Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Frontiers in pharmacology

Article Title: Edible Bird's Nest, an Asian Health Food Supplement, Possesses Moisturizing Effect by Regulating Expression of Filaggrin in Skin Keratinocyte.

doi: 10.3389/fphar.2021.685982

Figure Lengend Snippet: FIGURE 2 | Expressions of filaggrin and filaggrin-2 are evaluated by real- time PCR. (A) The schematic diagrams of primers flanking the exons of human filaggrin (FLG) and filaggrin-2 (FLG2) genes on chromosome 1q21 are indicated. (B) Filaggrin and filaggrin-2 mRNA levels in cultured differentiated (upper panel; 1.8 mM Ca2+) and undifferentiated (lower panel; low Ca2+) keratinocytes after 24-h treatments of EBN extract and digest, in doses of 1, 10, 100 μg/mL, as indicated. CaCl2 at 0.16 mM was adopted as a positive control. Values are expressed as the percentage of increase in relative to normalized basal expression set at 0, in mean ± SEM, n 4. Statistically significant results are marked with * p < 0.05, ** p < 0.01, and *** p < 0.001 against the control.

Article Snippet: Primary antibodies used: mouse and rabbit anti-filaggrin antibody at 1:100 (Santa Cruz Biotechnology, Dallas, United States, catalog number: sc-66192 and sc-30229), rabbit anti-filaggrin-2 antibody at 1:1,000 (Bethyl Laboratories, Montgomery, United States, catalog number: A305-861A-M), rabbit anti-p38 MAPK antibody at 1:1,000 (Cell Signaling Technology, Danvers, United States, catalog number: 8690S), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) antibody at 1: 1,000 (Cell Signaling Technology, catalog number: 4511S), rabbit anti-GATA3 antibody at 1:1,000 (Cell Signaling Technology, catalog number: 5852S), rabbit anti-phospho-GATA3 (Ser308) antibody at 1:1,000 (Thermo Fisher Scientific, catalog number: MA5-32144), rabbit anti-PPARα + PPARβ antibody at 1:2,000 (Abcam Ltd., Cambridge, United Kingdom, catalog number: ab1788865), rabbit anti-PPARγ antibody at 1:1,000 (Cell Signaling Technology, catalog number: 2435S), rabbit anti-CASP14 antibody at 1:1,000 (Cell Signaling Technology, catalog number: 8519S), and mouse antiα-tubulin antibody at 1:10,000 (Sigma-Aldrich, catalog number: 3873S).

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Positive Control, Expressing, Control

Journal: Frontiers in pharmacology

Article Title: Edible Bird's Nest, an Asian Health Food Supplement, Possesses Moisturizing Effect by Regulating Expression of Filaggrin in Skin Keratinocyte.

doi: 10.3389/fphar.2021.685982

Figure Lengend Snippet: FIGURE 3 | EBN induces the activity of pFLG2-eGFP and filaggrin expression. (A) The fluorescence images of pFLG2-eGFP transfected keratinocytes after 24-h treatments of CaCl2 (0.16 mM, a positive control), EBN extract (100 μg/mL), and EBN digest (100 μg/mL) are shown, n 3. Bar 100 μm. The pFLG2-eGFP plasmid was composed of FLG2 promoter tagged with EGFP reporter. (B) The quantifications from (A) are expressed as the percentage of increase in fluorescence intensity measured under excitation/emission wavelength 488/509 nm to normalized basal activity set at 0 in mean ± SEM, n 6. (C) Cytosolic expression of FLG protein in HaCaT keratinocytes after 24-h treatments of EBN extract (100 μg/mL) and EBN digest (100 μg/mL) are shown, in representative confocal images. CaCl2 at 0.16 mM was adopted as a positive control. n 3. Bar 20 μm. (D) The quantifications from (C) are expressed as the percentage increase in filaggrin fluorescence intensity to normalized basal activity set at 0 in mean ± SEM, n 3. The filaggrin fluorescence intensity of each cell was normalized to total cell area for quantification. Statistically significant results are marked with * p < 0.05, ** p < 0.01, and *** p < 0.001 against the control.

Article Snippet: Primary antibodies used: mouse and rabbit anti-filaggrin antibody at 1:100 (Santa Cruz Biotechnology, Dallas, United States, catalog number: sc-66192 and sc-30229), rabbit anti-filaggrin-2 antibody at 1:1,000 (Bethyl Laboratories, Montgomery, United States, catalog number: A305-861A-M), rabbit anti-p38 MAPK antibody at 1:1,000 (Cell Signaling Technology, Danvers, United States, catalog number: 8690S), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) antibody at 1: 1,000 (Cell Signaling Technology, catalog number: 4511S), rabbit anti-GATA3 antibody at 1:1,000 (Cell Signaling Technology, catalog number: 5852S), rabbit anti-phospho-GATA3 (Ser308) antibody at 1:1,000 (Thermo Fisher Scientific, catalog number: MA5-32144), rabbit anti-PPARα + PPARβ antibody at 1:2,000 (Abcam Ltd., Cambridge, United Kingdom, catalog number: ab1788865), rabbit anti-PPARγ antibody at 1:1,000 (Cell Signaling Technology, catalog number: 2435S), rabbit anti-CASP14 antibody at 1:1,000 (Cell Signaling Technology, catalog number: 8519S), and mouse antiα-tubulin antibody at 1:10,000 (Sigma-Aldrich, catalog number: 3873S).

Techniques: Activity Assay, Expressing, Transfection, Positive Control, Plasmid Preparation, Control

Journal: Frontiers in pharmacology

Article Title: Edible Bird's Nest, an Asian Health Food Supplement, Possesses Moisturizing Effect by Regulating Expression of Filaggrin in Skin Keratinocyte.

doi: 10.3389/fphar.2021.685982

Figure Lengend Snippet: FIGURE 4 | EBN induces the protein levels of filaggrin and filaggrin-2. (A) The protein levels of filaggrin (FLG) and filaggrin-2 (FLG2) in cultured differentiated keratinocytes, after 24-h treatments of EBN extract and digest, were determined in doses of 1, 10, 100 μg/mL, as indicated. CaCl2 at 0.16 mM was adopted as a positive control. (B) The total filaggrin (summation of the isomeric intermediates) and (C) filaggrin-2 protein levels, relative to α-tubulin protein, are expressed as the percentage increase to normalized basal expression set at 0 in mean ± SEM, n 3. Statistically significant results are marked with * p < 0.05, ** p < 0.01, and *** p < 0.001 against the control.

Article Snippet: Primary antibodies used: mouse and rabbit anti-filaggrin antibody at 1:100 (Santa Cruz Biotechnology, Dallas, United States, catalog number: sc-66192 and sc-30229), rabbit anti-filaggrin-2 antibody at 1:1,000 (Bethyl Laboratories, Montgomery, United States, catalog number: A305-861A-M), rabbit anti-p38 MAPK antibody at 1:1,000 (Cell Signaling Technology, Danvers, United States, catalog number: 8690S), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) antibody at 1: 1,000 (Cell Signaling Technology, catalog number: 4511S), rabbit anti-GATA3 antibody at 1:1,000 (Cell Signaling Technology, catalog number: 5852S), rabbit anti-phospho-GATA3 (Ser308) antibody at 1:1,000 (Thermo Fisher Scientific, catalog number: MA5-32144), rabbit anti-PPARα + PPARβ antibody at 1:2,000 (Abcam Ltd., Cambridge, United Kingdom, catalog number: ab1788865), rabbit anti-PPARγ antibody at 1:1,000 (Cell Signaling Technology, catalog number: 2435S), rabbit anti-CASP14 antibody at 1:1,000 (Cell Signaling Technology, catalog number: 8519S), and mouse antiα-tubulin antibody at 1:10,000 (Sigma-Aldrich, catalog number: 3873S).

Techniques: Cell Culture, Positive Control, Expressing, Control

Journal: Frontiers in pharmacology

Article Title: Edible Bird's Nest, an Asian Health Food Supplement, Possesses Moisturizing Effect by Regulating Expression of Filaggrin in Skin Keratinocyte.

doi: 10.3389/fphar.2021.685982

Figure Lengend Snippet: FIGURE 10 | EBN induces filaggrin in ex vivo mouse skin. (A) The expression of filaggrin (FLG) protein in 2-month-old mouse dorsal skin ex vivo after 24-h treatments of EBN extract (100 μg/mL) and EBN digest (100 μg/mL) were assessed by immunofluorescent staining. CaCl2 at 0.16 mM was adopted as a positive control, n 3. Bar 20 μm. Yellow dash lines indicate the fluorescent area of filaggrin. (B) The epidermal thickness of ex vivo mouse dorsal skin after 24-h treatments of CaCl2 (0.16 mM), EBN extract (100 μg/mL) and EBN digest (100 μg/mL) were assessed by H and E staining. Black dash lines include epidermis layer (right panel). The quantification of five randomly selected distance points were assessed for the epidermal thickness (left panel). n 3. Bar 100 μm.

Article Snippet: Primary antibodies used: mouse and rabbit anti-filaggrin antibody at 1:100 (Santa Cruz Biotechnology, Dallas, United States, catalog number: sc-66192 and sc-30229), rabbit anti-filaggrin-2 antibody at 1:1,000 (Bethyl Laboratories, Montgomery, United States, catalog number: A305-861A-M), rabbit anti-p38 MAPK antibody at 1:1,000 (Cell Signaling Technology, Danvers, United States, catalog number: 8690S), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) antibody at 1: 1,000 (Cell Signaling Technology, catalog number: 4511S), rabbit anti-GATA3 antibody at 1:1,000 (Cell Signaling Technology, catalog number: 5852S), rabbit anti-phospho-GATA3 (Ser308) antibody at 1:1,000 (Thermo Fisher Scientific, catalog number: MA5-32144), rabbit anti-PPARα + PPARβ antibody at 1:2,000 (Abcam Ltd., Cambridge, United Kingdom, catalog number: ab1788865), rabbit anti-PPARγ antibody at 1:1,000 (Cell Signaling Technology, catalog number: 2435S), rabbit anti-CASP14 antibody at 1:1,000 (Cell Signaling Technology, catalog number: 8519S), and mouse antiα-tubulin antibody at 1:10,000 (Sigma-Aldrich, catalog number: 3873S).

Techniques: Ex Vivo, Expressing, Staining, Positive Control

Journal: Ecotoxicology and environmental safety

Article Title: Betulinic acid attenuates cyclophosphamide-induced intestinal mucosa injury by inhibiting the NF-κB/MAPK signalling pathways and activating the Nrf2 signalling pathway.

doi: 10.1016/j.ecoenv.2021.112746

Figure Lengend Snippet: Fig. 6. BA decreased the intestinal inflammatory responses in CYP-challenged mice by inhibited the activation of NF-κB/MAPK signalling pathways. The mRNA expression of intestinal inflammatory cytokines IL-1β (A), IL-6 (B), TNF-α (C) and IL-10 (D) were analysed by RT-PCR in mice. The protein expression of p-NF-κB, NF- κB, p-IκBα, IκBα (E), p-p38, p38, p-JNK, JNK, p-ERK and ERK (H) in jejunum were detected by Western blot. The ratios of p-NF-κB/NF-κB (F), p-IκBα/IκBα (G), p-p38/ p38 (I), p-ERK/ERK (J) and p-JNK/JNK (K) protein bands for each region were quantified using densitometry and presented in the graph. The values are presented as mean ± SEM (n = 3), *p < 0.05, **p < 0.01 compared with the control group; #p < 0.05, ##p < 0.01 compared with the CYP group.

Article Snippet: Primary antibodies for NF-κB (4764S), phosphorylated NF-κB (p-NF-κB) (3033S), NF-κB inhibitor alpha (IκBα) (4812S), p-IκBα (2859S), extracellular signal-regulated kinase (ERK) (9102S), p-ERK (9101S), c-Jun N-terminal kinase (JNK) (9252S), p-JNK (9251S), p38 mitogen-activated protein kinase (p38) (9212S), p-p38 (9211S), B cell lymphoma/lewkmia-2 (Bcl-2) (2876S), Bcl-2 associated X protein (Bax) (2772S), caspase-3 (9662S), kelch-like ECH-associated protein-1 (Keap1) (8047S), Nrf2 (12721S), HO-1 (82206S) and β-actin (3700S) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Reproductive toxicology (Elmsford, N.Y.)

Article Title: A mixture of persistent organic pollutants detected in human follicular fluid increases progesterone secretion and mitochondrial activity in human granulosa HGrC1 cells.

doi: 10.1016/j.reprotox.2021.07.009

Figure Lengend Snippet: Fig. 5. Effects of the EDC mixture (Mix 0.1, Mix 1, and Mix 10) on (A) mRNA and (B) protein expression of GLUT1, (C) mRNA, and (D) protein expression of GLUT4, (E) glucose uptake, (F) mRNA expression of HK2, (G) mRNA expression of LDHA, (H) the PER, (I) basal glycolysis, and (J) the mitoOCR/glycoPER ratio in HGrC1 cells after 24 h (gene expression and the glycolytic rate) or 48 h (protein expression). mRNA expression of GLUT1 or GLUT4 in vehicle-treated cells was set to 1.0. RQ, relative quantity. Each bar represents the mean ± SEM of three independent experiments. C, control (0.01 % DMSO). RLU, relative luminescence units. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Expression levels of GLUT1 (SLC2A1; Hs00892681_m1), glucose transporter 4 (GLUT4; SLC2A4; Hs00168966_m1), CYP19A1 (Hs00240671_m1), 3βHSD (HSD3B1; Hs04194787_g1), hexokinase 2 (HK2; HK2; Hs00606086_m1), and LDHA (LDHA; Hs00855332_g1) after treatment with Mix 0.1, Mix 1, or Mix 10 for 24 h were measured by realtime PCR using the TaqMan Gene Expression Assay (Applied Biosystems/ThermoFisher Scientific, Waltham, MA, USA) as described in our previous study [13].

Techniques: Expressing, Gene Expression, Control

Journal: Reproductive toxicology (Elmsford, N.Y.)

Article Title: A mixture of persistent organic pollutants detected in human follicular fluid increases progesterone secretion and mitochondrial activity in human granulosa HGrC1 cells.

doi: 10.1016/j.reprotox.2021.07.009

Figure Lengend Snippet: Fig. 6. Effects of STF-31 (0.01, 0.05, 0.1, and 0.5 μM) on (A) mitochondrial activity, (B) mRNA and (C) protein expression of GLUT1, and (D) mRNA and (E) protein expression of GLUT4 in HGrC1 cells after 24 h (gene expression) or 48 h (protein expression and mitochondrial activity). mRNA expression of GLUT1 and GLUT4 in vehicle-treated cells was set to 1.0. RQ, relative quantity. Each bar represents the mean ± SEM of three independent experiments. C, control (0.01 % DMSO). RFU, relative fluorescence units. *P < 0.05 and ***P < 0.001.

Article Snippet: Expression levels of GLUT1 (SLC2A1; Hs00892681_m1), glucose transporter 4 (GLUT4; SLC2A4; Hs00168966_m1), CYP19A1 (Hs00240671_m1), 3βHSD (HSD3B1; Hs04194787_g1), hexokinase 2 (HK2; HK2; Hs00606086_m1), and LDHA (LDHA; Hs00855332_g1) after treatment with Mix 0.1, Mix 1, or Mix 10 for 24 h were measured by realtime PCR using the TaqMan Gene Expression Assay (Applied Biosystems/ThermoFisher Scientific, Waltham, MA, USA) as described in our previous study [13].

Techniques: Activity Assay, Expressing, Gene Expression, Control, Fluorescence

Journal: Signal transduction and targeted therapy

Article Title: VAV2 is required for DNA repair and implicated in cancer radiotherapy resistance.

doi: 10.1038/s41392-021-00735-9

Figure Lengend Snippet: Fig. 1 VAV2 functions as a radioresistant oncogene in ESCC. a Schematic diagram for the constructions of patient-derived xenograft (PDX) of esophageal squamous-cell carcinoma (ESCC), PDX-derived cells (PDC) and irradiation (IR) treatment. b Growth curves of PDXs from 6 individuals with ESCC, showing different sensitivity to IR. Data at each time point are mean ± SD from 3 mice. c Hematoxylin-eosin (H&E) and immunohistochemical (IHC) staining of Ki67 in PDX tumors with or without IR. Scale bars, 100 µm. d Proliferation curves of PDC-2 and PDC-5 cells with or without IR (4 Gy), showing different sensitivity to IR. Data are mean ± SEM from 3 independent experiments and most error bars are with the symbols. **P < 0.01 and ****P < 0.0001 of Student’s t-test. e Heat map showing the differentially expressed genes detected by RNA-sequencing in radiosensitive or radioresistant PDXs. f Venn diagram displaying the overlapped genes among 1,660 apparently overexpressed genes in 3 radioresistant PDXs and 149 amplified and overexpressed genes in 94 clinical ESCC specimens as described previously.16 g Heat map displaying the inhibitory effects of silencing 8 genes on the malignant phenotypes of two ESCC cell lines. The number presents the inhibitory efficiency and each cell line had 3 independent replications. h H&E and IHC analysis of VAV2 protein level in radiosensitive or radioresistant PDXs. Scale bars, 100 µm. i Box and bar plots comparing the VAV2 protein levels between ESCC and non-tumor tissue samples. P of Mann–Whitney test. j Kaplan–Meier curves of patient survival according to the VAV2 IHC score in tumors. High, IHC score > 6 and low, IHC score ≤6. Also present with Kaplan–Meier plot is the hazard ratio (HR) and 95% confidence interval (CI) from multivariate Cox proportional hazard models, including age, sex, tumor stage as covariates

Article Snippet: The antibody for coimmunoprecipitation of VAV2 (sc-271442) and Ku70 (sc-17789) were from Santa Cruz while the antibody against FLAG (F1804) or Ku80 (MA5-12933) was from Sigma-Aldrich or Thermo Fisher.

Techniques: Derivative Assay, Irradiation, Immunohistochemical staining, Immunohistochemistry, RNA Sequencing, MANN-WHITNEY

Journal: Signal transduction and targeted therapy

Article Title: VAV2 is required for DNA repair and implicated in cancer radiotherapy resistance.

doi: 10.1038/s41392-021-00735-9

Figure Lengend Snippet: Fig. 2 Irradiation induces ESCC cell radioresistance by evoking aberrant VAV2 overexpression. a, b Forced VAV2 overexpression in KYSE150 cells (a) or radiosensitive PDC-2 cells (b) caused resistance of cells to IR treatment. Left panels show proliferation curves of cells and right panels shows fractions of cell survival by limiting dilution assays. IR, irradiation (4 Gy). c Silencing VAV2 expression by siRNA in radioresistant PDC-5 cells significantly increased sensitivity of cells to IR treatment. Left panels show proliferation curves of cells with siRNA#1 (the results of siRNA#2 are shown in Supplementary Fig. S2g); middle and right panels show fractions of cell survival by limiting dilution assays. IR, irradiation (4 Gy). d, e Western blot analysis showed overexpression of VAV2 and γ-H2AX in ESCC cells treated with radiation (10 Gy), which is time- (d) and dose- dependent (e). f Comparison of VAV2 mRNA (left panel) and protein (right panel) levels in IR-induced radioresistant KYSE150 cells (KYSE150R) and its parental KYSE150 cells. Results are mean ± SEM from three independent determinations and each had three triplicates. P of Mann–Whitney test. g Tumor spheres of KYSE150 and KYSE150R cells treated with or without radiation (4 Gy). Left panel shows representative images of tumor spheres and right panel shows statistics. Scales bars, 100 μm. Data are mean ± SEM from at least three independent experiments and five fields were randomly selected for each experiment. *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001 of Student’s t-test

Article Snippet: The antibody for coimmunoprecipitation of VAV2 (sc-271442) and Ku70 (sc-17789) were from Santa Cruz while the antibody against FLAG (F1804) or Ku80 (MA5-12933) was from Sigma-Aldrich or Thermo Fisher.

Techniques: Irradiation, Over Expression, Expressing, Western Blot, Comparison, MANN-WHITNEY

Journal: Signal transduction and targeted therapy

Article Title: VAV2 is required for DNA repair and implicated in cancer radiotherapy resistance.

doi: 10.1038/s41392-021-00735-9

Figure Lengend Snippet: Fig. 3 VAV2 overexpression promotes DNA repair in ESCC cells. a, b Volcano plots displaying upregulated (fold-change > 1.3; P < 0.05) or downregulated genes (fold-change < 0.7; P < 0.05) in KYSE150 cells with VAV2 overexpression (a) or knockout (b). Data are from three experiments. c Volcano plot of gene expression changes in both cell lines with VAV2 overexpression (OE) or knockout (KO). Orange, genes upregulated in cells with VAV2 OE and downregulated in cells with VAV2 KO. Purple, genes downregulated in cells with VAV2 OE and upregulated and in cells with VAV2 KO. Blue, genes upregulated in cells with VAV2 OE or KO. Red, genes downregulated in cells with VAV2 OE or KO. Gray, genes unchanged in cells with VAV2 OE or KO. All P < 0.05 except for genes in gray color. d Gene set enrichment analysis (GSEA) of genes in orange or purple in c showed several pathways including DNA repair-related pathway positively (left panel) or negatively (right panel) correlated with VAV2 expression. e Verification of the expression changes of genes in DNA repair pathway identified by GSEA in (b) in cells with VAV2 OE or KO. The level of mRNA was determined by real-time-quantitative PCR and data are mean ± SEM from three independent determinations. **P < 0.01; ***P < 0.001 and ****P < 0.0001 of Student’s t-test. f–h DNA double-strand breaks expressed by γ-H2AX level in VAV2-overexpressing KYSE150 (f), KYSE450 (g), and KYSE30 (h) cells treated with or without irradiation (IR, 4 Gy). Left panels show γ-H2AX and VAV2 levels by western blot analysis in cells 2 h after IR. Middle panels show images of γ-H2AX foci in cells at various time points of IR as indicated. Scale bars, 20 µm. Right panels represent the statistics. Data are means ± SEM from three experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant of Student’s t-test. i DNA double-strand breaks detected by comet assays in PDC-5 cells with or without VAV2 silenced by siRNA and treated with or without IR (4 Gy). Left panel shows fluorescence images of comet assays. Scale bars, 100 µm. Right panel shows the statistics. Data are mean ± SEM from three replicates and ten fields were randomly selected for each experiment. ***P < 0.001 of Student’s t-test

Article Snippet: The antibody for coimmunoprecipitation of VAV2 (sc-271442) and Ku70 (sc-17789) were from Santa Cruz while the antibody against FLAG (F1804) or Ku80 (MA5-12933) was from Sigma-Aldrich or Thermo Fisher.

Techniques: Over Expression, Knock-Out, Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Irradiation, Western Blot

Journal: Signal transduction and targeted therapy

Article Title: VAV2 is required for DNA repair and implicated in cancer radiotherapy resistance.

doi: 10.1038/s41392-021-00735-9

Figure Lengend Snippet: Fig. 4 VAV2 is necessary in Ku70 and Ku80-mediated DNA NHEJ repair. a Fourteen top proteins potentially associate with VAV2 identified by mass spectrometry in ESCC cells ectopically overexpressing FLAG-tagged VAV2. Cell lysates were immunoprecipitated with antibody against FLAG and antibody against IgG was used as negative control. b Proteins potentially associate with VAV2 in KYSE30 and KYSE450 cells. Left panel is a Venn diagram showing 68 proteins identified in both cell lines. Right panel shows score of each protein. c–e Western blot detection of VAV2, Ku70 and Ku80 proteins by reciprocal immunoprecipitation with antibody against VAV2 (c), Ku70 (d) or Ku80 (e) in KYSE150 and KYSE450 cells. IgG was used as control. f Immunofluorescence analysis of VAV2 and Ku70 co-staining in KYSE150, KYSE450, PDC-4 and PDC-5 cells with or without IR (4 Gy), showing colocalization of VAV2 and Ku70. DAPI was used to label the nucleus. Scale bars, 20 µm. g Multiple immunofluorescences analysis of VAV2, Ku70, and Ku80 in clinical ESCC tumor tissues. All tumors (T) show strong colocalization signal of the 3 proteins while the corresponding non-tumor tissues (N) from patient 3, which had low VAV2, show very weak colocalization signal. Merge 1 represents the merge of VAV2 and Ku70, Merge 2 represents the merge of VAV2 and Ku80 and Merge 3 represents the merge of VAV2, Ku70, and Ku80. Scale bars, 100 µm. h Western blot analysis of FLAG-VAV2, Ku70 and Ku80 in ESCC cells ectopically expressing FLAG-tagged VAV2 and treated with siVAV2. Cell lysates were immunoprecipitated with antibody against FLAG or IgG. i Western blot analysis of VAV2, Ku70, and Ku80 in ESCC cells overexpressing VAV2. Cell lysates were immunoprecipitated with antibody against Ku70 or IgG. j Effects of increasing VAV2 expression and decreasing Ku70 expression on DNA damage in ESCC cell lines KYSE150 (upper panels) and KYSE450 (lower panels). Left panels show Ku70, Ku80, VAV2 and γ-H2AX levels detected by Western blot analysis in cells with different treatment. Middle panels are image of comet assays showing overexpression of VAV2 significantly diminished DNA damage, which could be reversed by knockdown of Ku70 expression. Right panel are the statistics of tail moments in comet assays. OE, overexpression. Data are mean ± SEM from 3 replicate experiments and 10 fields were randomly selected for each experiment. **P < 0.01; ****P < 0.0001 and ns, not significant of Student’s t-test

Article Snippet: The antibody for coimmunoprecipitation of VAV2 (sc-271442) and Ku70 (sc-17789) were from Santa Cruz while the antibody against FLAG (F1804) or Ku80 (MA5-12933) was from Sigma-Aldrich or Thermo Fisher.

Techniques: Mass Spectrometry, Immunoprecipitation, Negative Control, Western Blot, Control, Staining, Expressing, Over Expression, Knockdown

Journal: Signal transduction and targeted therapy

Article Title: VAV2 is required for DNA repair and implicated in cancer radiotherapy resistance.

doi: 10.1038/s41392-021-00735-9

Figure Lengend Snippet: Fig. 5 VAV2 overexpression excessively activates STAT1 signaling. a Scatter diagram shows the overlapped proteins in KYSE150 cells with VAV2 overexpression (OE) or knockout (KO). Orange, proteins upregulated in cells with VAV2 OE and downregulated in cells with VAV2 KO; Blue, proteins upregulated in cells with VAV2 OE or KO; Purple, proteins downregulated in cells VAV2 OE and upregulated in cells with VAV2 KO and Red, proteins downregulated in cells with VAV2 OE or KO. P < 0.05 of Student’s t-test compared with that in cells without VAV2 expression change (Control). The level changes of proteins in gray were not statistically significant between treated and control cells (P > 0.05). b Western blot analysis shows upregulation of STAT1, ATF7 and GTF2E1 in KYSE450 cells with VAV2 OE. p-STAT1, phosph-STAT1. c Western blot analysis of total STAT1, phosph-STAT1 (p-STAT1) and γ-H2AX levels in KYSE30 and KYSE150 cells with VAV2 OE or KO. d Comparison of VAV2, STAT1, p-STAT1, and γ-H2AX levels in KYSE150, KYSE450, irritation (IR)-induced radioresistant KYSE450R and KYSE450R cells. e Spearman correlation of STAT1 and VAV2 mRNA levels in clinical ESCC samples. f–i STAT1 inhibitor Fludarabine (Flud) significantly enhanced the radiosensitivity of radioresistant ESCC cells to IR in vitro. Shown are inhibitory effects of IR (2 Gy), Fludarabine (0.1 and 0.05 μM for cell growth or colony formation assays, respectively) or combination of IR and Fludarabine on cell growth detected by CCK-8 assays (f, h) and colony formation (g, i) of PDC-5 and KYSE150R. Data are mean ± SEM from 3 experiments and each had 3 replications. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 and ns, not significant of Student’s t-test. j STAT1 inhibitor Fludarabine (40 mg/kg) significantly enhanced the radiosensitivity of mouse xenografts derived from radioresistant PDC-5 to IR (10 Gy). Shown are curves of tumor growth overtime and tumors from each mouse in different groups are shown in Supplementary Fig. S5m. Data are mean ± SD from five mice. *P < 0.05; **P < 0.01 and ns, not significant of Student’s t-test. See methods for Fludarabine and IR treatment

Article Snippet: The antibody for coimmunoprecipitation of VAV2 (sc-271442) and Ku70 (sc-17789) were from Santa Cruz while the antibody against FLAG (F1804) or Ku80 (MA5-12933) was from Sigma-Aldrich or Thermo Fisher.

Techniques: Over Expression, Knock-Out, Expressing, Control, Western Blot, Comparison, In Vitro, CCK-8 Assay, Derivative Assay

Journal: Signal transduction and targeted therapy

Article Title: VAV2 is required for DNA repair and implicated in cancer radiotherapy resistance.

doi: 10.1038/s41392-021-00735-9

Figure Lengend Snippet: Fig. 6 VAV2 predicts radiotherapy vulnerability of ESCC. a Chemoradiotherapy efficacy in patients with ESCC (n = 31) as function of the VAV2 level in tumor samples detected by immunohistochemical (IHC) staining, showing ESCC having IHC score >6 were significantly more resistant to the therapy compared with ESCC having the score ≤6. b–d Correlation between the VAV2 and γ-H2AX levels in ESCC tumors before and after chemoradiotherapy. Images show computed tomography of tumors before and after treatment (left) and H&E, VAV2 and γ-H2AX IHC staining in tumors before (middle) and after (right) treatment in ESCC sensitive (b) or resistant (c) to chemoradiotherapy. Scale bars, 100 µm. d Shown are the Spearman correlations between VAV2 and γ-H2AX levels in tumors before and after treatment. e Kaplan–Meier curves of patient survival according to VAV2 level in ESCC tumor. VAV2 high, IHC score > 6; VAV2 low, IHC score ≤6. Also present with the Kaplan–Meier curves is the hazard ratio (HR) and 95% confidence interval (CI) from multivariate Cox proportional hazard models, including age, sex, tumor stage as covariates. Four of 31 patients lost follow-up and thus were excluded in the analysis

Article Snippet: The antibody for coimmunoprecipitation of VAV2 (sc-271442) and Ku70 (sc-17789) were from Santa Cruz while the antibody against FLAG (F1804) or Ku80 (MA5-12933) was from Sigma-Aldrich or Thermo Fisher.

Techniques: Immunohistochemical staining, Immunohistochemistry, Computed Tomography

Journal: Signal transduction and targeted therapy

Article Title: VAV2 is required for DNA repair and implicated in cancer radiotherapy resistance.

doi: 10.1038/s41392-021-00735-9

Figure Lengend Snippet: Fig. 7 The schematic illustration for the possible mechanisms of VAV2-mediated radioresistance of ESCC cells and Fludarabine as a radiosensitizer. Overexpressed VAV2 in cancer cells increases VAV2-Ku70/Ku80 complex formation and activates signal transducer and activator of transcription 1 (STAT1) signaling, which enhances the ability of cells to repair DNA damage caused by ionizing radiation (IR) and thus promotes cancer growth. However, Fludarabine can inhibit STAT1 activity and reverses the vulnerability of VAV2-overexpressing cancer cells to IR. On the other hand, cancer cells without VAV2 overexpression are relatively sensitive to IR

Article Snippet: The antibody for coimmunoprecipitation of VAV2 (sc-271442) and Ku70 (sc-17789) were from Santa Cruz while the antibody against FLAG (F1804) or Ku80 (MA5-12933) was from Sigma-Aldrich or Thermo Fisher.

Techniques: Activity Assay, Over Expression

Journal: Pharmaceutical biology

Article Title: Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl 4 /2-AAF.

doi: 10.1080/13880209.2021.1961820

Figure Lengend Snippet: Figure 3. YGJ inhibits the activation of M1 macrophages and non-canonical Wnt signalling pathways and inhibits the differentiation of hepatic progenitor cells into myofibroblasts in vivo. (a) STAT1, IRF3, IRF5, IRF8, and SOCS3 protein bands were depicted in the immunoblot images, and (b) the densitometric quantification of the protein bands presented as a histogram (n ¼ 5 per group). (c) The mRNA expressions of Wnt-4, -5 A, -5B, FZD-2, -3, and -6 in liver were measured by RT-PCR and nor- malized to GAPDH mRNA (n ¼ 5 per group). (d) Wnt5A, Wnt5B, and FZD2 protein bands were depicted in the immunoblot images, and (e) the densitometric quantifi- cation of the protein bands presented as a histogram (n ¼ 5 per group). (f) OV6, SOX9, EpCAM, CK19, and Hep mRNA expressions were measured by RT-PCR and normalized to GAPDH mRNA (n ¼ 5 per group). (g) EpCAM protein bands were depicted in the immunoblot images, and (h) the densitometric quantification of the protein bands presented as a histogram (n ¼ 5 per group). p < 0.05 and p < 0.01. N: untreated group (control); 2-AAF/CCl4: 2-acetylaminofluorene/carbon tetra- chloride-treated group; YGJ: 2-AAF/CCl4 þ Yiguanjian decoction-treated group; SORA: 2-AAF/CCl4 þ sorafenib-treated group.

Article Snippet: Rabbit polyclonal antibodies against Oval Cell Marker (OV6), cytokeratin (CK) 19, signal transducer and activator of transcription (STAT) 5A, STAT5B, STAT6, interferon regulatory factor (IRF) 3, IRF5, IRF8, frizzled (FZD) 2, and LDL receptor-related protein (LRP) 5 were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Activation Assay, In Vivo, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Pharmaceutical biology

Article Title: Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl 4 /2-AAF.

doi: 10.1080/13880209.2021.1961820

Figure Lengend Snippet: Figure 4. YGJ inhibits the differentiation of WB-F344 cells into myofibroblasts through inhibiting the activation of M1 macrophages. (a) a-SMA immunofluorescent staining (green) in WB-F344 cells (200) (the presented in vitro experiments were conducted in the same batch, the normal and model groups used the same pictures as published articles. The figures of WB-F344 co-cultured with inactivated macrophages and WB-F344 co-cultured with M1 macrophages were reused form a previous study [Ying Xu et al. 2018] and are reproduced with permission here). (b) a-SMA mRNA expression in WB-F344 cells was measured by RT-PCR and normalized to GAPDH mRNA (n ¼ 3 per group). (c) STAT1, NF-jB, IRF3, IRF5, IRF8, and SOCS3 mRNA expressions in RAW264.7 cells were measured by RT-PCR and normalized to GAPDH mRNA (n ¼ 3 per group); (d) STAT1, NF-jB, IRF3, IRF5, and SOCS3 protein bands were depicted in the immunoblot images, and (e) the densitometric quantification of the protein bands presented as a histogram (n ¼ 3 per group). p < 0.05 and p < 0.01. N: WB-F344 cells co-cultured with inactivated RAW264.7 cells; M: WB-F344 cells co-cultured with LPS (100ng/mL)-activated RAW264.7 cells (referred to as LPS-RAW264.7); YGJ: WB-F344 cells co-cultured with LPS-RAW264.7 treated with Yiguanjian decoction; WIF-1: WB-F344 cells co-cultured with LPS-RAW264.7 treated with Wnt inhibitory factor-1.

Article Snippet: Rabbit polyclonal antibodies against Oval Cell Marker (OV6), cytokeratin (CK) 19, signal transducer and activator of transcription (STAT) 5A, STAT5B, STAT6, interferon regulatory factor (IRF) 3, IRF5, IRF8, frizzled (FZD) 2, and LDL receptor-related protein (LRP) 5 were purchased from Proteintech Group Inc. (Chicago, IL, USA).

Techniques: Activation Assay, Staining, In Vitro, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot