Fig 1: (A) Representative example of RNA ISH single-plex assay performed on FFPP section of pancreatic tumor metastasized in skin using the positive control probe POLR2A. Brown dots show expression of POLR2A using the RNAscope 2.5 HD Reagent Kit-BROWN. (Magnification x20). (B) Squamous cell carcinoma with keratinization. SERPINB7 is expressed in the tumor cell layer beneath the keratin. In normal skin this layer would be compatible with stratum granulosum. Arrows are indicating tumor cells. (Magnification ×20). (C) Squamous cell carcinoma used as positive control. (Magnification ×8). (D) PDAC with expression of SERPINB7 in some tumor cells. Arrows are indicating tumor cells expressing SERPINB7. (Magnification ×20). (E) Arrows are indicating reactive pancreatic ducts which are embedded in pancreatic tumor tissue and express SERPINB7 (Magnification ×20). (F) Arrows are indicating some tumor cells expressing SERPINB7 (Magnification ×20).
Fig 2: DFS (A) and OS (B) of patients that received gem as adjuvant therapy. Univariate analysis showed that SERPINB7 was associated with a poor DFS (two-stage test, P = .01) and poor OS(log-rank test P = .002). Multivariate analysis confirmed the independent predictive value of the expression of SERPINB7 on OS (P = .006, HR: 3.47; 95% CI: 1.49–8.09) in the gem group.
Fig 3: SerpinB7 deficiency promotes proliferation and suppresses differentiation in keratinocytes.A NHEK cells were transfected with shCT and sh-SerpinB7. The mRNA level of SerpinB7 was measured by RT-PCR. B CaCl2-induced Filaggrin, Loricrin, and KRT10 expression in shCT and sh-SerpinB7 NHEKs at 0–72 h was measured by RT-PCR. C, D The proliferation of sh-SerpinB7 keratinocytes was evaluated with a CCK8 (Cell Counting Kit 8) assay and colony formation assay. E Identical fields scratch of undifferentiated sh-SerpinB7 and control NHEKs at 0 and 24 h. The dotted line represents the area of the scratch. F The mRNA level of SerpinB7 in Serpinb7-/- and Serpinb7+/+mice primary keratinocytes (MKs) was measured by RT-PCR. G The proliferation of SerpinB7+/+ and Serpinb7-/- MKs was evaluated with a CCK8 assay. H, I SerpinB7+/+ and Serpinb7-/- MKs homeostasis and treated for 24 h with 1.6 mM CaCl2+ were analyzed by RT-PCR for the expression of early (Krt1, Krt10) and late (Notch1, Loricrin, Filaggrin, Lce1a2, Lce1d, Lce1m) differentiation genes. J Morphology of SerpinB7+/+ and Serpinb7-/- MKs in response to Ca2+ induced differentiation, red arrow indicated typical undifferentiated keratinocyte state. Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed Student’s t-test (A, H, I), one-way ANOVA (B, C, F, G). All the data are representative of three independent experiments.
Fig 4: SerpinB7 affected keratinocytes differentiation and inflammation via regulating Ca2+ influx.A, B Ca2+ concentration in cytosol ([Ca2+]c) and C, D Ca2+ concentration in mitochondria ([Ca2+]m) of shSerpinB7 and shCT NHEKs homeostasis and treated for 24 h with 1.6 mM Ca2+ were measured by flow cytometry and fluorescence confocal imaging using Flou4-AM and Rhod2-AM, respectively. E [Ca2+]c of Ca2+-induced shSerpinB7 and shCT NHEKs treated with BAPTA-AM were measured by flow cytometry using Flou4-AM. F The mRNA level of keratinocyte differentiation markers in Ca2+-induced shSerpinB7 and shCT NHEKs treated with BAPTA-AM was measured by RT-PCR. G The mRNA level of chemokines and microbial peptides in Ca2+-induced shSerpinB7 and shCT NHEKs treated with 20 um/mL BAPTA-AM was measured by RT-PCR. Flou4-AM: intracellular calcium indicator, Rhod2-AM mitochondrial Calcium indicator, BAPTA-AM, Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed Student’s t-test (A–D), one-way ANOVA (E–G). All the data are representative of three independent experiments.
Fig 5: Ablation of SerpinB7 leads to exacerbation of IMQ-induced Psoriasis model.A SerpinB7+/+ and SerpinB7-/- mice were treated with IMQ for 5 days, n = 5/group. Representative images of the dorsal skin from mice (left) and mice PASI scores were depicted (right). B Representative H&E staining section from the skin tissues of SerpinB7+/+ (n = 5) and SerpinB7-/- (n = 5) mice. Parakeratosis is indicated by the triangle, Munro’s microabscess is indicated by the arrows. Right: statistical analysis of skin epidermal thickness and inflammatory cell infiltration. C The RNA levels of keratinocytes differentiation-related genes were assessed by qPCR. D The RNA levels of chemokines and antimicrobial peptides were assessed by qPCR. E Immunofluorescent staining of Krt10, Filaggrin, and Loricrin in the dorsal skin of SerpinB7+/+ (n = 5) and Serpinb7-/- (n = 5) mice. Right: statistical analysis of staining intensity in the epidermis. Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Two-tailed Student’s t-test (A–E). All the data are representative of three independent experiments.
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