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. Author manuscript; available in PMC: 2020 Nov 26.
Published in final edited form as: FASEB J. 2020 Jun 29;34(8):10657–10667. doi: 10.1096/fj.202000770RR

Autophagy plays a positive role in induction of epidermal proliferation

Junyi Wang 1,2,#, Nihal Kaplan 1,#, Sijia Wang 3, Wending Yang 1, Liqiang Wang 2, Congcong He 4, Han Peng 1
PMCID: PMC7688513  NIHMSID: NIHMS1628277  PMID: 32598088

Abstract

Autophagy is a multistage catabolic process that mediates stress responses. However, the role of autophagy in epidermal proliferation, particularly under conditions when the epidermis becomes “activated” (hyperproliferative), remains unclear. We have shown that inhibition of Beclin 1, a key activator in the initiation phase of autophagy, attenuates imiquimod (IMQ)-induced epidermal hyperplasia in adult mice as well as naturally occurring hyperproliferation in neonatal mouse epidermis. Inhibition of Beclin 1 did not change the levels of several key inflammatory molecules or the numbers of immune cells in lesional skins. This indicates that autophagy does not affect inflammatory regulators in IMQ-treated mouse skin. Bioinformatic analysis combined with gene expression quantitative assays, revealed that a deficiency in autophagy decreases the expression of PDZ Binding Kinase (PBK), a regulator of the cell cycle, in mouse epidermis and human epidermal keratinocytes (HEKs). Interestingly, the decrease in PBK results in inhibition of proliferation in HEKs and such reduced proliferation can be rescued by activation of p38, the downstream signaling of PBK. Collectively, autophagy plays a positive role in epidermal proliferation, which is in part via regulating PBK expression.

Keywords: Beclin 1, imiquimod, psoriasis, PBK, skin

1 |. INTRODUCTION

Autophagy is critical for mediating intrinsic and extrinsic cellular stress-related situations in tissues.1–8 Upon stimulation (eg, starvation, UV radiation), this self-cannibalistic process starts with activation of the Beclin 1-containing class III phosphatidylinositol 3-kinase (PI3K-III) complex. This lipid kinase complex is required for recruitment of autophagy-related gene (ATG) proteins and ultimately formation of LC3II, a mature form of LC3 protein, on the double membranes of autophagosomes.9 Autophagosomes fuse with lysosomes to form autolysosomes, where degradation occurs. End-stage autophagy is when autolysosomes are recycled to reform lysosomes via autophagic lysosome reformation (ALR).10–12 Autophagy is a dynamic multistage process, known to regulate numerous physiological and pathological conditions, in which cellular components need to be degraded and recycled.9 Thus, autophagy is critical for maintaining tissue homeostasis. For example, in the epidermis, it has been known for almost half a century that autophagy plays a role in epidermal homeostasis via contributing to epidermal terminal differentiation.13–15

Psoriasis is a common skin disease caused by a complex interplay between the immune system and the epidermis, which results in a multifaceted process that includes dysregulation of inflammatory responses and an “activated” or hyperproliferative epidermis.16–20 However, regulation of epidermal hyperproliferation in psoriasis remains poorly understood. Recently, studies revealed a relationship between abnormal autophagy and psoriasis.7,21–26 For instance, in psoriasis, there was an upregulation of the negative marker of autophagy, p62 in the lesional epidermis.21 Furthermore, mutation of the psoriasis risk gene AP1S3 resulted in compromised autophagy and accumulation of p62 in keratinocyte cultures.22 However, the true status of autophagy in psoriatic epidermis remains unclear. Several single nucleotide polymorphisms (SNPs) in the ATG16L1 gene, which interacts with ATG5/ATG12 to mediate the lipidation of LC3, a key component of autophagosome biogenesis,9 are associated with a susceptibility to psoriasis.23 Whether these SNPs are related to activation or inhibition of autophagy remains to be investigated. There have been reports of the exacerbation of psoriasis following 4-10 weeks treatment of chloroquine (Chlo),27 a pharmacological inhibitor of autolysosomes.9 It is unclear that whether the effect of long-term treatment of Chlo is due to its autophagy-dependent or non-specific autophagy-independent properties.

Conversely, other studies indicate that autophagy may be positively associated with psoriasis. Eight-methoxypsoralen in combination with UVA radiation, an effective treatment for psoriasis, inhibited autophagy in HaCaT cells.24 The expression of Progranulin as well as microRNAs-103/107 (miRs-103/107) were increased in psoriatic lesions25,28–30 and both positively regulate autophagy in keratinocytes.25,31

Here, we show that inhibition of autophagy attenuates hyperproliferation in mouse epidermis. We demonstrated that IMQ-induced epidermal hyperproliferation was attenuated by inhibition of Beclin 1 in a keratinocyte autonomous manner without involvement of immune cells. Moreover, inhibition of Beclin 1 negatively regulates epidermal proliferation via targeting PBK in keratinocytes. Collectively, this study strongly suggests an association between autophagy and psoriasis, one of the skin diseases with a hyperproliferative epidermis.

2 |. MATERIALS AND METHODS

2.1 |. Mice

To investigate the effects of autophagy in epidermis, healthy Beclin 1 het (Beclin 1 +/−) mice,32,33 which have compromised autophagy,33 were utilized. We also used Beclin 1 flox mouse 34 and generated Beclin 1 flox/flox; keratin 14 (Krt14)-cre mice, which have conditional depletion of Beclin 1 (Beclin 1-cKO) in epidermis and compromised autophagy. Mice at 8 weeks of age received a topical treatment of IMQ cream (5%) (62.5 mg) daily on the shaved back for six consecutive days. In order to label actively proliferating cells, mice were injected with Bromodeoxyuridine/5-bromo-2′-deoxyuridine (BrdU) (50 mg/kg) intraperitoneally 1 hours prior to euthanasia. Skin samples were dissected out and embedded in Optimal Cutting Temperature (OCT) medium immediately (Sakura Tissue-Tek) or embedded in paraffin blocks. To obtain total RNAs, skin samples were dissolved in Qiazol (Qiagen). Animal experiments were approved by the Northwestern University Animal Care and Use Committee.

2.2 |. Immunostaining

OCT sections (5 μm) were fixed in 4% of paraformaldehyde (PFA) at room temperature for 10 minutes, and then, incubated for one hour at room temperature with a universal protein block (Dako). Following was an overnight incubation with an antibody against Ki67 (SP6-1:500 dil; Sigma), Keratin 15 (MA5-11244-1:100 dil; Thermo Scientific), CD3 (17617-1-AP–1:100 dil; Proteintech), CD11c (17342-1-AP–1:100 dil; Proteintech), and PBK (16110-1-AP–1:100 dil; Proteintech) at 4°C. After washing, slides were incubated with Alexa Fluor –555 and –488-linked secondary Immunoglobulin G (IgG). 2-[4-(Aminoiminomethyl)phenyl]-1H-Indole-6-carboximidamide hydrochloride (DAPI) (Sigma-Aldrich) was used to counterstain nuclei.31,35,36 Fluorescent images were acquired using EC Plan-Neofluar 20×/0.5 on an epifluorescence microscope system (AxioVision Z1; Carl Zeiss) fitted with a slide module (Apotome; Carl Zeiss) and a digital camera (AxioCam MRm; Carl Zeiss). To visualize BrdU + cells in paraffin sections, immunohistochemical (IHC) staining was conducted as described previously.35 For BrdU staining, sections were incubated overnight with antibody recognizing BrdU (G3G4-1:10; DSHB). Sections were counterstained with hematoxylin to visualize morphology. Images were taken using a Zeiss Axioplan 2 microscope system (Carl Zeiss). AxioVision software (Carl Zeiss, Germany) was used to acquire and analyze the images. Ki67, BrdU, CD3, and CD11c positive cells were counted and relative fluorescence was measured by ImageJ.

2.3 |. Real time qPCR

Total RNAs were isolated and purified by a miRNeasy kit (Qiagen, Hilden, Germany). Real-time quantitative polymerase chain reaction (qPCR) was performed on a Roche LightCycler 96 System using the Roche FastStart Essential DNA Green Master (Roche) according to the manufacturer’s instructions.

2.4 |. Cell cultures

Primary cultures of HEKs were isolated from neonatal foreskin by NU Skin Disease Research Center - Skin Tissue Engineering Core (SBDRC-STE) and maintained in medium 154 (Cascade Biologics) containing HKGS growth supplements and 0.07 mM of CaCl2. To silence gene expression, siRNAs (20 nM) targeting ATG3, ATG7, Beclin 1, PBK, or a scrambled negative control (Invitrogen) were transiently transfected into cells using RNAiMAX (Invitrogen). To activate p38, cells were treated with U46619 (5 μM) for 2 days.

2.5 |. Western blotting

Western blots were performed as described previously.37 The following antibodies were used: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), Beclin 1, p-p38 (Cell Signaling Technology, Danvers, MA, USA), and PBK (Proteintech, Rosemont, IL, USA).

2.6 |. WST-1 assay

Cell growth was measured by WST-1 assay (Sigma) according to the manufacturer’s instructions. All groups were followed up to 6 days.

2.7 |. Statistical analysis

Unpaired t test was performed to determine statistical significance. The data are shown as means ± standard deviation (SD). The differences were considered significant for P values of < .05. All experiments were replicated at least three times. The bar graphs and box graph with jitters were drawn using a R package ggpubr.

3 |. RESULTS

3.1 |. Beclin 1 het attenuates IMQ-induced hyperproliferation

Initiation of autophagy requires ATG genes including Beclin 1.9 Homozygous Beclin 1−/− mice die in early embryogenesis. Heterozygous Beclin 1+/− (Beclin 1 het) mice, a model for attenuated initiation of autophagy,33 are developmentally normal and show reduced autophagy activity.38,39 To explore whether manipulation of the initiation stages of autophagy impacts hyperproliferation in the epidermis, we used a mouse model of imiquimod (IMQ)-induced psoriasiform dermatitis.40–45 IMQ treatment dramatically increases the number of proliferating keratinocytes, indicating that the epidermis becomes hyperproliferative.40–45

Interestingly, we analyzed the data set series GSE1335546 and found that Beclin 1 expression was significantly increased in lesional biopsies of patients with psoriasis than the healthy control skin (Supplemental Figure 1). Accordingly, following IMQ treatment, the epidermis of Beclin 1 het mice had a markedly thinner epidermis than wild-type (WT) littermates (Figure 1). To assess the proliferative rate, we measured two well-accepted proliferation markers: BrdU labeling and Ki67. Consistent with a thinner epidermis, inhibition of Beclin 1 reduced BrdU-labeled and Ki67 positive cells compared with WT littermates (Figure 2). These results suggest that autophagy may be necessary for certain aspects of proliferation in epidermis.

FIGURE 1.

FIGURE 1

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Loss of Beclin 1 inhibits IMQ-induced epidermal hyperproliferation. Wild-type and Beclin 1 het mice were treated with IMQ for five consecutive days. A-D, Representative hematoxylin and eosin (H&E)-stained back skin sections showed that Beclin 1 het reduced epidermal thickness after IMQ treatment (IMQ+). E, Epidermal thickness was measured using ImageJ. n ≥ 5 animals per group. *P < .05

FIGURE 2.

FIGURE 2

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Loss of Beclin 1 reduces BrdU labeling and Ki67 + cells in IMQ-treated mouse skin. Wild-type and Beclin 1 het mice were treated with IMQ for five consecutive days. Immunostaining with an antibody that recognizes BrdU A, B, and Ki67 D, E, was conducted. Black and white dotted lines demarcate the basement membrane region. Quantification of BrdU+ C, and Ki67+ F, cells in epidermis was performed using ImageJ. n ≥ 5 animals per group. *P < .05

3.2 |. Beclin 1 het doesn’t alter inflammation in IMQ-treated mouse skin

It has been shown that loss of Atg7, a gene involved in autophagosome maturation,9 modulates the immune response and reduces hyperplasia in ultraviolet (UV)-treated mouse skin.7 Thus, to understand how autophagy regulates epidermal proliferation in the context of IMQ treatment, we asked whether Beclin 1 het affects inflammation in IMQ-treated mouse skin. We examined the expression of several key inflammatory molecules that play central roles in psoriasis such as Tnfa, Il22r, Illra, Il12rb1, Tlr7, and Depb2.47–55 After IMQ treatment, the levels of these key inflammatory molecules did not change in lesional skin of the Beclin 1 het mice (Supplemental Figure 2A–F). Furthermore, Beclin 1 het did not alter inflammatory cell levels in lesional skin after IMQ treatment. (Supplemental Figure 2G,H). This validates the idea that the effects of Beclin 1-dependent autophagy in hyperproliferative epidermis of IMQ-treated skins are independent of inflammatory status.

3.3 |. Conditional knockout of Beclin 1 in epidermis attenuates IMQ-induced hyperproliferation

To investigate whether autophagy has a direct effect on epidermal proliferation, we specifically deleted Beclin 1 in mouse epidermis by Cre under the control of Keratin 14 promoter (Krt14-Cre) (Beclin 1-cKO). After IMQ treatment, BrdU labeling (Figure 3A–C) and Ki67 positive cells (Figure 3D–F) were markedly reduced in the epidermis with Beclin 1 depletion (Figure 3G–H), indicative of a direct role of autophagy in epidermal proliferation in the context of IMQ-induced dermatitis.

FIGURE 3.

FIGURE 3

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Epidermal specific knockout of Beclin 1 attenuates IMQ-induced epidermal hyperproliferation. Wild-type and Beclin 1 conditional knockout (cKO) mice were treated with IMQ for five consecutive days. Immunostaining with an antibody that recognizes BrdU A, B, Ki67 D, E, as well as Beclin 1 G, H, was conducted. Quantification of BrdU+ C, and Ki67+ F, cells in epidermis was performed using ImageJ. n ≥ 5 animals per group. *P < .05. White dotted lines demarcate the basement membrane region

3.4 |. Beclin 1 het attenuates naturally occurring hyperproliferation

To determine whether autophagy directly affects the proliferation of epidermal keratinocytes in an inflammation-independent situation, we compared skins of WT and Beclin 1 het neonatal mice (postnatal day 2). Neonatal mouse skin can be used as a model of spontaneous epidermal hyperproliferation since proliferation rate of basal keratinocytes in 2-5 day old mouse epidermis is ~ 5-fold higher than in adults.56,57 BrdU labeling and evaluation of Ki67 positive cells showed that proliferation in epidermis of newborn Beclin 1 het mice was markedly decreased compared with WT littermate controls (Figure 4).

FIGURE 4.

FIGURE 4

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Loss of Beclin 1 decreases proliferation in neonatal mouse epidermis. Representative images of immunostaining for BrdU A, B, and Ki67 D, E, in 2 day old epidermis of wild-type and Beclin 1 het mice. BrdU+ C, and Ki67+ F, cells were counted using ImageJ. n ≥ 5 animals per group. *P < .05. White dotted lines demarcate the basement membrane region

3.5 |. Depletion of key ATG genes specifically in human epidermal keratinocytes attenuates proliferation

To explore whether autophagy has a direct effect on proliferation of human epidermal keratinocytes (HEKs), we knocked down Beclin 1, ATG3, and ATG7, which are essential for autophagy.9 We found that knockdown of these key autophagy-related genes reduced BrdU labeling (Figure 5A) and attenuated cell growth (Figure 5B) in HEKs. This strongly suggests that autophagy plays a key role in regulating epidermal hyperproliferation in a cell autonomous way.

FIGURE 5.

FIGURE 5

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Knockdown of ATG genes inhibits proliferation of HEKs in submerged cultures. Submerged cultures of HEKs were transfected with a siRNA pool for ATG3 (siATG3), ATG7 (siATG7), Beclin 1 (siBeclin 1), or siControl. BrdU analysis A, and WST-1 assay B, were conducted to measure cell proliferation in submerged cultures. The bar graphs with jitters were drawn using a R package ggpubr. *P < .05

3.6 |. Loss of Beclin 1 reduces the expression of PBK

To investigate the underlying mechanisms responsible in the regulation of proliferation in keratinocytes by autophagy, we analyzed our single cell RNA sequencing (scRNA-seq) data of stem/transit amplifying cell population in corneal epithelium from Beclin 1 het versus WT mice58 and identified several cell cycle-related genes that were regulated by Beclin 1 in proliferating keratinocytes.59–62 Among them, the expression of PDZ-binding kinase 1 (PBK) was reduced in Beclin 1 het mouse epidermis (Figure 6A–E), HEKs lacking Beclin 1 (Figure 6F,G), and Beclin 1-cKO mouse epidermis (Figure 6K,L). Interestingly, knockdown of PBK significantly reduced cell growth in HEKs (Figure 6I). PBK facilitates cell proliferation by mediating p38 activation.63,64 Since knockdown of Beclin 1 reduced phospho-p38 (p-p38) levels (Figure 6F,H), we examined whether p-p38 signaling was required for the role of Beclin 1 in HEK proliferation. HEKs transfected with siBeclin 1 were treated with U46619, which can activate p38 and promote cell proliferation.65,66 Consistently, U46619 dramatically increased cell growth in HEKs transfected with the siControl (Figure 6J). More importantly, the cell growth of HEKs treated with U46619 and siBeclin 1 increased to a level similar to HEKs transfected with siControl (Figure 6J). Since U46619 is not a direct activator of p38, we cannot directly attribute the effects of U46619 to a rescue of p38 activity. However, our results are consistent with known roles of U46619 in promoting p38 activation as well as a role of p38 in promoting keratinocyte proliferation.67–69 This suggests that the inhibitory effect of Beclin 1 knockdown on HEK proliferation may be due to activation of p38 signaling.

FIGURE 6.

FIGURE 6

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Beclin 1 affects HEK proliferation via regulating PBK signaling. A-E, Immunostaining with an antibody that recognizes PBK and Keratin 15 was conducted. White dotted lines demarcate the basement membrane region. Quantification of PBK E, cells in epidermis was performed using ImageJ. n ≥ 5 animals per group. *P < .05. F-H, HEKs were transfected a siRNA pool for Beclin 1 (siBeclin 1) or siControl for 3 days. Western blotting showed that knockdown of Beclin 1 in HEKs decreased PBK expression and p-p38. Densitometry analysis of the Western blots of PBK G, and p-p38 H, was normalized to GAPDH. I, HEKs were transfected a siRNA pool for Beclin 1 (siBeclin 1) or siControl. After 6 days of transfection, WST-1 assay showed that knockdown of Beclin 1 and PBK reduced the relative OD in WST-1 assays, an indicator for cell growth. n = 5. *: P < .05. J, HEKs were transfected a siRNA pool for Beclin 1 (siBeclin 1) or siControl. And then, these cells were treated with U46619 (5 μM) or vehicle. Cell growth was measured by WST-1 assay. n = 5. *P < .05. K-L, Western blotting for proteins isolated from epidermis of Beclin 1-cKO or control mice showed that knockout of Beclin 1 in epidermis decreased PBK expression. Densitometry analysis was normalized to GAPDH (1). n = 7. *P < .05

4 |. DISCUSSION

Psoriasis is a multifaceted skin disease with a hyperproliferative epidermis and involves dysregulation of inflammatory responses.16–20 Whether autophagy significantly participates in the regulation of the hyperproliferative state characteristic of psoriasis is unclear and many unanswered questions remain. For example, 1. whether autophagy is necessary for cytokine-induced (eg, IL-23) hyperproliferation; 2. which regulatory factors affect autophagy in psoriatic skin lesions; 3. whether inhibition of autophagy at late stages blocks epidermal hyperproliferation in the context of psoriatic-like dermatitis. Therefore, further exploration of novel regulators of autophagy in epidermis is necessary. We have addressed some of these concerns by demonstrating that genetic inhibition of autophagy in the epidermis attenuates IMQ-induced epidermal hyperproliferation, which is a key feature of psoriasis. Such attenuation is keratinocyte autonomous and is not associated with the immune response. Our single cell RNA seq data suggest that attenuation of Beclin 1 expression inhibits the expression of PBK, a cell cycle-related gene.58 We validated this observation in mouse epidermis. Similar to loss of ATG genes, knockdown of PBK attenuated cell growth in HEKs, suggesting that inhibition of Beclin 1 negatively regulates epidermal proliferation via PBK in keratinocytes in the context of an activated epidermis.

Recent studies revealed unexpected roles for autophagy and selective autophagic degradation in the process of cell division.70,71 Activation of autophagy is preferentially linked to specific cell-cycle phases and such activation can promote cell cycle progression via regulating cell cycle-related proteins.70,71 For example, inhibition of autophagy in MCF-7 cells induces the stabilization of a subset of cyclin A2, whereas induction of autophagy accelerates the degradation of cyclin A2. In the data set GSE84935 from GEO of NCBI, ATG7 depletion in mouse keratinocytes increases the expression of ckap2 (a cell cycle inhibitor72) and decreases Atf5 (a cell cycle promoter73,74). These investigations indicate that autophagy can influence cell cycle machinery directly. In agreement with these investigations, we show that inhibition of Beclin 1 reduces the expression of PBK, a regulator of the cell cycle, and that such downregulation results in retarded cell growth. This supports the idea that attenuation of IMQ-induced hyperproliferation in Beclin 1 het mouse epidermis is due, in part, to blocking a portion of the cell cycle machinery in keratinocytes.

We show that Beclin 1 het in IMQ-treated skins does not alter levels of the inflammatory regulators that play key roles in inflammation-related skin diseases (eg, psoriasis; Supplemental Figure 2) as well as the numbers of CD3 + T cells and CD11c + cells (Supplemental Figure 2). This indicates that inflammation does not contribute to the effects of autophagy on epidermal proliferation. Furthermore, inhibition of autophagy specifically in keratinocytes attenuates keratinocyte proliferation (Figures 3 and 5). These observations suggest that canonical autophagy, which requires Beclin 1,9 regulates epidermal proliferation via cell autonomous mechanisms. In contrast, a recent study has shown that Atg7, another critical regulator of initiation of autophagy, regulates UV-induced proliferation and such regulation can be via a non-cell autonomous way.7 This raises interesting questions: whether in the context of UV-induced hyperplasia, Atg7 regulates proliferation in an autophagy-independent way or functions in noncanonical autophagy, which does not require Beclin 1. If the later is the case, how is noncanonical autophagy regulated?

Immunostaining and immunoblotting have shown that Beclin 1 protein is depleted in epidermis of Beclin 1 flox/flox; Krt14-cre mice (Figures 3G,H and 6K). These Beclin 1 flox/flox; Krt14-cre mice have normal life span. However, Beclin 1 flox/flox; K5-cre mice died within a day after birth.75 It has been reported that the human K5 promoter-driven Cre protein can be sporadically expressed in ova.76 Thus, it is possible that when using K5-cre, Beclin 1 was deleted at earlier developmental stage than K14-cre, which may result in such neonatal death. In support of this idea, Atg7flox/flox; K14-Cre mice, another autophagy deficient model, also did not show any abnormality in life span.7 Nonetheless, the discrepancy in life span between Beclin 1 flox/flox; Krt14-cre and Beclin 1 flox/flox; K5-cre mice is a very interesting topic which is worth investigating in future.

Taken together, autophagy can directly affect keratinocyte proliferation. Thus, identification of novel regulators of autophagy in the epidermis has potential for the development of unique therapeutic approaches in skin diseases with hyperproliferation. For example, we have shown that subcutaneously injection of antagomir-103 and antagomir-107 inhibits autophagy flux in keratinocytes in vivo.77 Since the expression of microRNAs-103/107 (miRs-103/107) has been reported to be increased in psoriatic lesions,28–30 delivery of these two inhibitors of miR-103 and miR-107 has a potential to block hyperproliferation in psoriatic lesions.

Supplementary Material

2

ACKNOWLEDGMENTS

The NU-SBDRC Morphology and Phenotyping Core facility assisted in morphologic analysis. The NU-SBDRC is supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR057216. This research is supported by a Dermatology Foundation research grant and Career Development Award (to HP); Eversight Eye Bank research grants (to HP); National Key R&D Program of China Grant 2017YFA0103204 (to LW); National Institutes of Health Grants EY028560 and EY019463 (to Robert, M. Lavker); National Institutes of Health grant DK094980, Northwestern Alzheimer’s Disease Center Pilot Award NIA AG13854, American Federation for Aging Research New Investigator Award in Alzheimer’s Disease (to CCH).

Funding information

Dermatology Foundation (DF); Eversight; HHS | National Institutes of Health (NIH), Grant/Award Number: DK094980; American Federation for Aging Research (AFAR)

Abbreviations:

ATG

autophagy-related gene

BrdU

Bromodeoxyuridine/ 5-bromo-2′-deoxyuridine

DAPI

2-[4-(Aminoiminomethyl)phenyl]-1H-Indole-6-carboximidamide hydrochloride

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

H&E

hematoxylin and eosin

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HEKs

human epidermal keratinocytes

IgG

immunoglobulin G

Ki67

marker of proliferation Ki-67

Krt14

keratin 14

OCT

optimal cutting temperature

PBK

PDZ Binding Kinase

PFA

paraformaldehyde

qPCR

quantitative polymerase chain reaction

Footnotes

CONFLICT OF INTEREST

No conflict of interest.

SUPPORTING INFORMATION

Additional Supporting Information may be found online in the Supporting Information section.

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