EMBO/soda-nel huggingface dataset:
The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours. (2015)
Figure 2

Characterisation of stroma IMCs

Giemsa staining of isolated stroma IMCs. These cells show round or oval-shaped nuclei that lack indentations, and have an abundant basophilic cytoplasm without showing prolonged cytoplasmic processes, suggestive of immature myeloid-lineage cells that are morphologically distinct from monocytes/DCs. Scale bar, 25 μm.

Cell surface marker analysis of IMCs showing lack of common hematopoietic marker expression except for CD11b, which is expressed at low levels (overlaid with isotype control histograms shown in grey).

Cell surface marker analysis (dot plots) of IMCs demonstrating CD11c+ IMCs do not express macrophage (F4/80), DC (CD24) or M1-polarisation (CD86) markers, but weakly express the M2 marker CD206. Insets show isotype control staining.

Morphological evaluation of IMCs cultured for 2 weeks without additional cytokines. The cells grown on culture plates were imaged by phase-contrast microscopy (left). Then, the cells were trypsinised and smeared on glass slides for Giemsa staining (right). These cells are intermediate to large sized, round or amoeboid cells with cytoplasmic vacuolation, consistent with a macrophage morphology. Scale bars, 50 μm.

Cell surface marker analysis of cultured IMCs showing differentiation into F4/80+ macrophages with CD11bhighCD11c+CD86+CD206low surface phenotype (overlaid with isotype control histograms shown in light grey).

PCR detection of Braf recombination in purified IMCs, AT2 cells and tumour cell aggregates.

Source data are available online for this figure.
The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours. (2015)
Figure 3

IMCs promote growth and EMT of lung tumour cells

CMT64 cells (1 × 104/well in 12-well plates) were co-cultured with 1.5–3 × 105 IMCs for 4 days. Cells were collected by trypsinisation to exclude most of the IMCs since they are resistant to trypsin due to their strong adhesion to the culture plate. Harvested cells were subjected to counting (left). Any contaminating IMCs were separately counted according to their morphological distinction from CMT64 cells. The bar graph (left) shows CMT64 growth (cell counting) data pooled from two independent experiments. Phase-contrast images of CMT64 cells cultured without IMCs (CMT64 only) or co-cultured with IMCs (CMT64/IMC co-culture) are also indicated in the middle and right. A higher magnification image of co-cultured CMT64 cells (right) highlights IMCs migrating into CMT64 cells (arrow heads). Scale bars are 200 μm, except for the high magnification image (right) in which the scale bar is 100 μm.

(Left) Growth and EMT-like morphological changes of CMT64 cells cultured with 25% IMC-CM. CMT64 cells cultured for 4 days at low-density in DMEM/10%FCS without IMC-CM (-IMC-CM) or with 25% IMC-CM (+IMC-CM) were counted and normalised to the average of control as 100%. The data in the bar chart represent mean + SD of five cultures utilising IMC-CM obtained from three independent sources. (Middle) Phase-contrast images to indicate representative morphologies of CMT64 cells maintained without IMC-CM (control) or cultured with 25% IMC-CM (+IMC-CM) for 2 weeks. (Right) E-cadherin immunofluorescence of CMT64 cells cultured as above. Scale bars are 200 μm (phase contrast) and 10 μm (immunofluorescence).

Co-culture of AT2 cells and autologous IMCs from BVE mice (8- to 10-week-old) for 48 h using the Transwell® culture system. In vitro BrdU incorporation (flow cytometry, left and middle) and EMT marker expression (RT–PCR, right) in co-cultured AT2 cells are indicated. Representative flow cytometry plots are indicated on the left, and the bar chart in the middle indicates % BrdU+ AT2 cell numbers normalised to control cultures without IMCs (n = 4, mean + SD). Gapdh serves as a loading control for the RT–PCR on the right.

Morphological alterations and intracellular signalling in AT2 cells cultured with IMC-CM. On the left and middle, primary AT2 cells from 3-week-old BVE mice were cultured for 48 h and then incubated in serum-free IMC-CM (AT2 with IMC-CM) or DMEM (AT2 w/o IMC-CM, serving as a control) for another 48 h, followed by immunostaining for E-cadherin (red) and vimentin (green) and confocal laser scanning microscopy (CLSM) imaging. The 3× zoomed image in the middle right highlights dividing vimentin+ cells that show internalised E-cadherin (arrows). Scale bars, 50 μm. On the right, primary AT2 cells from 10-week-old BVE mice were cultured for 48 h, serum starved for 5 h and treated with or without serum-free IMC-CM for 30 min. Phosphorylation of AKT, SMAD3, MEK and ERK were analysed by immunoblotting.

Source data are available online for this figure.
The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours. (2015)
Figure 4

CCR1 inhibition suppresses IMC recruitment and tumour development <italic>in vivo</italic>

CCR1 immunofluorescence of primary IMCs (left, co-stained with Mac2) and AT2 cells (right, co-stained with E-cadherin) detected by CLSM. Scale bars, 10 μm. CCR1 staining was mainly detected in cytoplasmic vesicles within IMCs, but some CCR1 fluorescence was also detected at the plasma membrane along with Mac2 (inset in the left microphotograph).

CCR1 immunohistochemistry of lung sections from BVE mice showing CCR1 expression in stroma IMCs. Scale bar, 50 μm.

CT imaging of lungs of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (top) or CCR1 inhibitor (bottom). H: heart; * indicates a tumour region accompanied by atelectasis.

H&E staining of lung sections from vehicle (left) or CCR1 inhibitor (right) treated AdCre-infected Braf+/LSL−V600E mice. Scale bars, 250 μm (top) or 50 μm (bottom).

Flow cytometry analysis of CD11c+ (top, green) and SPC+ (bottom, red) cells in the lung of AdCre-infected Braf+/LSL−V600E mice treated with vehicle (left) or CCR1 inhibitor (right).

Lung CD11c+ (left) and SPC+ (right) cell numbers (per left lobe) were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).

% Lung CD4+ (left) and CD8+ (right) T lymphocytes were quantitated in AdCre-infected Braf+/LSL−V600E mice treated with CCR1 inhibitor or vehicle (V) (n = 3, mean + SD).

Source data are available online for this figure.
The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours. (2015)
Figure 5

<sup>V600</sup><sup>E</sup>BRAF-driven lung tumours secrete NPC2

NPC2 immunoblots of concentrated whole lung CM (top) and whole lung lysates (bottom) generated from Braf+/+;CreER™ (WT) or BVE (VE) mice. Whole lung lysates were obtained from 3-to 8-week-old mice.

NPC2 immunoblots of un-concentrated CM (containing 10% FCS) from fractionated IMC and AT2 cultures. Total protein staining of the membrane by Amido black (bottom) serves as a loading control showing serum albumin at 70 kDa, derived from FCS in the media.

Npc2 mRNA expression in the purified IMC and AT2 populations as determined by quantitative RT–PCR. Mean + SD (n = 3) of Npc2 expression normalised to Gapdh is indicated.

NPC2 immunoblots of cell lysates from freshly isolated IMCs and AT2 cells.

NPC2 immunoblots of cell lysates from freshly isolated or cultured (48 h in serum-free DMEM) IMCs.

NPC2 immunoblots of cell lysates from primary IMCs cultured with or without 50 μg/ml bNPC2 for 48 h.

CLSM imaging of NPC2-Alexa488 taken up by IMCs. (Left) Freshly isolated IMCs were incubated for 2 h with (+NPC2-Alexa488) or without (-NPC2-Alexa488) 82.5 nM NPC2-Alexa488, chased in NPC2-free media for additional 2 h and live imaged by CLSM. Single-colour (top) and DIC-merged (bottom) fluorescence images are indicated. Scale bar, 20 μm. (Right) Zoomed images (DIC-merged and single-colour) from different fields showing vesicular and reticular distribution of incorporated NPC2-Alexa488. Scale bar, 10 μm.

Npc2 mRNA expression in IMCs cultured for 48 h with or without 50 μg/ml bNPC2 as determined by qRT–PCR. Mean ± SD (n = 3) of Npc2 expression normalised to Hprt1 is indicated.

The diagram indicates our hypothetical model for the source of NPC2 in IMCs, based on our ex vivo observations.

Source data are available online for this figure.
The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours. (2015)
Figure 6

<italic>Npc2</italic> haploinsufficiency causes stroma IMC accumulation

Shortened survival of BVE mice following reduction in Npc2 gene dosage. Genotypes of mice are as follows: +/+ = Braf+/LSL−V600E;CreER+/0;Npc2+/+, +/hypo = Braf+/LSL−V600E;CreER+/0,Npc2+/hypo, hypo/hypo = Braf+/LSL−V600E;CreER+/0;Npc2hypo/hypo. Median survival time (MST) for each genotype is indicated.

Representative H&E staining of lung sections from 5-week-old BVE mice on the Npc2+/+ (+/+) or Npc2+/hypo (+/hypo) background is shown. Scale bars, 500 μm.

Stroma IMC quantification per lung in Npc2+/+ (+/+) or Npc2+/hypo (+/hypo) BVE mice at different ages (39–109 days p.p.).

Ki67/Mac2 immunofluorescence analysis of Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE lung sections at 5 weeks of age. Merged images for Ki67 (green), Mac2 (red) and DAPI (blue) are indicated. Arrows indicate dual positive cells for nuclear Ki67 and Mac2. Scale bars, 50 μm. The bar chart indicates % Ki67 in Mac2+ lung stroma cells (mean + SD), obtained from 4 independent Npc2+/+ and Npc2+/hypo BVE pairs.

In vivo BrdU incorporation of CD11c+ IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice are shown. Representative flow cytometry plots of three independent experiments are indicated.

Ki67 (green)/Mac2 (red) dual immunofluorescence staining of lung sections from 5-week-old Npc2+/hypo BVE mice with nuclear DAPI (blue) staining, focusing on intratumour Mac2+ cell clusters. Scale bar, 50 μm. Enlarged images of the boxed areas 1 and 2 highlight Ki67+Mac2− cells associated with Mac2+ cells (arrows).

Lung SPC+/CD11c+ cell numbers of Braf+/LSL−V600E;Npc2+/+ (+/+, n = 3) and Braf+/LSL−V600E;Npc2+/hypo (+/hypo, n = 7) mice at 10 weeks post-AdCre infection.

Source data are available online for this figure.
The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours. (2015)
Figure 7

NPC2 reduces intracellular cholesterol accumulation in IMCs

Intracellular un-esterified cholesterol distribution as detected by filipin staining in freshly isolated Npc2+/+ and Npc2+/hypo IMCs. Scale bars, 25 μm. Filipin-stained cells were graded from 0 to 3+ as indicated in the microphotographs (see Materials and Methods for criteria), and % of each grade in IMCs from Npc2+/+ (+/+) and Npc2+/hypo (+/hypo) BVE mice is shown in the bar chart. The data were obtained by analysing 176 Npc2+/+ and 242 Npc2+/hypo IMCs from three independent Npc2+/+ and Npc2+/hypo pairs.

CLSM imaging of fresh Npc2+/hypo IMCs stained with filipin (cyan) and LAMP2 immunofluorescence (purple). Scale bars, 5 μm. Coarse structures strongly stained with filipin are surrounded by cytoplasmic membranous staining of LAMP2. Single-colour greyscale images are also demonstrated in the middle (filipin) and right (LAMP2).

Intracellular un-esterified cholesterol distribution as detected by filipin staining in Npc2+/hypo IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 25 μm. Quantitative data in the bar graph were obtained by analysing 265 bNPC2-untreated (-bNPC2) and 252 bNPC2-treated (+bNPC2) cells from three independent cultures using the method described in (A).

Source data are available online for this figure.
The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours. (2015)
Figure 8

NPC2 inhibits IMC secretion of CCL6

Immunoblot (left) and ELISA (right, n = 3) analyses of CCL6 in IMC-CM from primary Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h (immunoblots) or 72 h (ELISA). The data in the bar graph (ELISA) represent mean + SD.

Quantification of secreted CCL6 into IMC-CM during the initial 24 h (0–24 h) and subsequent 48 h (24–72 h) of IMC culture by ELISA (n = 3, mean + SD).

Immunoblot and qRT–PCR analyses of CCL6 expression in Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. The data in the bar graph (qRT–PCR) represent mean + SD; n = 3.

CLSM imaging of CCL6 (green)/LAMP1 (red) immunofluorescence of Npc2+/+ IMCs cultured with or without 50 μg/ml bNPC2 for 48 h. Scale bars, 10 μm. The right bottom image highlights the boxed area of the merged image of bNPC2-treated IMCs showing partial co-localisation of LAMP1 and CCL6.

Immunoblot analysis of intracellular and secreted (CM) CCL6 in bafilomycin-treated Npc2+/+ IMC culture. IMCs without (left) or with (right) 50 μg/ml bNPC2 pre-loading for 30 min were chased in serum-free DMEM for 3 h and then treated with 200 nM bafilomycin A1 for 24 h.

Source data are available online for this figure.
Disrupted Hypothalamic CRH Neuron Responsiveness Contributes to Diet-induced Obesity (2020)
Figure 1

Figure 1. HFD blunted PVH CRH neuron responsiveness.

(A-C) CRH-Cre::Ai9 reporter male mice fed on chow or 4 weeks on HFD were used to examine c-Fos expression in the PVH in 24hr fasting or refed after 24hr fasting. (A) Representative images showing CRH neurons (red in the first colomn), c-Fos expression (green in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed, chow versus HFD) are labelled on the left. Scale bar=200µm. (B-C) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (B, **p=0.0032: chow fasting versus chow refed and *p=0.023, HFD fasting versus HFD refed) and total number of c-Fos positive CRH neurons (C, **p<0.0001: chow fasting versus chow refed and p=0.7158: HFD fasting versus HFD refed), n=3-4 each, data presented as mean +/-SEM, two way ANOVA tests.

(D-G) Effects of HFD feeding on diurnal rhythmicity in feeding (D and E) and energy expenditure (F and G). The measurement was collected with male mice fed chow or after 4 weeks HFD feeding, n=6 each, data presented as mean +/- SEM, **p=0.0066 in E and **p=0.0.0007 in G, unpaired Student's t tests.

(H-M) CRH-Cre mice received delivery of AAV-Flex-GCaMP6m viral vectors to bilateral PVH and optic fiber implantation targeting PVH neurons, and were used for in vivo live recording. (H-I) Representative traces showing recordings of activity changes in CRH neuron in response to stressor (water spray) in chow-fed controls (H), and 4 weeks after HFD (I), (J) Comparison of average responses in the two conditions, n=4 each, **p=0.0006, paired Student's t test. (K-L) Representative traces showing recordings of activity changes in CRH neurons in response to Dexamethasone (Dex) injections in chow-fed controls (K) and after 4 weeks on HFD (L). (M) Comparison of average responses in the two conditions, n=4 each, data presented as mean +/- SEM, **P=0.0012, paired Student's t tests.

Data information: 3V: the third ventricle; downward arrows indicating onset of stimuli or Dex injections.
Disrupted Hypothalamic CRH Neuron Responsiveness Contributes to Diet-induced Obesity (2020)
Figure 2

Figure 2. Clamping PVH CRH neuron activity at high levels disrupted neuron responsiveness.

(A-C) CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age and recordings were made at least 4 weeks after viral delivery on neurons with NachBac (one side) or control neurons (contralateral side) of the same animal (A); typical action potentials (APs) in controls (B, top panel) and typical NachBac-mediated APs showing lower threshold and long duration (B, bottom, the inset highlighting the long duration) in NachBac mice; and the distribution of the membrane potential and firing cells in the two groups (C, ratios in the bracket denoting neuron number out of total number recorded with the indicated behavior).

(D-F) CRH-Cre male mice were injected with either AAV-Flex-GFP or AAV-EF1a-Flex-EGFP-P2A-mNachBac to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing GFP or NachBac (GFP in the top 2 rows and NachBac in the bottom 2 rows of the first colomn), c-Fos expression (red in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left. (E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p<0.0001: fasting versus refed; **p=0.0026: fasting versus fasting/NachBac; **p<0.0001: fasting/NachBac versus refed/NachBac) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; **p<0.0001: fasting versus fasting/NachBac; p=0.3423: fasting/NachBac versus refed/NachBac), n=3-4 each, data presented as mean +/- SEM, two way ANOVA tests.

(G-J) CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (G), and comparison of responsiveness to water spray between NachBac and control mice (H). (I-J) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (I), and comparison of responsiveness to water spray between NachBac and control mice (J).Gray traces showed Ca2+ independent signal for system stability and red ones showed Ca2+ dependent signals for neuron activity, N=5-7 each, males, data presented as mean +/-SEM, **p<0.0001 in both (H and J), unpaired Student's t tests.

Data information: All mice were fed chow unless otherwise noted. 3V: the third ventricle; downward arrows indicating onset of stimuli or Dex injections. Scale bar=200µm.
Disrupted Hypothalamic CRH Neuron Responsiveness Contributes to Diet-induced Obesity (2020)
Figure 4

Figure 4. Clamping PVH CRH neuron activity at low levels disrupted neuron responsiveness.

(A-C) CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors and AAV-Flex-GFP at 7-8 weeks of age and recording was made at least 4 weeks after viral delivery on neurons with Kir2.1 (one side) or control GFP neurons (contralateral side) of the same animal (A); typical spontaneous neuron activity recorded in GFP controls (B, top panel) and Kir2.1 neurons (B, bottom); and comparison in resting membrane potentials between the two groups (C, **p<0.0001, unpaired Student's t test).

(D-F) CRH-Cre male mice were injected with either AAV-Flex-mCherry or AAV-EF1a-DIO-Kir2.1-P2A-dTomato to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing mCherry or Kir2.1 (mCherry in the top 2 rows and Kir2.1 in the bottom 2 rows of the second colomn), c-Fos expression (green in the first colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left. (E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p=0.0001: fasting versus refed; *p=0.0497: refed versus refed/Kir2.1; **p=0.0025: fasting/Kir2.1 versus refed Kir2.1) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; p=0.9586: fasting/Kir2.1 versus refed/Kir2.1), n=3-4 each, two way ANOVA tests. All data presented as mean +/- SEM.

(G-J) CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in Kir2.1 mice (G), and comparison of responsiveness to water spray between Kir2.1 and control mice (H). (I-J) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in Kir2.1 mice (I), and comparison of responsiveness to water spray between Kir2.1 and control mice (J).Gray traces showed Ca2+ independent signal for system stability and red ones showed Ca2+ dependent signals for neuron activity, N=5 each, males, data presented as mean +/-SEM, **p<0.0001 in both (H and J), unpaired Student's t tests.

Data information: All mice were fed chow unless otherwise noted. 3V: the third ventricle; downward arrows indicating onset of stimuli or Dex injections. Scale bar=200µm.
Disrupted Hypothalamic CRH Neuron Responsiveness Contributes to Diet-induced Obesity (2020)
Figure 5

Figure 5. Clamping PVH CRH neuron activity at low levels causes diet-induced obesity.

(A-I) CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A, *p<0.0001, unpaired Student's t test), measurements of fat mass (B, **p=0.0006, unpaired Student's t test) and lean mass (C, p=0.3728, unpaired Student's t test) at 15 weeks after viral delivery. (D-F) Measurements of energy expenditure during the 7th week after viral delivery. Comparison of real time O2 consumption traces during the 4-day measurement period (D), of average O2 consumption between the 2 groups (E, *p=0.0007, unpaired Student's t test), and of the difference between day and night periods between the 2 groups (F, p=0.0791, unpaired Student's t test). (G-I) Measurements of feeding during the 7th week after viral delivery. Comparison of real time feeding c traces during the 4-day measurement period (G), of average food intake between the 2 groups (H, **p=0.0019, unpaired Student's t test), and of the difference in food intake between day and night periods between the 2 groups (I, *p=0.0363, unpaired Student's t test). n=6 each. All data presented as mean +/- SEM.

Expanded View Figure Lengends: