atcc 53255 strains  (ATCC)

Journal: bioRxiv

Article Title: Keratin 16 spatially inhibits type I interferon responses in stressed skin

doi: 10.1101/2024.12.27.630544

Figure Lengend Snippet: (A) Analysis of differential expression between KRT16 high and KRT16 low expressing cells in single-cell RNA datasets from PSOR and HS patients. Differentially expressed genes are plotted according to “% Expression in the population” (Y-axis) vs Avg Log2 fold change (X-axis). Interferon antiviral genes are highlighted in red. (B-C) Immunostaining for K16 (green) and nuclei (DAPI, blue) in tissue sections from non-lesional (B) or lesional skin (C) of individuals with psoriasis. (D) Analysis of IP/MS data from 3xflag-tagged K16 expression in HaCat keratinocytes. Highlighted are the type II keratin K6A (blue) and proteins related to interferon signaling (red). (E) Results of statistical overrepresentation analysis using Panther API (Reactome dataset). Interferon responsive genes and pathways are highlighted in red. (F) Gene network map for highest interacting proteins identified in the IP/MS that showed SAINT score >0.8. (G) Statistical overrepresentation test for differentially expressed pathways in microarray data from lesional skin of PC patients with mutations in KRT6 or KRT16 (source: ). (H) Statistical overrepresentation test for differentially expressed pathways in microarray data from lesional skin of Krt16 null mice .

Article Snippet: Samples were bound to Dyanbeads protein G (Fisher Scientific) with 2μg anti-K16 antibody (Santa Cruz Biotechnology, #sc-53255), and immunoprecipitated following manufacturer instructions.

Techniques: Expressing, Immunostaining, Microarray

Journal: bioRxiv

Article Title: Keratin 16 spatially inhibits type I interferon responses in stressed skin

doi: 10.1101/2024.12.27.630544

Figure Lengend Snippet: (A) Protocol of IMQ application used to induce psoriasiform disease in mice (see Methods). (B-E) Images of WT and Krt16 null mice prior to treatment (day 0; B,D) and following the last treatment (day 4; C,E). Insets highlight the treated area. (F-I) H&E staining of back skin sections from WT mice (F,G) or Krt16 null mice (H,I) treated with Vaseline control or IMQ, respectively. Images presented are maximum projection across 5um depth. (J-L) Indirect immunofluorescence for K16 (green), and K14 (red), and staining for nuclei (DAPI, blue) in back skin sections from WT mice treated with Vaseline (J) or IMQ (K), or Krt16 null treated with IMQ (L). White dashed lines delineate the basement membrane. (M) Quantification of epidermis thickness from Vaseline– or IMQ-treated WT and Krt16 null mice. (N-P) Indirect immunofluorescence for Ki67 (green) and K14 (red), and staining for nuclei (DAPI, blue) in back skin sections WT mice treated with Vaseline (J), IMQ (K), or Krt16 null treated with IMQ. White dashed line delineates the basement membrane. (Q) Quantification of mitotic index (%Ki67-positive cells) from Vaseline– or IMQ-treated WT and Krt16 null mice. (R-S’) Indirect immunofluorescence for OAS1 (green), and staining of nuclei (DAPI, blue) in back skin sections following IMQ treatment of WT (R,R’) and Krt16 null mice (S-S’). Yellow boxes in (R) and (S) are shown at higher magnification in (R’) and (S’). White dashed lines delineate the basement membrane, and red dashed lines depict the interface between basal and suprabasal compartments. (T) Quantification of OAS1 signal intensity (normalized from 0-1 per section) from the dermo-epidermal interface to the stratum corneum (x=0) in WT (blue) and Krt16 null mice (red). Lines represent the mean intensity per um, with standard error across animals shown in light blue or red, respectively. One-way ANOVA with Tukey’s multiple comparisons test was used to compare conditions in (M) and (Q).

Article Snippet: Samples were bound to Dyanbeads protein G (Fisher Scientific) with 2μg anti-K16 antibody (Santa Cruz Biotechnology, #sc-53255), and immunoprecipitated following manufacturer instructions.

Techniques: Staining, Control, Immunofluorescence, Membrane

Journal: bioRxiv

Article Title: Keratin 16 spatially inhibits type I interferon responses in stressed skin

doi: 10.1101/2024.12.27.630544

Figure Lengend Snippet: (A) Protocol for TPA topical treatment of mouse ear skin (see methods). (B-D) Indirect immunofluorescence of mouse ear skin sections for neutrophil marker Ly6G (red), K16 (green), K14 (yellow), and staining for nuclei (DAPI, blue), at 6h after single TPA application to WT (B) and Krt16 null mice (C), or dual TPA application to WT (D) or Krt16 null ear skin (E). Dashed white lines delineate the cartilage (c) and arrow depicts dorsal to ventral direction. (F-F’) Quantification of neutrophil area (F) and distribution (F’) across the dermis of dorsal ear skin at 6h after single TPA treatment. (G-G’) Quantification of neutrophil area (G) and distribution (G’) across the dermis of dorsal ear skin at 6h after dual TPA treatments. (H-I) Indirect immunofluorescence for OAS1 (green), and staining of nuclei (DAPI, blue) in ear skin sections after dual acetone (H-H’; K-K’) or dual TPA treatments (I-I’; L-L’). Yellow boxes are region of interests magnified in frames H’, I’, K’, and L’. White dashed lines delineate the basement membrane, and red dashed lines depict the interface between basal and suprabasal compartments. (J) Quantification of OAS1 levels in ear epidermis of WT or Krt16 null mice after dual treatment with Acetone or TPA. (M) Quantification of OAS1 signal intensity (normalized from 0-1 per section) relative to distance from the dermo-epidermal interface (x=0) in WT (blue) and Krt16 null mice (blue). Lines represent the mean intensity per um, with standard error across animals shown in light blue or red respectively. One-way ANOVA with Tukey’s multiple comparisons test was used to compare conditions in (F) and (G), and Holm-Šídák’s multiple comparisons test used to compare conditions in (J).

Article Snippet: Samples were bound to Dyanbeads protein G (Fisher Scientific) with 2μg anti-K16 antibody (Santa Cruz Biotechnology, #sc-53255), and immunoprecipitated following manufacturer instructions.

Techniques: Immunofluorescence, Marker, Staining, Membrane

Journal: bioRxiv

Article Title: Keratin 16 spatially inhibits type I interferon responses in stressed skin

doi: 10.1101/2024.12.27.630544

Figure Lengend Snippet: (A) Schematic depicting the mode of keratinocyte activation and downstream interferon response by dsRNA (PolyIC). (B) Western blot analysis of K16, ISG15 and p-IRF7 levels in WT and KRT16 KO bilayer N/TERT keratinocytes following (1) untreated, (2) polyIC treatment without recovery and (3) polyIC treatment followed by 24 hours of recovery. Presented are two biological replicates per condition (WT #1,#2; KO #1, #2). HSP90 was used as a loading control for K16 and ISG15. Cofilin was used as a loading control for p-IRF7. (C-E) Quantification of K16 (C) p-IRF7 (D) and ISG15 (E) levels across polyIC treatment conditions in WT, KRT16 KO, or MOCK N/TERT keratinocytes. Each dot represents a biological replicate. (F-I) Indirect immunofluorescence for ISG15 (green) and staining for nuclei (DAPI, magenta) in WT (F-G) or KRT16 KO (H-I) N/TERT keratinocytes untreated (F,H) or 24h after treatment with polyIC (G,I). (J) quantification of mean ISG15 fluorescence intensity per cell in untreated or polyIC recovered N/TERT keratinocytes. (K-N) Indirect immunofluorescence for p-IRF7 (grey) in WT (K-L) or KRT16 KO (M-N) N/TERT keratinocytes untreated (K,M) or 24 hours after treatment with polyIC (L,N). (O) Quantification of mean nuclear p-IRF fluorescence intensity per cell in untreated or polyIC recovered N/TERT keratinocytes. (P) Illustration depicting human neutrophil migration assay (see method). (Q) Quantification of % neutrophil migration towards conditioned media from WT (green) or KRT16 KO (magenta) N/TERT keratinocytes, untreated or at 24h after treatment with polyIC. (R) Quantification of % neutrophil migration towards conditioned media from WT (green) or KRT16 KO (magenta) N/TERT keratinocytes at 24 h after acetone or TPA treatment. In (Q) and (R), each dot represents a single biological replicate. One-way ANOVA with Šídák’s multiple comparisons test was used to compare conditions in (C-E). Tukey multiple comparisons test was used to compare conditions in (J) and (O). Repeated Measures one way ANOVA with Tukey’s multiple comparisons test was used for statistics presented in panels (Q-R).

Article Snippet: Samples were bound to Dyanbeads protein G (Fisher Scientific) with 2μg anti-K16 antibody (Santa Cruz Biotechnology, #sc-53255), and immunoprecipitated following manufacturer instructions.

Techniques: Activation Assay, Western Blot, Control, Immunofluorescence, Staining, Fluorescence, Migration

Journal: bioRxiv

Article Title: Keratin 16 spatially inhibits type I interferon responses in stressed skin

doi: 10.1101/2024.12.27.630544

Figure Lengend Snippet: (A) Schematic depicting the regulation of RIG1/MDA5 and MAVS signaling by 14-3-3ε and TRIM proteins. (B) Western blot analysis of 14-3-3ε levels in WT and KRT16 KO bilayer N/TERT keratinocytes following (1) no treatment (control), (2) polyIC treatment and (3) at 24h after polyIC treatment. Two biological replicates per condition are reported (WT #1,#2; KO #1,#2). HSP90 was used for loading control. (C-D) Indirect immunofluorescence for K16 (green) and staining for nuclei (DAPI, blue) in the basal layer (C) and suprabasal layer (D) of post-confluent N/TERT keratinocytes. Blue rectangle in D depict the location of the magnified images in panel D’. (D’-D’’) Airyscan confocal microscopy for K16 (green), 14-3-3ε (red) and nuclei (blue) in suprabasal N/TERT keratinocytes. Yellow indicates areas of colocalization between the two proteins. Yellow rectangle in D’ is magnified in D’’. (E-I) Proximity ligation assay (PLA) signal between K16 and 14-3-3ε (green) in basal and suprabasal layers of WT, untreated (E-F) and polyIC-treated (F-G), and of Krt16 KO, untreated (H) and polyIC treated (I), N/TERT keratinocytes. Nuclei are stained with DAPI (grey). (J) Quantification of the K16:14-3-3ε PLA signal across N/TERT keratinocytes layers, and in KRT16 KO controls in untreated or at 24h after polyIC treatment. (K) Immunoprecipitation of endogenous K16 from WT or KRT16 KO cells. Lysates and immunoprecipates fractions were blotted for K16 and 14-3-3ε. Input (2ug total) was used to validate genotype and assess for 14-3-3ε levels across conditions. K16 pulldown (IP from total 35 ug) was verified in the IP conditions, and high exposure was used to detect 14-3-3ε pulldown in the IP samples across conditions. (L) Predicted 14-3-3 binding sites in human and mouse K16. Each residue is colored by rank provided by 14-3-3 pred interface. Highlighted in red are the S44 and S40 orthologs in human and mouse, and their ranking in parentheses. (M) Illustration of approach used to generate a mass spectrometry phospho-proteomic profile of K16 following 0, 6, 12, 24 and 48 h after a single TPA treatment of ear skin in WT mice (n= 3 animals). (N) Results from MS analysis of in WT mouse ear proteins identify an increase in K16 levels across time after TPA treatment. (O) Levels of phosphorylated S40 residue in K16 across time after TPA treatment. One-way ANOVA with Tukey’s multiple comparisons test was used to compare conditions in (J).

Article Snippet: Samples were bound to Dyanbeads protein G (Fisher Scientific) with 2μg anti-K16 antibody (Santa Cruz Biotechnology, #sc-53255), and immunoprecipitated following manufacturer instructions.

Techniques: Western Blot, Control, Immunofluorescence, Staining, Confocal Microscopy, Proximity Ligation Assay, Immunoprecipitation, Binding Assay, Residue, Mass Spectrometry

Journal: International Journal of Molecular Sciences

Article Title: GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples

doi: 10.3390/ijms25116084

Figure Lengend Snippet: ( A ) Expression of S100A7 and KRT16 in three melanoma cell lines (CHL-1, A375, and MEL224), after transfection (TF) with GLI1, GLI2, and GLI3 expression plasmids. ( B ) Validation of S100A7 , KRT16 , and a known target of GLI proteins, PTCH1 , in cell lines CHL-1 and MEL224 resistant to inhibitor GANT61 (GANTR), a model for pathway downregulation. ( C ) Validation of S100A7 and KRT16 gene expression in the CHL-1 cell line with stable overexpression of SHH, a model of autocrine pathway upregulation. The relative gene expression of S100A7 and KRT16 , as well as the PTCH1 gene, is increased, but not statistically significant for S100A7 and KRT16 . ( D ) Validation of S100A7 and KRT16 gene expression in spheroid culture (sph) of the A375 cell line. Dark blue columns show adherent A375 cell line (adh), light purple columns show non-transfected spheroids of A375 cell line, and pink columns show spheroids transfected with GLI1 after 72 h. Data are presented with mean value ± standard deviation (SD), *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: Primary antibodies were used at the following dilutions: GLI1 (Novus Biologicals, NB600-600) 1:100, GLI2 (sc-271786, Santa Cruz, Dallas, TX, USA) 1:100, GLI3 (GTX104362, Gene Tex, Irvine, CA, USA) 1:200, S100A7 (47C1068, Novus Biologicals, Centennial, CO, USA) 1:100, KRT16 (sc-53255, Santa Cruz, Dallas, TX, USA), 1:100.

Techniques: Expressing, Transfection, Over Expression, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples

doi: 10.3390/ijms25116084

Figure Lengend Snippet: Proportion of samples with staining intensities (no expression, weak or strong) for ( A ) GLI1, ( B ) GLI3, ( C ) KRT16, and ( D ) S100A7 in different melanoma stages (TIS-T4). For each sample, the intensity was measured at three locations: tumor mass (TUMOR), epidermis bordering the tumor mass (BORDER), and epidermis overlying the tumor mass (CENTRAL). In general, the staining intensities for all proteins were stronger in the epidermis (both border and central) compared to the tumor mass.

Article Snippet: Primary antibodies were used at the following dilutions: GLI1 (Novus Biologicals, NB600-600) 1:100, GLI2 (sc-271786, Santa Cruz, Dallas, TX, USA) 1:100, GLI3 (GTX104362, Gene Tex, Irvine, CA, USA) 1:200, S100A7 (47C1068, Novus Biologicals, Centennial, CO, USA) 1:100, KRT16 (sc-53255, Santa Cruz, Dallas, TX, USA), 1:100.

Techniques: Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples

doi: 10.3390/ijms25116084

Figure Lengend Snippet: Representative staining of the border area of the T4 tumor for ( A ) GLI1, ( B ) GLI3, ( C ) KRT16, and ( D ) S100A7. ( E ) Negative control (no primary antibody), and ( F ) higher magnification of GLI1 staining in the tumor. The scale bar for ( A – E ) was 200 μm and for ( F ) was 50 μm.

Article Snippet: Primary antibodies were used at the following dilutions: GLI1 (Novus Biologicals, NB600-600) 1:100, GLI2 (sc-271786, Santa Cruz, Dallas, TX, USA) 1:100, GLI3 (GTX104362, Gene Tex, Irvine, CA, USA) 1:200, S100A7 (47C1068, Novus Biologicals, Centennial, CO, USA) 1:100, KRT16 (sc-53255, Santa Cruz, Dallas, TX, USA), 1:100.

Techniques: Staining, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples

doi: 10.3390/ijms25116084

Figure Lengend Snippet: Comparison of protein expressions ( A ) GLI1 vs. GLI3, ( B ) GLI1 vs. KRT16, ( C ) GLI1 vs. S100A7, and ( D ) S100A7 vs. KRT16 in the same location (epidermis bordering the tumor = BORDER). There is a significant association between GLI1 expression and expressions of all other tested proteins for border vs. border ( p < 0.0001). ( E – H ) Expression of the same proteins in BORDER vs. tumor mass (TUMOR). GLI1 ( E ), GLI3 ( F ), and S100A7 expression ( G ) in the tumor tissue is significantly associated with the expression in the border epidermis ( p < 0.0001 for all three proteins), while KRT16 expression ( H ) is not significantly associated ( p = 0.107).

Article Snippet: Primary antibodies were used at the following dilutions: GLI1 (Novus Biologicals, NB600-600) 1:100, GLI2 (sc-271786, Santa Cruz, Dallas, TX, USA) 1:100, GLI3 (GTX104362, Gene Tex, Irvine, CA, USA) 1:200, S100A7 (47C1068, Novus Biologicals, Centennial, CO, USA) 1:100, KRT16 (sc-53255, Santa Cruz, Dallas, TX, USA), 1:100.

Techniques: Comparison, Expressing

Journal: International Journal of Molecular Sciences

Article Title: GLI Transcriptional Targets S100A7 and KRT16 Show Upregulated Expression Patterns in Epidermis Overlying the Tumor Mass in Melanoma Samples

doi: 10.3390/ijms25116084

Figure Lengend Snippet: Distribution of intensities of staining for GLI1 ( A ), GLI3 ( B ), KRT16 ( C ), and S100A ( D ) in the epidermis bordering the tumor (BORDER) by stage (TIS, T1, T2, T3, and T4). All the stained proteins show significant differences between different stages ( p < 0.0001 for all).

Article Snippet: Primary antibodies were used at the following dilutions: GLI1 (Novus Biologicals, NB600-600) 1:100, GLI2 (sc-271786, Santa Cruz, Dallas, TX, USA) 1:100, GLI3 (GTX104362, Gene Tex, Irvine, CA, USA) 1:200, S100A7 (47C1068, Novus Biologicals, Centennial, CO, USA) 1:100, KRT16 (sc-53255, Santa Cruz, Dallas, TX, USA), 1:100.

Techniques: Staining

Journal: Cell Death Discovery

Article Title: DNA damage signalling histone H2AX is required for tumour growth

doi: 10.1038/s41420-024-01869-9

Figure Lengend Snippet: A–D Representative immunofluorescence of H2AX WT or KO epidermis for keratin K5 (red) and K10 (green; A ), involucrin ( B , green), filaggrin ( C , green), keratin K16 ( D ,green). DNA in blue by DAPI. Broken line for the basement membrane. Scale bars: 20 µm.

Article Snippet: The following antibodies were used: anti-Ki67 (ab16667, Abcam; IF), anti-BrdU (347580, Biosciences; IF), anti-filaggrin (PRB-417, Covance; IF), anti-involucrin (RINVOL, 924401, Biologend; IF), anti-K5 (SAB45016501, Sigma-Aldrich; IF), anti-K10 (sc-23877, Santa Cruz Biotechnology; IF), anti-K16 (sc-53255, Santa Cruz Biotechnology; IF), anti-Cyclin A (sc-751, Santa Cruz Biotechnology; IF), anti-pH3 (sc-8656-R, Santa Cruz Biotechnology; IF), anti-p-H2AX (sc-517348, Santa Cruz Biotechnology; IF), anti-p40 (deltaNp63, 24-8626-RBP1, ARP; IF), anti-Cyclin E1 (sc-248, Santa Cruz Biotechnology; WB), anti-PCNA (sc-56, Santa Cruz Biotechnology; WB) and anti-GAPDH (sc-32233, Santa Cruz Biotechnology; WB).The following secondary antibodies were used: Alexa Fluor® 488-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Jackson; IF), Alexa Fluor® 594-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Jackson; IF) and DyLight TM 800-conjugated goat anti-mouse IgG antibody (Invitrogen; WB).

Techniques: Immunofluorescence, Membrane

Journal: Cell Death Discovery

Article Title: DNA damage signalling histone H2AX is required for tumour growth

doi: 10.1038/s41420-024-01869-9

Figure Lengend Snippet: A Representative double immunofluorescence for the epidermal stem cell marker ∆p63 (green) and differentiation marker Keratin K10 (red) in WT or KO mice, as indicated. DNA in blue by DAPI. Bar histogram represents the percent of ∆p63 positive basal nuclei cells of the epidermis. Broken line for the basement membrane. *** p value <0.001, Student´s t -test. Data were mean ± SEM of five representative fields (more than 500 nuclei). Scale bar: 20 µm. B Model for the induction of differentiation upon H2AX inactivation in the epidermis. In normal tissue, the lack of H2AX deregulates the cell cycle of stem cells, which leads to the accumulation of DNA damage due to increased replication stress (RS) and decreased DNA repair. Unknown signals recognise the damage and trigger terminal squamous differentiation (Diff.). This impairs the growth of rising tumour cells. Additional mutations in the signals blocking mitosis (AM) may lead to genomic instability and cancer progression.

Article Snippet: The following antibodies were used: anti-Ki67 (ab16667, Abcam; IF), anti-BrdU (347580, Biosciences; IF), anti-filaggrin (PRB-417, Covance; IF), anti-involucrin (RINVOL, 924401, Biologend; IF), anti-K5 (SAB45016501, Sigma-Aldrich; IF), anti-K10 (sc-23877, Santa Cruz Biotechnology; IF), anti-K16 (sc-53255, Santa Cruz Biotechnology; IF), anti-Cyclin A (sc-751, Santa Cruz Biotechnology; IF), anti-pH3 (sc-8656-R, Santa Cruz Biotechnology; IF), anti-p-H2AX (sc-517348, Santa Cruz Biotechnology; IF), anti-p40 (deltaNp63, 24-8626-RBP1, ARP; IF), anti-Cyclin E1 (sc-248, Santa Cruz Biotechnology; WB), anti-PCNA (sc-56, Santa Cruz Biotechnology; WB) and anti-GAPDH (sc-32233, Santa Cruz Biotechnology; WB).The following secondary antibodies were used: Alexa Fluor® 488-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Jackson; IF), Alexa Fluor® 594-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Jackson; IF) and DyLight TM 800-conjugated goat anti-mouse IgG antibody (Invitrogen; WB).

Techniques: Immunofluorescence, Marker, Membrane, Blocking Assay