <i>GLI3</i> transcript in human donor or ALD samples. Mean±S.E.M. Significant (*) difference between means (One-sided student t-test, **<i>p</i><0.005). Western blot for full-length GLI1 protein (>150 kDa) in donor (Don) or ALD patient samples. Densitometry analysis with GLI1 normalised to GAPDH (Image J). Mean±S.E.M; **<i>p</i> = 0.0093 (Two-sided student t-test). <b>(C)</b> Nuclear GLI2 (green) expression in EpCAM⁺ (red) LPCs in donor, ALD, PBC and NASH liver. <b>(D)</b> Nuclear GLI2 (green) expression demonstrated within CD31⁺ (red) ECs, CK18⁺ (red) hepatocytes, CD45⁺ (red) leukocytes and vimentin⁺ (red) HSCs/myofibroblasts, in ALD. 63x objective. <b>(E)</b> Maximum intensity projection illustrating close physical association between EpCAM⁺ LPCs (green) and vimentin⁺ HSCs/myofibroblasts (red), both of which express GLI2 (grey), in ALD tissue. Arrows indicate myofibroblasts directly contacting LPCs. Confocal microscopy, 63x objective. Quantitation (%) of EpCAM⁺ GLI2⁺ cells and vimentin⁺ GLI2⁺ cells within the same FOV (<i>n</i> = 3 ALD samples, 8 FOV/sample).</p