EMBO/soda-nel huggingface dataset:
Loss of AhR potentiates FoxM1 signaling to enhance self-renewal of colonic stem/progenitor cells (2020)
Figure 5

Figure 5. Effects of AhR KO on colonic cell proliferation.

(A and B) Proliferating Lgr5+ stem cells marked by EdU in the distal colon from AhR WT (GT) and KO (HGT) mice. Arrowhead and star symbols denote proliferating stem cells (GFP+EdU+) and static stem cells (GFP+EdU-), respectively, during a 2 h EdU pulse, n=3 per treatment.

(C) Total number of EdU+ cells per crypt in the distal colon of AhR WT and KO (HGT) mice, n=3 mice per treatment. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.

(D&E) Crypt cell proliferative zone analysis in AhR WT and KO (HGT) mice, presented as the distance from the crypt base to the highest EdU+ cell divided by total crypt length. Red lines between crosses show the measurement of distance. Each symbol represents one crypt, n=3 mice per treatment, scale bar 20 µm. Grey line represents mean.

(F&G) AhR KO promoted cell proliferation 5 d post DSS treatment. AhR WT (AhRf/f [H], n=4) and AhR KO (AhRf/f X Villin-Cre [HV], n=4) mice were used. Data represent mean ± SEM, quantified as the ratio of the EdU+ area relative to the DAPI+ area using KeyenceTM software.

(H) Increased cell proliferation (EdU+ cells) observed in AhR KO vs WT organoids derived from HGC and GC mice, respectively (n=6 from 3 mice per group).

(I) Effect of AhR KO on cell cycle S and G2-M phases (n=3 mice per group).

(J). FoxM1 inhibition reduced the number of colonic stem cells and cell proliferation. Mice were intraperitoneally injected with the FoxM1 inhibitor Thiostrepton (50 mg/kg) for 2 consecutive days, n=80~100 crypts from 4 mice per group. Scale bar, 50 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles. Bars with different letters are significantly different (p<0.05).

Data information: Data were shown as mean ± SD in (B)(G)(H) and (I). Two tailed Student's t-test was performed in (B)(C)(E)(G)(H) and (I). A one-way ANOVA with Tukey's multiple comparisons test was carried out in (J).
Loss of AhR potentiates FoxM1 signaling to enhance self-renewal of colonic stem/progenitor cells (2020)
Figure 6

Figure 6. AhR KO promotes colorectal tumor growth in AOM/DSS treated mice.

(A) Schematic regimen for AOM/DSS-induction of colon tumorigenesis. At 8~12 wk of age, AhR WT (H, n=10) and KO (HV, n=10) mice were treated with a single dose of AOM (10 mg/kg) by s.c injection, followed by 3 cycles of 2% DSS in the drinking water for 5 d. Mice were terminated 6 wk after the third cycle of DSS.

(B) Representative colon images from AhR WT and AhR KO mice following AOM/DSS treatment. Scale bar, 9 mm.

(C) H&E staining of representative colon tumor sections from AhR WT and KO mice, scale bar 200 μm.

(D) Incidence of colon adenomas, adenocarcinomas and both combined (tumor) in AhR WT and KO mice, *p<0.05.

(E, F) (E) Adenoma and (F) adenocarcinoma multiplicity in AhR WT and KO mice.

(G) Tumor volume in AhR KO and WT mice. Grey line indicates mean.

(H) FoxM1 mRNA expression in normal (uninvolved) crypts adjacent to colonic tumors. Images were quantified and expressed as FoxM1 positive area per crypt (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.

(I) FoxM1 mRNA expression in colon tumors. FoxM1 mRNA ISH area was averaged per tumor on each slide (n=5 mice per group). Scale 200 µm. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.

Data information: Data were shown as mean ± SD in (E)(F) and (I). Chi Square analysis was performed in (D). One-tailed Student's t-test was determined in (E) and (F). Mann-Whitney U test was utilized in (G). Two-tailed Student's t-test was carried out in (H) and (I).
Loss of AhR potentiates FoxM1 signaling to enhance self-renewal of colonic stem/progenitor cells (2020)
Figure 7

Figure 7. Assessment of AhR signaling in human colonic organoids.

(A-B) mRNA expression of human colonic stem cell markers LGR5 and OLFM4, n=5 or 6 independent human samples. Paired student t-test.

(C) Representative images of LGR5 ISH in human organoids treated with DMSO or TCDD. Scale bar 50 µm.

(D-G) mRNA expression of target genes, n=5 or 6 independent human samples. Paired student t-test.

(H) Representative immunoblots for FoxM1 and PLK1 protein derived from organoids treated with DMSO or TCDD from n=6 independent samples. Total protein was used as a loading control.

(I) Determination of luciferase reporter activity of human FoxM1 in SW48 and Caco2 cell lines treated with DMSO or TCDD. n=3-4 per group.

(J) FOXM1 and CYP1A1 mRNA expression in different AhR WT and CRISPR KO clones treated with TCDD for 1 d. C56 and C61 designate different human subjects.

(K) Validation of CRISPR KO clones was assessed by PCR amplification using primers flanking the targeted AhR exon. Subsequent sequencing revealed indels at the expected locations.

(L) The association of FoxM1 and AhR mRNA expression in human normal colon biopsies (n=304). AhR low (n=152) is defined as below its median expression; AhR high (n=152) is defined as above its median expression. Dashed line indicates median and dotted lines indicate 25th/75th quartiles.

(M) Correlation between FoxM1 and Cyp1b1 mRNA expression in human normal colon biopsies (n=303) analyzed by Spearman rank correlation.

(N) Representative brightfield images of human colonic organoids from sorted live cells at day 6, scale bar 200 μm.

(O, P) (O) Human organoid forming efficiency, and (P) organoid diameter from sorted individual cells from human colonic organoids in the presence of DMSO, TCDD and CH223191 (10 μM) for 6 d. n=5 per group, 3 independent experiments. Grey line indicates mean in (P). Treatments not sharing a common letter are significantly different, p<0.05.

(Q) Representative brightfield images of human colonic organoids treated with DMSO or FDI-6. FoxM1 inhibition suppressed human colonic organoid growth in a dose-dependent manner, scale bar 200 µm.

Data information: Data were shown as mean ± SD in (I) and (O). Paired Student's t-test was performed in (A)(B)(D-G). Two-tailed Student t-test was carried out in (I) and (L). One-way ANOVA with Tukey's multiple comparisons was determined in (O) and (P).

EXPANDED VIEW LEGENDS
Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells (2020)
Figure 1

Figure 1: USP28 is highly abundant in human squamous tumours and correlates with poor prognosis.

Expression of USP28 (left) and TP63 (right) in human lung squamous cell carcinomas (SCC, n=498), adenocarcinomas (ADC, n=513) and normal non-transformed tissue (normal SCC=338, normal ADC=348). Xena UCSC software. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots.

Correlation of mRNA expression of USP28 and TP63 in lung SCC (left, n=498), ADC (right, n=513) and normal, non-transformed tissue (normal SCC=338, normal ADC=348). R: Spearmans correlation coefficient; m=Slope. Xena UCSC software.

IHC analysis of USP28 and ∆Np63 protein abundance in lung cancer and non-transformed human samples (n=300). The staining intensity was quantified in arbitrary units from 0 up to 3 by three independent pathologists. In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR and outliers are marked as dots. p-values were calculated using two-tailed t-test.

Kaplan-Meier estimator of NSCLC patients stratified by USP28 (left, n=1145) and TP63 (right, n=1926) expression. p-values were calculated using log-rank test. HR: hazard ratio. Kmplot software.

Kaplan-Meier estimator of lung SCC patients stratified by USP28 expression (n=271). The p-value was calculated using a logrank test. HR: hazard ratio. Kmplot software.

Data information: *p-value < 0.05; **p-value < 0.01; *** p-value < 0.001; **** p-value < 0.001. See also Figure EV1 and Appendix Table S3 (Exact P-values and statistical test used)
Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells (2020)
Figure 2

Figure 2: ∆Np63 stability is regulated by USP28 via its catalytic activity.

Co-immunoprecipitation of exogenous HA-USP28 and FLAG-ΔNp63 in HEK293 cells. Either HA-USP28 or FLAG-ΔNp63 were precipitated and blotted against FLAG-ΔNp63 or HA-USP28. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control).

Ni-NTA His-ubiquitin pulldown in control transfected or HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pull down. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.

Ni-NTA His-ubiquitin pulldown K48 or K63 in control and HA-USP28 overexpressing HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using VINCULIN for normalization.

Co-immunoprecipitation of exogenous FLAG-USP28 C171A and FLAG-ΔNp63 in HEK293 cells. ΔNp63 was precipitated and blotted against FLAG-USP28 or ΔNp63. The input corresponds to 10% of the total protein amount used for the IP (ACTIN as loading control).

Ni-NTA His-ubiquitin pulldown in control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells, followed by immunoblot against exogenous ΔNp63. The input corresponds to 10% of the total protein amount used for the pulldown. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.

CHX chase assay (100ug/ml) of control, FLAG-USP28 or FLAG-USP28 C171A transfected HEK293 cells for indicated time points. Representative immunoblot analysis of FLAG (USP28) and ∆Np63 as well quantification of relative protein abundance (ACTIN as loading control).

Immunoblot of USP28 and ∆Np63 in transfected HEK293 cells upon treatment with either DMSO or indicated concentrations of PR-619 for 24 hours. Relative protein abundance was calculated ACTIN as loading control.

Data information: Western blots shown are representative of 3 independent experiments (n=3). All quantitative graphs are represented as mean ± SD of three experiments (n=3). p-values were calculated using two-tailed t-test statistical analysis; *p-value < 0.05; **p-value < 0.01; see also Appendix Figure S1 and Appendix Table S3 (Exact P-values and statistical test used).
Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells (2020)
Figure 3

Figure 3: USP28 regulates ∆Np63 protein stability in SCC tumour cell lines

Immunoblot of endogenous USP25, USP28 and ∆Np63 immunoprecipitated (IP) from A-431 cells and co-precipitated ∆Np63 or USP28. Beads were coupled to specific USP25, USP28 and ∆Np63 antibodies or non-specific rabbit IgG as control. The input corresponds to 10% of the total protein amount used for the IP (TUBULIN as loading control).

Inducible depletion of USP28 in A-431 upon treatment with doxycycline (1µg/ml) for 96h, western blot (left, VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN (right) was performed.

Tandem ubiquitin binding entity (TUBE) pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon DOX depletion of USP28. Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.

Cycloheximide (CHX) chase assay (100µg/ml) of control or inducible sh-USP28 A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot (left, VINCULIN as loading control) of USP28 and ∆Np63 and quantification of relative protein abundance (right).

Doxycycline induced murine USP28 overexpression (EtOH or 1ug/ml dox for 96 hours) in A-431 cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28 and ∆Np63. For qPCR, human USP28 and murine USP28 (mUSP28) primers were used. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping).

TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon overexpression of mUSP28 for 96 hours (EtOH or 1µg/ml dox). Relative ubiquitination of the representative immunoblot was calculated using ACTIN for normalization.

CHX chase assay (100µg/ml) of control or inducible mUSP28 overexpressing A-431 cell line (EtOH or 1µg/ml dox) for indicated time points. Representative immunoblot analysis of USP28 and ∆Np63 as well quantification of relative protein abundance (VINCULIN as loading control).

Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63 and USP28 protein abundance in LUDLU-1adh (VINCULIN as loading control), followed by qPCR analysis of USP28 and ∆Np63 expression relative to ACTIN.

Doxycycline induced mUSP28 overexpression (EtOH or 1µg/ml dox for 96 hours) in LUDLU-1adh cells followed by immunoblot (VINCULIN as loading control) and qPCR analysis of USP28, mUSP28 and ∆Np63. Relative mRNA was calculated using ∆∆Ct analysis for human USP28 and ∆Ct for mUSP28 (ACTIN as housekeeping for the analysis).

Data information: Western blots shown are representative of three independent experiments (n=3). All quantitative graphs are represented as mean ± SD of three experiments (n=3). p-values were calculated using two-tailed t-test statistical analysis; *p-value < 0.05; See also Appendix Figure S2 and Appendix Table S3 (Exact P-values and statistical test used).
Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells (2020)
Figure 5

Figure 5: SCC depend on USP28 expression for tumour induction and maintenance.

Schematic diagram of CRISPR/Cas9 mediated tumour modelling and targeting of p53∆; Lkb1∆; KRasG12D(KPL) or Usp28∆; p53∆; Lkb1∆:KRasG12D(KPLU) mouse lines in Rosa26Sor-CAGG-Cas9-IRES-GFP mice,

Representative haematoxylin and eosin (H&E) staining of tumour bearing animals 12 weeks post intratracheal infection. Boxes indicate highlighted tumour areas in (C) (a, b, a' and b'). Scale bar = 2000µm; nKPL= 6 and nKPLU = 5. H=heart.

Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 abundance in KPL (n=6) and KPLU (n=5) lung tumours. Scale bar: 20µm.

Quantification of % tumour area (normalised to total lung area) in KPL (n=6) and KPLU (n=5) animals.

Quantification of lung tumour numbers in KPL (n=6) and KPLU (n=5) animals.

Immunoblot of endogenous LKB1, USP28 and ∆Np63 in KPL and KPLU tumours. (VINCULIN as loading control). Western blot shown are representative of three independent experiments (n=3).

Representative immunoblot of endogenous NOTCH1, c-MYC and c-JUN in KPL and KPLU (ACTIN as a control). n=3

Kaplan-Meier survival curves comparing KPL (n=6) and KPLU (n=5) animals upon AAV intratracheal infection. The p-value was calculated using a log-rank test.

Schematic diagram of generating murine SCC tumour cells from the classic KP (lsl-KrasG12D: p53fl/fl) mouse model;

Representative qPCR of SCC and ADC marker expression in two independent KP lung tumour clones, resulting in KPADC and KPSCC; (ACTIN served as loading control). Quantitative graph is represented as mean ± SD of three experiments (n=3).

Immunoblot against endogenous USP28 in the murine KPSCC cell line upon targeting with two independent shRNA sequences. ACTIN served as loading control.

Schematic diagram of orthotopic re-transplantation of cell lines into the lung of recipient wild type C57BL/6J mice.

Representative haematoxylin and eosin (H&E) images of tumour bearing animals 8 weeks post intratracheal transplantation of 2 × 105 cells/animal. KPSCC sh-NTC (n=3); KPSCC sh-USP28 (n=3), Scale bar = 5000µm

Quantification of % tumour area (top, normalised to total lung area) and lung tumour numbers (bottom) on KPSCC sh-NTC (n=6) and KPSCC sh-USP28 (n=6) animals.

Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 and GFP abundance in KPSCC sh-NTC (n=3) and KPSCC sh-USP28 (n=3) lung tumours. Scale bar =50 µm

Data information: In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR. p-values were calculated using two-tailed t-test. **p-value < 0.01; ****p-value < 0.0001. See also Figure EV3 and Appendix Table S3 (Exact P-values and statistical test used).
Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells (2020)
Figure 6

Figure 6: ∆Np63-driven SCC cells of various tissues are vulnerable to USP28 depletion

Analysis of occurring TP63 genetic alterations in lung squamous (LUSC), cervical (CESC), oesophagus (ESCA), head and neck (HNSC) and pancreatic (PAAD) tumours. Cbioportal.

Expression of TP63 (left) and USP28 (right) in human Lung (n=498), Cervix (n=254), Oesophagus (n=96) and HNSC (n=522) SCC tumours and normal non-transformed tissue (nLung=338 nCervix=3, nOesophagus=11 and nHNSC=44). In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR. p-values were calculated using two-tailed t-test statistical analysis. Xena UCSC software.

Correlation of mRNA expression of KRT14 / KRT7 and TP63 in ADC and SCC tumours for Lung (nADC=513 and nSCC=498), Cervix (nADC=47 and nSCC=254) and Oesophagus (nADC=89 and nSCC=96). Blue dots: ADC; Red dots: SCC; R= Spearmans correlation coefficient; m: Slope. Xena UCSC.

Spearman's Correlation values of the gene list '' Squamous cancer markers'' and TP63, c-JUN, c-MYC or NOTCH1 in Lung (n=1011), Cervix (n=301), Oesophagus (n=185), HNSC (n=522) and PAAD (n=178) tumours. Intense blue: Low correlation; Intense red: Strong correlation. Gepia software.

Immunoblot of control (sh-NTC) and two independent shRNA targeting USP28 (sh-USP28#1 and #2) for ∆Np63, KRT14 and USP28 protein abundance in H1299, LUDLU-1adh, HELA, SiHa, Ca Ski, PANC-1 and BXPC-3 (ACTIN as loading control).

Cells were seeded at equal cell density and counted after five days, Brigth field images of control or sh-USP28#2 infected H1299, LUDLU-1adh, Hela, CaSKI, PANC-1 and BXPC-3 cells before quantification. Scale bar = 30µm

Relative number of H1299, LUDLU-1adh, Hela, CaSKI, SiHa, PANC-1 and BXPC-3 sh-USP28#2 cells compared with sh-NTC control cells. p-values were calculated using two-tailed t-test statistical analysis. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63.

Data information: All quantitative graphs are represented as mean ± SD of three experiments (n=3). p-values were calculated using two-tailed t-test statistical analysis; *p-value < 0.05, **** p-value < 0.0001. See also Appendix Figure S3 and Appendix Table S3 (Exact P-values and statistical test used).
Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells (2020)
Figure 7

Figure 7: Pharmacological inhibition of USP28 with the first-generation inhibitor AZ1 regulates ΔNp63 stability via de-ubiquitylation and shows a selective antiproliferative response of SCC cells

Immunoblot of endogenous USP28 in A-431 cells upon treatment with warheads (WH) and either DMSO or indicated concentrations of AZ1 (USP28 Inh.) for 24 hours. USP28 Upper band: Active USP28; USP28 lower band: Inactive USP28, VINCULIN as loading control.

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control. TP63 and USP28 half maximal inhibitory protein abundance (IC50) was calculated. Graphs are represented as mean ± SD (n=3).

TUBE pulldown of endogenous ubiquitylated ∆Np63 in A-431 cells upon treatment with either DMSO or indicated concentrations of AZ1 for 24 hours. VINCULIN served as loading control for relative Ubiquitination.

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in A-431 cells treated for 18 hours with either DMSO or 15µM AZ1 followed by 6 hours of treatment with 20µM proteasomal inhibitor MG-132 (20µM) or DMSO. VINCULIN served as loading control.

A-431 cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. Scale bar = 250µm. n= 30 fields analysed

Immunoblot of endogenous USP28, ∆Np63 and KRT14 in LUDLU-1adh, H1299, Ca Ski, Hela and SiHa cells treated for 24 hours with either DMSO or indicated concentrations of AZ1. VINCULIN served as loading control

LUDLU-1ADH, H1299, Ca Ski, Hela and SiHa cells were seeded at equal cell density and cultured in the presence of either DMSO, 1µM, 10µM or 30µM AZ1 for 48h. Scale bar for LUDLU-1adh and H1299 = 500µm. Scale bar for Ca Ski, Hela and SiHa = 250µm. SiHa* = Notably, the human SCC cell line SiHa was negative for ΔNp63.

Number of cells were quantified with the Operetta imaging system using Hoechst staining. 50% growth inhibition (GI50) was calculated. n=30 fields analysed

Data information: See also Figure EV4.
Maintaining protein stability of ∆Np63 via USP28 is required by squamous cancer cells (2020)
Figure 8

Figure 8: Pharmacologic inhibition of USP28 with AZ1 reduces tumour growth in an orthotopic model of lung SCC tumours.

Immunofluorescence (IF) staining against endogenous ∆Np63 in KP and KPL cell lines (blue: DAPI [staining of nuclei] and red/magenta: ∆Np63), Scale bar = 32µm.

Schematic diagram of AZ1 treatment. After first 28 days without treatment, tumours were treated with an AZ1 dosage every 3 days until day 46.

GFP Fluorescent images of lungs treated with either PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Tumour cells are positive for GFP (green arrows). n=3

Quantification of % tumour area (normalised to total lung area) in control (n=5), 125 mg/kg AZ1 (n=4) and 375 mg/kg AZ1 (n=4) treated animals.

Representative staining of H&E and IHC for USP28 and ∆Np63 of SCC tumours from mice treated with PBS/DMSO/0,1% Tween80 or indicated concentrations of AZ1. Boxes indicate highlighted tumour areas. First line Scale bars = 500µm; Lower scale bars = 20µm. n=3

∆Np63 staining intensity of lung tumours treated with PBS/DMSO/0,1%Tween80 or indicated concentrations of AZ1. nControl = 33712 cells ; nAZ1 125mg/kg = 7382 cells; nAZ1 375mg/kg = 4967 cells. Cells were analysed from three independent animals.

Schematic model

Data information: In box plots, the centre line reflects the median, the cross represents the mean and the upper and lower box limits indicates the first and third quartile. Whiskers extend 1.5x the IQR. p-values were calculated using two-tailed t-test. *p-value < 0.05; ****p-value < 0.0001. See also Figure EV5 and Appendix Table S3 (Exact P-values and statistical test used).
Loss of TREM2 rescues hyperactivation of microglia, but not lysosomal deficits and neurotoxcity in models of progranulin deficiency (2021)
Figure 1

Figure 1 - TSPO-PET imaging indicates rescue of microglial hyperactivation in Double-/- mice

A. Axial slices as indicated below (A1, A2, A3) show %-TSPO-PET differences between Grn-/-, Trem2-/- or Double-/- mice and WT at the group level. Images adjusted to an MRI template indicate increased microglial activity in the brain of Grn-/- mice (hot color scale), compensated microglial activity in the brain of Double-/- mice and decreased microglial activity in the brain of Trem2 -/- mice (cold color scale), each in contrast against age-matched WT mice.

B. Scatter plot illustrates individual mouse TSPO-PET values derived from a whole brain volume of interest. 8-15 female mice per group at an average age of 11.1 ± 1.6 months (Grn-/- (n = 8), Trem2-/- (n = 9), Double-/- (n = 10), WT (n= 15)). Data represent mean ± SD. For statistical analysis one-way ANOVA with Tukey post hoc test was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ns, not significant.