Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Microarray heatmap shows P2ry14 receptor expression in p75 + /EGFR + SCP-like tumor-initiating cells derived from human plexiform neurofibroma tumor cells compared to p75 + /EGFR - SCP-like cells. ( B ) Western blot of human Schwann cells and neurofibroma SCP shows the latter has a 1.9-fold increase in P2ry14 protein expression. ( C ) Immunohistochemistry of human neurofibroma shows P2ry14 expression (DAB staining: brown [P2ry14 positive cells] blue [cell nuclei]). ( D ) Representative fluorescence-activated cells sorting (FACS) plot shows live sorted human plexiform neurofibroma tumor cells. ( E ) Representative FACS plot shows human plexiform neurofibroma tumor cells sorted into p75 + /EGFR + SCP-like tumor-initiating cells (pink square). ( F ) Representative FACS plot shows p75 + /EGFR + SCP-like tumor-initiating cells further sorted into p75 + /EGFR + / P2ry14 - (left, purple square) and P75 + /EGFR + / P2ry14 + (right, blue square). ( G ) Photomicrographs of human neurofibromas dissociated using FACS to yield: unsorted, p75 + /EGFR + / P2ry14 - and P75 + /EGFR + / P2ry14 + cells. ( H ) Quantification of unsorted, p75 + /EGFR + / P2ry14 - and P75 + /EGFR + / P2ry14 + cells plated in sphere medium. (n = 3; two-way ANOVA; primary: **p = 0.0057, ****p < 0.0001; secondary: *p = 0.0487; **p < 0.0024, ****p < 0.0001; tertiary: *p = 0.0321, ***p = 0.0006).

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques: Microarray, Expressing, Derivative Assay, Western Blot, Immunohistochemistry, Staining, Fluorescence

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Quantification of percent of sphere forming cells in mouse wild-type (WT) and Nf1 -/- SCPs treated with the selective P2ry14 inhibitor (300 nM 4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid [PPTN]) (primary, secondary, tertiary passage) (n = 3; two-way ANOVA; primary: *p = 0.0375, ***p = 0.0001, ****p < 0.0001; secondary: *p = 0.0101, **p = 0.0015, ***p = 0.0009; tertiary: *p = 0.0101, **p = 0.0050, ***p = 0.0005). ( B ) P2ry14 mRNA expression in WT and Nf1 -/- E12.5 mouse SCP treated with sh non-target (shNT) control and sh P2ry14 (****p < 0.0001). ( C ) Western blot of WT and Nf1 -/- SCPs treated with shNT and sh P2ry14 showing P2ry14 knockdown. WT sh P2ry14 show a 0.4-fold decrease of P2ry14 protein compared to WT shNT. Nf1 -/- sh P2ry14 show a 0.5-fold decrease compared to Nf1 -/- . ( D ) Quantification of percent of sphere forming cells in mouse WT and Nf1 -/- SCPs treated with shNT and sh P2ry14 (n = 3; two-way ANOVA; primary: *p = 0.0288, ****p < 0.0001; secondary: **p = 0.0029,***p = 0.0005, ****p < 0.0001; tertiary: *p = 0.0154, ***p = 0.0007). ( E ) Western blot of WT and Nf1 -/- Schwann cell (SC) spheres shows changes in pPKA substrate phosphorylation after sh P2ry14 knockdown. WT sh P2ry14 shows a 1.43-fold increase in pPKA after P2ry14 knockdown. Nf1-/- cells have a 1.31-fold increase in pPKA expression after P2ry14 knockdown. ( F ) Quantification of percent of sphere forming cells in Nf1 -/- mouse SCPs treated with 1 µM rolipram or 300 nM PPTN (n = 3; two-way ANOVA; primary: ***p = 0.0002, ****p < 0.0001; secondary **p = 0.0030, ****p < 0.0001; tertiary: *p = 0.0476, ***p = 0.0004).

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques: Expressing, Control, Western Blot, Knockdown, Phospho-proteomics

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Photomicrographs of WT and Nf1 -/- mouse Schwann cell (SC) spheres treated with the selective P2ry14 inhibitor (PPTN; 300 nM). ( B ) Photomicrographs of WT and Nf1 -/- mouse SC spheres treated with sh non-target (shNT) and sh P2ry14 (09). ( C ) Photomicrographs of NF1 -/- SCPs treated with vehicle and 1 µM of rolipram.

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques:

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Photomicrographs of WT and Nf1 -/- mouse Schwann cell (SC) spheres treated with the P2ry14 inhibitor (PPTN; 30 nM). ( B ) Photomicrographs of WT and Nf1 -/- mouse SC spheres treated with the P2ry14 inhibitor (PPTN; 100 nM). ( C ) Photomicrographs of WT and Nf1 -/- mouse SC spheres treated with the P2ry14 inhibitor (PPTN; 500 nM). (Note: for A-C , the same WT and Nf1 -/- vehicle controls photomicrographs are used for each dose). ( D ) Quantification of the percent of sphere forming cells after treatment with the P2ry14 inhibitor (PPTN; 30 nM) in WT and Nf1 -/- mouse SC spheres (two-way ANOVA: primary and secondary passage: ****p < 0.0001, tertiary passage: *p = 0.0168, **p = 0.0074). ( E ) Quantification of percent sphere forming cells after treatment with the P2ry14 inhibitor (PPTN; 100 nM) in WT and Nf1 -/- mouse SCP (two-way ANOVA: primary, secondary, and tertiary passage: ****p < 0.0001). ( F ) Quantification of percent sphere forming cells after treatment with the P2ry14 inhibitor (PPTN; 500 nM) in WT and Nf1 -/- mouse SCP; results show that this concentration was toxic to the cells (two-way ANOVA: primary, secondary, and tertiary passage: ****p < 0.0001).

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques: Concentration Assay

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Photomicrographs of WT and Nf1 -/- SCP treated with sh P2ry14 (64). ( B ) Quantification of the percent of sphere forming cells in WT and Nf1 -/- SCP after treatment with sh P2ry14 (64) (n = 3; two-way ANOVA: primary: *p = 0.0184, ****p < 0.0001; secondary: *p = 0.0270, **p = 0.0024; tertiary: *p = 0.0270, **p = 0.0078). ( C ) Photomicrographs of WT and Nf1 -/- SCP treated with sh P2ry14 (84). ( D ) Quantification of the percent of sphere forming cells in WT and Nf1 -/- SCP after treatment with sh P2ry14 (84) (n = 3; two-way ANOVA: primary: ***p = 0.0005, ****p < 0.0001; secondary: *p = 0.0358; tertiary: *p = 0.0245, **p = 0.0030). (Note: for A & C , the same WT and Nf1 -/- sh non-target (shNT) control photomicrographs are used). ( E ) WT and Nf1 -/- E12.5 mouse Schwann cells (SCs) cyclic AMP (cAMP) levels measured at baseline and after activation of adenylate cyclase (AC) via forskolin stimulation (n = 3, multiple t-test: vehicle *p = 0.0407, forskolin (5 µM) *p = 0.0045).

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques: Control, Activation Assay

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Schematic of generation of neurofibroma mice breeding of P2ry14 -/- mice with Nf fl/fl mice to obtain P2ry14 +/- ; Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ; Nf1 fl/fl ;Dhh Cre littermates after several crosses. ( B ) Genotyping confirmation of wild-type (WT) and P2ry14 knockout (KO) alleles. ( C ) Western blot of sciatic nerve and neurofibroma tumors of Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre mice show decrease in P2ry14 expression upon P2ry14 knockdown (1- to 0.1-fold decrease in sciatic nerve and 1- to 0.2-fold decrease in neurofibroma tissue). ( D ) Spinal cord (SC) immunofluorescent staining of mouse embryos at embryonic day 12.5 (E12.5) shows P2ry14 expression (β-galactosidase) co-localization with SOX-10 SCs at the dorsal root ganglion (DRG) and ventral roots (VR). ( E ) Spinal cord (SC) immunofluorescent staining of mouse embryos at E12.5 shows P2ry14 expression (β-galactosidase) co-localization with SOX-10 SCs at DRG. E1 and E2 insets show enlarged sections of the DRG. ( F ) Immunofluorescent staining of 7-month-old mouse sciatic nerve shows β-galactosidase positive staining as a confirmation of P2ry14 knock-in; co-labeling of β-galactosidase and CNPase shows that P2ry14 co-localizes with SCs (inset). ( G ) DAB staining of 7-month-old WT nerve and Nf1 fl/fl ;Dhh Cre mouse neurofibromas (DAB staining: brown [ P2ry14 positive cells] blue [cell nuclei]).

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques: Knock-Out, Western Blot, Expressing, Knockdown, Staining, Knock-In, Labeling

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Kaplan-Meier survival plot of Nf1 fl/fl ;Dhh Cre (red line; n = 8); P2ry14 +/- ; Nf1 fl/fl ;Dhh Cre (black line, n = 14); P2ry14 -/- ; Nf1 fl/fl ;Dhh Cre (blue line; n = 13); Nf1 fl/+ control (green line, n = 11) (*p = 0.0256) shows P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre have increased survival. ( B ) Representative image of gross dissection of Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre mice at 7 months of age. ( C ) Neurofibroma tumor number quantification at 7 months of age (unpaired t-test ****p < 0.0001). ( D ) Neurofibroma diameter quantification at 7 months (unpaired t-test ***p = 0.0004) (for C and D : Nf1 fl/fl ;Dhh + n = 8 mice, 48 neurofibroma tumors; P2ry14-/-;Nf1 fl/fl ;Dhh+ n = 8 mice, 11 neurofibroma tumors). ( E ) Ki67 staining of mouse dorsal root ganglion (DRG) and neurofibroma tumor tissue at 7 months of age. ( F ) Quantification of Ki67+ cells in mouse DRG and neurofibroma tumor tissue at 7 months of age (one-way ANOVA; multiple comparisons ***p = 0.0008; ****p < 0.0001). ( G ) Anti-p-PKA substrate staining in wild-type (WT), Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ; Nf1 fl/fl ;Dhh Cre mice. p-PKA substrate phosphorylation labeling co-localized with CNPase Schwann cell (SC) marker (insets).

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques: Control, Dissection, Staining, Phospho-proteomics, Labeling, Marker

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Representative image of gross dissection of Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre mice at 4 months. ( B ) Neurofibroma tumor number quantification at 4 months of age (unpaired t-test **p=0.0052). ( C ) Neurofibroma tumor diameter quantification at 4 months (unpaired t-test **p=0.0489) (for figures B and C : Nf1 fl/fl ;Dhh Cre n=5 mice, 19 neurofibroma tumors; P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre n=5 mice; 5 neurofibroma tumors). ( D ) Ki67+ staining of Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre neurofibromas at 4 months of age. ( E ) Quantification of percent of Ki67 positive cells in Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre 4-month-old mice (t-test; n.s.). ( F ) H&E staining at 4 months in Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ; Nf1 fl/fl ;Dhh Cre mice. ( G ) Ki67 immunofluorescence co-labeling with CNPase (Schwann cell [SC] marker) in 7-month sciatic nerve. ( H ) H&E tumor staining at 7 months in Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre mice.

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques: Dissection, Staining, Immunofluorescence, Labeling, Marker

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Electron micrograph of saphenous nerve of 4-month-old wild-type (WT) , Nf1 fl/fl ;Dhh Cre , P2ry14 -/- ; Nf1 fl/fl ;Dhh Cre and P2ry14 -/- mice. ( B ) Electron micrograph of saphenous nerve of 7-month-old WT , Nf1 fl/fl ;Dhh Cre and P2ry14 -/- ;Nf1 fl/fl ;Dhh Cre mice. ( C ) Remak bundle quantification at 4 months of age (n = 3; two-way ANOVA: ****p < 0.0001). ( D ) Remak bundle quantification at 7 months of age (n = 3; two-way ANOVA: **p = 0.0027, ****p < 0.0001).

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques:

Journal: eLife

Article Title: P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis

doi: 10.7554/eLife.73511

Figure Lengend Snippet: ( A ) Rolipram drug treatment experimental design. ( B ) Tumor lysates of vehicle and rolipram treated Nf1 fl/fl ;Dhh Cre mice show changes in p-PKA substrate. ( C ) Ki67 staining at 9 months of age in vehicle and rolipram treated mice. ( D ) Quantification of Ki67+ cells in vehicle treated versus rolipram treated mice (unpaired t-test: ****p < 0.0001; n = 3). ( E ) P2ry14 inhibitor (4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid [PPTN]) drug treatment experimental design. ( F ) Immunofluorescent staining of sciatic nerve of 4-month-old wild-type (WT), Nf1 fl/fl ;Dhh Cre (vehicle) and Nf1 fl/fl ;Dhh Cre (PPTN treated) shows increased p-PKA expression after PPTN treatment. ( G ) Ki67+ staining of Nf1 fl/fl ;Dhh Cre (vehicle) and Nf1 fl/fl ;Dhh Cre (PPTN treated) neurofibroma tissue. ( H ) Quantification of Ki67+ cells after PPTN treatment in neurofibroma tissue.

Article Snippet: For cell sorting, we incubated cell suspensions with anti- P2ry14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018; RRID: AB_2039847 ) on ice for 30 min, washed with PBS twice.

Techniques: Staining, Expressing