Fig 2: Effects of TC-PTP deficiency on STAT3 dephosphorylation in keratinocytes following TPA treatment.Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for STAT3 and phosphorylated STAT3 (p-STAT3). (A) 3PC keratinocytes were treated with 10, 20, 50 and 100 nM of TPA for 1 h. (B) 3PC keratinocytes were cultured in the presence of 5, 10, 20 and 50 µM Na3VO4 for 6 h. (C) 3PC keratinocyte were cultured with 50 µM of Na3VO4 for 2 h before treatment with 100 nM TPA. Cells were collected 6 h after TPA treatment. (D) 3PC keratinocytes were transfected with siRNA specific for Ptpn2. Cells were collected 1 h after TPA (100 nM) treatment. (E) 3PC keratinocytes were treated with 20, 50 and 100 nM of TPA for 1 h. Cells were collected at the indicated time after TPA treatment. (F–G) Control (scrambled) and TC-PTP-knockdown keratinocytes were treated with TPA (20 nM) for 1 h. Following TPA treatment, cells were collected at the indicated time. (G) *At 3 h of incubation, control and TC-PTP-knockdown keratinocytes were re-treated with TPA for 1 h. Cells were then collected 4 h after second TPA treatment.

Fig 3: Generation of epidermal-specific TC-PTP knockout mice.(A) Schematic diagram of targeted disruption of the Ptpn2 gene within the epidermis using the Cre-LoxP system. (B) PCR analysis of the Ptpn2 deletion using genomic DNA obtained from the mouse tail. Allele-specific PCR was performed using the three primers described in Materials and Methods to evaluate the efficiency of the Ptpn2 deletion. (C) Western blot analysis of TC-PTP in the epidermis from K14Cre.Ptpn2w/w and K14Cre.Ptpn2fl/fl mice. Epidermis was collected and total cell lysates from both genotypes were resolved by SDS-PAGE and immunoblotted with antibodies specific for TC-PTP. (D) Immunohistochemical staining of TC-PTP in the epidermis from both genotypes. (E) Western blot analysis of TC-PTP in non-epidermal tissues from K14Cre.Ptpn2fl/fl mice. Brain, heart, liver, lung, and stomach were collected from K14Cre.Ptpn2fl/fl mice and total cell lysates were prepared.

Fig 4: Model: TRAF3 suppresses GITR expression and signaling in T cells. GITR transcription is stringently regulated by activity of NF?B2. In normal T cells (left panel), the major upstream kinase for activation of NF?B2, NIK is constitutively polyubiquitinated (represented as chain of black dots attached to NIK) and targeted for degradation by an E3 ubiquitination complex containing TRAF2, TRAF3, and cIAP. Engagement of GITR with GITRL leads to recruitment of TRAF3 and disassembly of the TRAF2/TRAF3/cIAP E3 complex, which releases NIK and activates downstream NF?B2. In addition, phosphatases PTPN2 and PTPN22 attenuate the signaling strength of the GITR complex independent of TRAF3. Negative regulation of GITR signaling by TRAF3 may also involve competition with TRAF2 and TRAF5 (yellow and blue ovals), both of which can promote GITR signaling (59). In the absence of TRAF3 (right panel), NF?B2 is constitutively active and upregulates GITR transcription. Increased levels of the GITR receptor and lack of TRAF3 regulation of the GITR signaling cascade lead to elevated GITR signaling in TRAF3-deficient T cells.

Fig 5: Effect of TC-PTP deficiency on TPA-induced epidermal proliferation.(A) Representative photomicrographs of primary keratinocytes from K14Cre.Ptpn2w/w and K14Cre.Ptpn2fl/fl mice after days 1–3 of culture. Equivalent number of primary keratinocytes from both genotypes were seeded and cultured for 3 days. (B) Proliferation of primary keratinocytes was measured using the WST-assay. *p < 0.05 by Mann-Whitney U test. (C) Primary keratinocytes were treated with TPA (40 nM) for 1 h. Proliferation of primary keratinocytes was measured using WST-assay. (D–I) Groups of 7 weeks old mice (n = 3/group) were treated topically with single application of TPA (6.8 nmol) and sacrificed 24 or 48 h after treatment. (D) Representative hematoxylin and eosin staining of epidermis from both genotypes of mice. Scale bar: 100 µm. (E) Quantification of epidermal thickness from both genotypes treated with TPA. *p < 0.05 by Mann-Whitney U test. (F) Representative BrdU staining of epidermis from both genotypes of mice. Scale bar: 100 µm. (G) Quantification of BrdU-labled keratinocytes from epidermis of both genotypes. *p < 0.05 by Mann-Whitney U test. (H) Representative PCNA staining of epidermis from both genotypes of mice. Scale bar: 100 µm. (I) Quantification of PCNA-positive keratinocytes from epidermis of both genotypes. *p < 0.05 by Mann-Whitney U test.