Journal: Molecular Cell

Article Title: The ribotoxic stress response drives acute inflammation, cell death, and epidermal thickening in UV-irradiated skin in vivo

doi: 10.1016/j.molcel.2024.10.044

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-ZAK , Proteintech , Cat#14945-1-AP; RRID: AB_1064269.

Techniques: Recombinant, Reverse Transcription, In Situ, Knock-Out, Variant Assay, Software, Purification, Blocking Assay

Journal: Disease Models & Mechanisms

Article Title: Sox11 regulates mammary tumour-initiating and metastatic capacity in Brca1 -deficient mouse mammary tumour cells

doi: 10.1242/dmm.046037

Figure Lengend Snippet: Sox11 is expressed during early stages of embryonic mammary gland development. Representative images of Sox11 immunohistochemistry at different development stages (E12.5 to P1) of BALB/c embryos. Scale bar: 100 μm.

Article Snippet: sgRNAs were designed using the CRISPOR algorithm ( www.crispor.tefor.net ). sgRNA sequences targeting Sox11 (Gene ID, 20666) were cloned into the Lenti-CRISPR–EGFP plasmid (Addgene, 75159 ) using a BsmBI enzyme site (sgRNA-1, 5′-TGGACGAGAGCGACCCGGAC-3′; sgRNA-2, 5′-GAGCCCCGACAAGAGCGCGG-3′).

Techniques: Immunohistochemistry

Journal: Disease Models & Mechanisms

Article Title: Sox11 regulates mammary tumour-initiating and metastatic capacity in Brca1 -deficient mouse mammary tumour cells

doi: 10.1242/dmm.046037

Figure Lengend Snippet: Sox11 regulates stem cell proliferation, survival and lineage marker status of mouse embryonic mammary progenitor cells. (A) Embryonic mammary progenitor cells with varying levels of Sox11 were grown in low attachment 96-well plates for 3 days before the addition of Matrigel or BME. Image analysis of mammospheres were performed at 10 days. N =3 independent experiments. Three to four replicates per group per experiment. (B) Expression of Sox11 in eG2E5 ‘stem-like cells’ after CRISPR/Cas9 gene editing. Western blot showing knockdown of Sox11 with guide #1 and guide #2. (C) Colony formation assay results from eG2E5 cells when Sox11 levels are reduced. N =3 independent experiments. Two replicates per group per experiment. (D) Sphere formation assay after knockdown of Sox11. eG2E5 cells were grown in low attachment 96-well plates for 10 days before image analysis. N =3 independent experiments. Eight replicates per group per experiment. (E) CellTiter-Glo results from eG2E5 cells when Sox11 levels are reduced. N =3 independent experiments. Eight replicates per group per experiment. (F) Immunofluorescence (IF) analysis of K8 in sg-Sox11-1 compared to sg-control spheres of eG2E5 cells. Sections were projected on one plane with maximum intensity ( z stack). Green, K8; red, SMA. Magnification 20×. N =3 independent experiments. Average of three replicates per group per experiment. Data are mean±s.d. Paired two-tailed Student's t -test. Scale bars: 1000 μm (A,D); 200 μm (F). NS, not significant; RLU, relative light unit.

Article Snippet: sgRNAs were designed using the CRISPOR algorithm ( www.crispor.tefor.net ). sgRNA sequences targeting Sox11 (Gene ID, 20666) were cloned into the Lenti-CRISPR–EGFP plasmid (Addgene, 75159 ) using a BsmBI enzyme site (sgRNA-1, 5′-TGGACGAGAGCGACCCGGAC-3′; sgRNA-2, 5′-GAGCCCCGACAAGAGCGCGG-3′).

Techniques: Marker, Expressing, CRISPR, Western Blot, Knockdown, Colony Assay, Tube Formation Assay, Immunofluorescence, Control, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Sox11 regulates mammary tumour-initiating and metastatic capacity in Brca1 -deficient mouse mammary tumour cells

doi: 10.1242/dmm.046037

Figure Lengend Snippet: Sox11 regulates stem cell activity and lineage status of Brca1 .3 mouse mammary tumour cells. (A) Expression of Sox11 in Brca1 .3 cells after CRISPR/Cas9 gene editing. qRT-PCR showing knockdown of Sox11 with guide #1 and guide #2 compared to sg-control. N =3 independent experiments. Three replicates per group per experiment. (B) Colony formation assay results from Brca1 .3 cells when Sox11 levels are reduced. N =3 independent experiments. Two replicates per group per experiment. (C) Sphere-forming capacity of sg-control and sg-Sox11-1 cells. Single cells were grown in the presence of 4% Matrigel for 10 days before image analysis. N =3 independent experiments. Six replicates per group per experiment. (D) Western blot showing fibronectin, N-cadherin, E-cadherin, Mex3A and Rcor2 after knockdown of Sox11 compared to sg-control. N =3 independent experiments. (E) Immunofluorescence (IF) analysis of K8 and K14 in Brca1 .3 cells. Sections were projected on one plane with maximum intensity ( z stack). K8 in sg-Sox11-1 spheres compared to sg-control spheres. Green, K8; red, K14. Magnification 20×. N =3 independent experiments. Three to five replicates per group per experiment. Data are mean±s.d. Paired two-tailed Student's t -test. Scale bars: 2000 μm (C); 200 μm (E). NS, not significant.

Article Snippet: sgRNAs were designed using the CRISPOR algorithm ( www.crispor.tefor.net ). sgRNA sequences targeting Sox11 (Gene ID, 20666) were cloned into the Lenti-CRISPR–EGFP plasmid (Addgene, 75159 ) using a BsmBI enzyme site (sgRNA-1, 5′-TGGACGAGAGCGACCCGGAC-3′; sgRNA-2, 5′-GAGCCCCGACAAGAGCGCGG-3′).

Techniques: Activity Assay, Expressing, CRISPR, Quantitative RT-PCR, Knockdown, Control, Colony Assay, Western Blot, Immunofluorescence, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Sox11 regulates mammary tumour-initiating and metastatic capacity in Brca1 -deficient mouse mammary tumour cells

doi: 10.1242/dmm.046037

Figure Lengend Snippet: Sox11 regulates stem cell activity and lineage status of Brca1 .1516 mouse mammary tumour cells . (A) Expression of Sox11 in Brca1 .1516 cells after CRISPR/Cas9 gene editing. qRT-PCR showing knockdown of Sox11 with guide #1 and guide #2 compared to sg-control. N =3 independent experiments. Three replicates per group per experiment. (B) Colony formation assay results from Brca1 .1516 cells when Sox11 levels were reduced. N =3 independent experiments. Two replicates per group per experiment. (C) Sphere-forming capacity of sg-control and sg-Sox11-1 cells. Single cells were grown in the presence of 4% Matrigel for 10 days before image analysis. N =3 independent experiments. Two replicates per group per experiment. Scale bar: 2000 μm. (D) Western blot showing fibronectin, N-cadherin, E-cadherin, Mex3A and Rcor2 after knockdown of Sox11 (guide #1) compared to sg-control. N =3 independent experiments. Data are mean±s.d. Paired two-tailed Student's t -test.

Article Snippet: sgRNAs were designed using the CRISPOR algorithm ( www.crispor.tefor.net ). sgRNA sequences targeting Sox11 (Gene ID, 20666) were cloned into the Lenti-CRISPR–EGFP plasmid (Addgene, 75159 ) using a BsmBI enzyme site (sgRNA-1, 5′-TGGACGAGAGCGACCCGGAC-3′; sgRNA-2, 5′-GAGCCCCGACAAGAGCGCGG-3′).

Techniques: Activity Assay, Expressing, CRISPR, Quantitative RT-PCR, Knockdown, Control, Colony Assay, Western Blot, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Sox11 regulates mammary tumour-initiating and metastatic capacity in Brca1 -deficient mouse mammary tumour cells

doi: 10.1242/dmm.046037

Figure Lengend Snippet: Sox11 regulates proliferative status of Brca1 .3 tumour cells. (A) Sphere formation assay after knockdown of Sox11. Brca1. 3 cells were grown in low attachment 96-well plates for 10 days before image analysis of relative sphere area. N =4 independent experiments. Three replicates per group per experiment. (B) Cell viability as determined by CellTiter-Glo assay. N =3 independent experiments. Seven replicates per group per experiment. (C) Quantification of Ki67-expressing cells compared to control in spheroids. N =3 independent experiments. Two to four replicates per group per experiment. (D) Three-dimensional invasion assay after knockdown of Sox11. Brca1. 3 cells were grown in low attachment 96-well plates for 7 days before the addition of Matrigel for a further 7 days before image analysis. Relative invasive area was calculated by subtracting the sphere area of day 7 from day 14. N =4 independent experiments. Three replicates per group per experiment. Data are mean±s.d. Paired two-tailed Student's t -test. Scale bars: 1000 μm (A,D); 200 μm (C). NS, not significant. RLU, relative light unit.

Article Snippet: sgRNAs were designed using the CRISPOR algorithm ( www.crispor.tefor.net ). sgRNA sequences targeting Sox11 (Gene ID, 20666) were cloned into the Lenti-CRISPR–EGFP plasmid (Addgene, 75159 ) using a BsmBI enzyme site (sgRNA-1, 5′-TGGACGAGAGCGACCCGGAC-3′; sgRNA-2, 5′-GAGCCCCGACAAGAGCGCGG-3′).

Techniques: Tube Formation Assay, Knockdown, Glo Assay, Expressing, Control, Invasion Assay, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Sox11 regulates mammary tumour-initiating and metastatic capacity in Brca1 -deficient mouse mammary tumour cells

doi: 10.1242/dmm.046037

Figure Lengend Snippet: Sox11 regulates tumour growth, lineage status and metastasis of Brca1.3 cells in vivo . (A) Tumour volume curve of the experiment. Fifteen mice per cohort. (B) Bar graph of tumour volumes at 2 weeks post-injection. (C) Immunohistochemistry of primary tumours showing the expression markers SMA and K14 (three samples for sg-control immunohistochemistry for SMA). The others are eight samples per group. Scale bars: 5 mm (top panels) and 1 mm (lower panels). (D) The percentage of metastasis to each distant organ in each cohort. (E) Bioluminescence imaging and measurement (RLU) of secondary organs ex-vivo revealed the presence of tumour cells in brain, lung, spleen and liver at study endpoint. N =13 and 14 mice per group. (F) Serial dilutions of primary tumour sg-Sox11-1 and sg-control cells were resuspended in Matrigel, transplanted into the left and right inguinal mammary fat pads of NSG mice. n =5 mice, ten injected sites per group. (G) In vivo limiting dilution assay of Brca.3 sg-Sox11-1 and sg-control cells. Stem cell frequency was calculated using the ELDA software web interface. Data are mean±s.d. RLU, relative light unit.

Article Snippet: sgRNAs were designed using the CRISPOR algorithm ( www.crispor.tefor.net ). sgRNA sequences targeting Sox11 (Gene ID, 20666) were cloned into the Lenti-CRISPR–EGFP plasmid (Addgene, 75159 ) using a BsmBI enzyme site (sgRNA-1, 5′-TGGACGAGAGCGACCCGGAC-3′; sgRNA-2, 5′-GAGCCCCGACAAGAGCGCGG-3′).

Techniques: In Vivo, Injection, Immunohistochemistry, Expressing, Control, Imaging, Ex Vivo, Limiting Dilution Assay, Software

Journal: Nature Communications

Article Title: Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer

doi: 10.1038/ncomms4830

Figure Lengend Snippet: ( a ) ZAK gene model and protein domain structure. Thick bars: coding exons; thin bars: UTR. TV2 lacks the last nine exons and has a long terminal coding and non-coding exon. Blue and red indicate unique TV1 and TV2 sequences. Transcript variant 1 (TV1) encodes a longer protein product with a sterile alpha motif domain. ( b ) ZAK TV1 fraction is significantly higher in gastric tumours (left) and colon tumours (right), compared with normal adjacent tissues. The fraction of TV1 was measured by the ratio between the number of reads uniquely assignable to TV1 and the number of reads mapped to the entire ZAK gene. To account for smooth muscle contamination in normal tissues, we fit a linear model with smoothelin expression as predictor and the log isoform fraction as response, and used the residuals of the model as the ‘adjusted isoform fraction’. Dots represent samples. Grey lines connect matched tumour and normal samples. The boxes in the box-and-whisker plots represent the interquartile range between the first and third quartiles; the dashed lines (whiskers) extend to the most extreme data points, which is no more than 1.5 times the interquartile range from the box. ( c ) ZAK isoform fractions derived from RNAseq data correlate with quantitative PCR (qPCR) measurements. For nine gastric cancer cell lines in our study, we quantified the ratio between ZAK total expression and ZAK TV1 expression using qPCR, and compared the measurements with the isoform fraction we derived from the RNAseq data. The two measurements have significant correlation (Pearson’s correlation coefficient r =0.91, P -value=0.00077). ( d ) ZAK isoform expression in six TCGA data sets where there are >10 normal samples. Normal samples are represented by blue dots and tumour samples by red dots. ZAK TV1 fraction is significantly higher (adjusted P -value <0.001 and fold change >2) in breast and bladder cancer data (marked by the green asterisks).

Article Snippet: The TaqMan probe and primer assays for ZAK (Hs00370448_m1 for entire gene, Hs00928997_m1 for TV1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH Hs00266705_g1) were obtained from Life Technologies, CA.

Techniques: Variant Assay, Sterility, Expressing, Whisker Assay, Derivative Assay, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer

doi: 10.1038/ncomms4830

Figure Lengend Snippet: ( a ) Immunoblots of ZAK TV1 and TV2 expressions show that protein level of TV1 is higher in gastric tumours and cell lines, compared with normal stomach tissues. ZAK TV1 was detected with Bethyl α-ZAK antibody and TV2 with Sigma α-ZAK antibody (see Methods). ( b ) ZAK TV1, but not TV2, can stimulate multiple transcriptional programs related to cancer pathways. Transcription reporter assay in 293 cells transfected with empty vector, TV1 or TV2 along with the indicated firefly luciferase reporter construct (AP1, NFkB and TCF/LEF). Activity is normalized to cell number using CellTiter-Glo. Immunoblot shows relative ZAK isoform expression from 293 cells transfected with the indicated construct. ZAK was detected with Sigma α-ZAK antibody. ( c ) Depletion of ZAK from gastric cancer cell lines inhibits cell growth. In cell lines where ZAK knockdown led to reduced viability, there was consistently high TV1 expression, while TV2 expression was marginal and variable (for example, IM-95m cell line, see for example, panel a ). Cell viability analysis was carried out 6 days after infection of gastric cancer cell lines with independent ZAK shRNAs. Cell number is normalized to shNTC-infected cells. Immunoblot indicates the level of ZAK-TV1 depletion 4 days after infection. ZAK was detected with Bethyl α-ZAK antibody.

Article Snippet: The TaqMan probe and primer assays for ZAK (Hs00370448_m1 for entire gene, Hs00928997_m1 for TV1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH Hs00266705_g1) were obtained from Life Technologies, CA.

Techniques: Western Blot, Reporter Assay, Transfection, Plasmid Preparation, Luciferase, Construct, Activity Assay, Expressing, Knockdown, Infection