Dsg3 deficiency in keratinocytes does not dramatically affect localization or solubility of other desmosomal components, but does compromise cell-cell adhesion.

<p>A) Immunofluorescence analysis to examine localization of various desmosomal components in <i>Dsg3+/+, Dsg3+/−, Dsg3−/−</i> mouse keratinocyte monolayers after 48 hours of treatment with 2 mM Ca²⁺. DAPI is used as a nuclear marker (abbreviations: Dsg3 =  Desmoglein 3, Dsg1+2 =  Desmoglein 1 and 2, Dsg1 =  Desmoglein1, Ecad =  E-cadherin, Pkp3 =  Plakophilin 3, Pg =  Plakoglobin, Dsp1+2 =  Desmoplakin 1 and 2). B) Western blot analysis showing both the Triton X-100-soluble and urea-only soluble fractions of <i>Dsg3+/+</i> and <i>Dsg3−/−</i> mouse keratinocyte monolayers after 48 hours of treatment with 2 mM Ca²⁺. 1 and 2 denote two different keratinocyte samples. Gapdh serves as a loading control for the Triton X-100-soluble pool, while Keratin 14 serves as a loading control for the urea fraction. C) Graph indicating the average percentage (+/− SD) of fragments released from <i>Dsg3−/−</i> keratinocyte monolayers after mechanical stress relative to wild-type controls in a mechanical dissociation assay. Experiments were performed in triplicate. p<0.0001, Student’s t-test.</p

Dsg3 deficiency does not affect UVB-induced apoptosis or immune cell infiltration <i>in vivo.</i>

<p>A) Graph indicating the average number of cleaved Caspase 3 (CC3) positive cells +/− SD per cm of epidermis in <i>Dsg3+/+</i> and <i>Dsg3−/−</i> mice 24 hours after 2.5 kJ/m² UVB. n = 3 for each condition. B) Representative images of cleaved Caspase 3 immunohistochemistry in the epidermis of mice analyzed in A). Arrows indicate the apoptotic cells. C) Graph indicating the average number of mast cells +/− SD in the skin of <i>Dsg3+/+</i> and <i>Dsg3−/−</i> mice after 52 weeks of UVB treatment. n = 12 for <i>Dsg3+/+</i> mice and n = 5 for <i>Dsg3−/−</i> mice. D) Representative toluidine blue staining of the skin of <i>Dsg3+/+</i> and <i>Dsg3−/−</i> mice after 52 weeks of UVB treatment. Arrows indicate mast cells stained by toluidine blue. Insets show higher magnification images of toluidine blue-stained skins, highlighting mast cells.</p

Dsg3 does not affect tumor incidence, tumor volume, or tumor grade in UVB-induced skin carcinogenesis.

<p>A) Representative hematoxylin and eosin (H and E)-stained sections of <i>Dsg3+/+</i> and <i>Dsg3−/−</i> mouse tongue epithelium. Arrow indicates the presence of blisters in the <i>Dsg3−/−</i> tongue epithelium. B) Photograph of an adult <i>Dsg3−/−</i> mouse, showing hair loss typical of <i>Dsg3</i> deficiency. C) Schematic diagram illustrating the experimental design and timeline for the UVB-induced SCC model used. D) Kaplan-Meier analysis of tumor-free survival of <i>Dsg3+/+</i> and <i>Dsg3−/−</i> mice subjected to chronic UVB treatment. p = 0.17, log rank test. E) Graph indicating the tumor volume in each <i>Dsg3+/+</i> (24 tumors collected) and <i>Dsg3−/−</i> (8 tumors collected) mouse after 52 weeks of UVB treatment. p = 0.65, Student’s t-test. F) Histological analysis by H and E staining, showing the percentages of sarcomas and squamous cell carcinomas among the tumors observed in mice of each genotype. A representative H and E-stained sarcoma is shown. G) Analysis of the percentages of SCCs of different grades in <i>Dsg3−/−</i> and <i>Dsg3+/+</i> mice. Photographs show representative H and E-stained SCCs with different levels of differentiation (highly, moderately, and poorly differentiated).</p

Dsg3 facilitates transformed keratinocyte allograft tumor growth.

<p>A) Experimental design for allograft tumor assays. B) Western blot analysis of <i>p53−/−;Dsg3+/−</i> and <i>p53−/−;Dsg3−/−</i> mouse keratinocytes transduced with HRasV12 lentiviruses confirms efficient HRasV12 expression relative to uninfected <i>Dsg3+/−</i> mouse keratinocytes. C) Graph displaying the volume of each tumor formed five weeks after implantation of <i>HRasV12;p53−/−;Dsg3+/−</i> (n = 16) and <i>HRasV12</i>;<i>p53−/−;Dsg3−/−</i> (n = 16) keratinocytes. p = 0.018, Student’s t-test. D) Representative hematoxylin and eosin (H and E)-stained sections of <i>HRasV12;p53−/−;Dsg3+/−</i> and <i>HRasV12;p53−/−;Dsg3−/−</i> tumors.</p

Rb inactivation does not change the mRNA expression or protein levels of cell cycle markers in MYC-induced HCC.

<p><b>A.</b> RT-qPCR for several cell cycle genes on RNA from <i>MYC</i> (black, n = 3) and <i>MYC/Rb</i> (grey, n = 3) mutant HCC. <i>Rb^lox/lox MYC^Off</i> non-tumor liver is used as a control (CTRL) (white, n = 2). There is not statistical difference for any of these gene expression levels between <i>MYC</i> and <i>MYC/Rb</i> tumors. <b>B.</b> Representative immunoblot analysis for several cell cycle markers on protein extracts from <i>MYC</i> and <i>MYC/Rb</i> mutant HCC compared to wild-type liver. The concentration of the protein extracts was quantified using a Bradford assay and similar amounts of proteins were loaded in each lane. Actin serves as a loading control; note that CTRL liver cells express less Actin per µg of protein extract than tumor cells.</p

MYC activation and Rb deletion drive HCC development in the liver of adult mice.

<p><b>A.</b> Strategy to produce <i>MYC</i> and <i>MYC/Rb</i> mutant tumors using <i>Rb^lox/lox</i> and TRE-<i>MYC</i> LAP-<i>tTA</i> mice. MYC is activated in the liver by the removal of doxycycline from the drinking water while <i>Rb</i> is specifically deleted in the liver by splenic injection of Ad-Cre. Ad-GFP is used as a control. <b>B.</b> RT-qPCR analysis of <i>MYC</i> RNA levels in control livers (<i>Rb^lox/lox MYC^Off</i>, n = 2) and in <i>MYC</i> and <i>MYC/Rb</i> mutant tumors (n = 3). <b>C.</b> Immunoblot analysis of MYC protein levels in control livers (<i>MYC^Off</i>) and in <i>MYC</i> and <i>MYC/Rb</i> mutant tumors (two independent tumors each, T1 and T2). Actin serves as a loading control. <b>D.</b> Genomic PCR analysis for the deleted allele of <i>Rb</i> (<i>Rb^Δ</i>) using DNA from <i>MYC</i> and <i>MYC/Rb</i> mutant tumors. <i>Actin</i> serves as a positive PCR control. <b>E.</b> RT-qPCR analysis of <i>Rb</i> RNA levels in <i>MYC</i> (black, n = 3) and <i>MYC/Rb</i> (grey, n = 3) mutant tumors.</p

Survival analysis of Myc and Myc/Rb mutant mice.

<p><b>A.</b> Kaplan-Meier survival analysis of <i>MYC</i> (n = 27) and <i>MYC/Rb</i> (n = 19) mutant mice when both tumorigenic events were triggered simultaneously (time 0) in adult mice (8–12 weeks after birth) by removal of doxycycline and Ad-Cre infection. <b>B.</b> Survival analysis of <i>MYC/Rb</i> mutant mice developing HCC when both tumorigenic events were triggered simultaneously in adult mice (8–12 weeks after birth) (n = 19) and when <i>Rb</i> deletion by Ad-Cre was performed 4 weeks before MYC activation (8–12 weeks and 12–16 weeks after birth, respectively) (n = 15); time 0 is the time of MYC activation.</p

Rb inactivation does not change the histology and differentiation status of MYC-induced HCC.

<p><b>A.</b> Histology of non-tumor liver tissue from <i>MYC</i> mutant mice (left) and tumor tissue from <i>MYC</i> (center) and <i>MYC/Rb</i> (right) mutant mice. <b>B.</b> RT-qPCR analysis of <i>Albumin</i> (a marker of hepatocytes), <i>CK19</i> (a marker of bile ducts), and <i>Afp</i> (a marker of HCC) in control livers (white, n = 2), <i>MYC</i> mutant tumors (black, n = 3), and <i>MYC/Rb</i> mutant tumors (grey, n = 3). Note that the p-value for <i>Afp</i> between control livers and <i>MYC/Rb</i> mutant tumors is 0.06, just below the significance threshold.</p

Additional file 1: of Non-inflammatory tumor microenvironment of diffuse intrinsic pontine glioma

Table S1. Patient characteristics of early post-mortem DIPG autopsy cases. (XLSX 55 kb

Additional file 6: of Non-inflammatory tumor microenvironment of diffuse intrinsic pontine glioma

Figure S4. DIPG cells do not express significant levels of cytokines (a-b) FPKMs of cytokine (left), chemokine (middle) and other factors (right) expressed by patient-derived DIPG cell cultures (a) or in bulk primary DIPG tissue (b) Horizontal line represents FPKM = 5 (c) Violin plots of single-cell DIPG expression of cytokines, chemokines, and other factors from primary DIPG biopsy tissue. Horizontal line represents log(tpm + 1) = 1. (TIF 1442 kb