Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: White matter microglia/macrophages are activated in the dorsal funiculi of male Plp1 mutant mice. Cervical spinal cord sections from ( A ) mildly-affected P16 rsh ( B ) severely-affected P16 msd ( C ) subclinical P25 Plp1 #66 heterozygous ( D ) moderately-affected P25 Plp1 #66 homozygous mice labeled with antibodies against Iba-1 (red) and CD68 (green). Many Iba-1 positive microglia/macrophages with swollen cell bodies and short processes are labeled with anti-CD68 antibodies in rsh and msd mice, which is indicative of activated cells. The insets in these panels show the canonical activated morphology revealed with anti-Iba-1 antibodies and strong CD68 staining. In contrast, there is little evidence for broad CD68 labeling in Plp1 #66 heterozygous mice in panel C, which is consistent with previous data that immune cells are rarely activated . Activation of microglia/macrophages is apparent for Plp1 #66 homozygous mice in panel D, which exhibit an intermediate level of CD68 labeling. Scale bar: 95 μm, insets 15 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Mutagenesis, Labeling, Staining, Activation Assay

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Proportion of cervical spinal cord Iba-1 + microglia/macrophages in dorsal column, ventral column and lateral funiculus that are activated (CD68 + ).

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques:

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Oligodendrocytes in gray matter from P16 rsh and msd mice do not undergo a UPR and microglia/macrophages exhibit resting morphology. ( A – C ) Iba1 (red) and CD68 (green) antibody labeling in dorsal spinal cord white matter tracts of P16 wild type ( A ) rsh ( B ) and msd ( C ) mice. Although the morphology and CD68 staining is characteristic of resting microglia/macrophages in wild type mice (white arrowheads), these cells are activated in the Plp1 mutants. ( D – F ) In contrast to white matter regions, microglia/macrophages have a resting state phenotype in the adjacent substantia gelatinosa (gray matter) from wild type and mutant mice (black arrowheads). ( G – I ) A major difference between these regions in rsh and msd mice is the relative abundance of oligodendrocytes undergoing an unfolded protein response (UPR), which is evident by the large number of CHOP + cells (green) in white matter (above the dotted line) compared to gray matter (below). We have previously shown that 100% of CHOP + cells in these mutants are oligodendrocytes . Dotted lines mark the white/gray matter boundary. Scale bar in I: 100 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Antibody Labeling, Staining, Mutagenesis

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Activation of rsh and msd microglia/macrophages in optic nerve at P16. ( A ) Longitudinal section from wild type mouse reveals Iba-1 + microglia/macrophages (red) with non-activated morphology (arrowheads). Most of these cells do not express CD68 or express the protein at low levels (green). ( B ) DAPI staining showing the nuclei of the microglia/macrophages in ( A ). ( C , E ) Iba-1 + microglia/macrophages from rsh ( C ) and msd ( E ) mice exhibit an activated morphology with enlarged cell bodies and thickened processes. Most of these cells express CD68 at high levels, which is localized to perinuclear regions. ( D , F ) DAPI staining of the fields in ( C , E ). Scale bar in F: 30 μm.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Activation Assay, Staining

Journal: Brain Sciences

Article Title: Potential for Cell-Mediated Immune Responses in Mouse Models of Pelizaeus-Merzbacher Disease

doi: 10.3390/brainsci3041417

Figure Lengend Snippet: Expression fold changes of other non-chromosome 17 interferon-induced genes in microarray data for rsh and msd mice.

Article Snippet: Antibodies used were: mouse anti-MBP (1:1000, SMI99, Sternberger Monoclonal Inc., Baltimore, MD, USA); rabbit anti-Iba-1; monoclonal anti-human CD68 antibody (clone 514H12, AbD Serotec, Oxford, UK).

Techniques: Expressing, Microarray

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Workflow showing the selection strategy for KHDRBS1 among DNA-damage response genes transcriptionally activated by Myc and significantly associated to breast cancer prognosis. Venn diagram showing the overlap between Myc-transcriptionally activated genes, DNA-damage response genes and genes associated to breast cancer. Specifically, genes were retrieved from: (i) microarray data of Myc-overexpressing mammospheres (M2) (GSE86407); (ii) published dataset (MD Anderson Human-DNA Repair Genes, https://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repair-genes.html ), BioRad DNA-damage signaling pathway (SAB Target List H96) and recently published DNA-damage-associated genes (Supplementary Table ); and (iii) breast cancer versus normal breast tissues TCGA BRCA and GTeX gene expression data (Supplementary Table ). Genes were further selected for association to the worse relapse-free survival probability in breast cancer (Supplementary Table ) and novelty in the field, excluding known genes associated with BRCAness . B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA ( n = 1212) and GTeX ( n = 179) gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. C Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients stratified by high or low KHDRBS1 expression levels. D GSEA of DNA-repair gene signatures in IMEC-WT versus M2 ( n =3). E Scheme showing MYC and H3K4me3 PCR amplicons localization (red box) on IMEC-WT and M2 cells and layered H3K27ac signals on KHDRBS1 ( SAM68 ) promoter from ENCODE. Chromatin state was assessed by ChromHMM from ENCODE. MYC-MAX binding on multiple cell lines was assessed by ChIP-seq from ENCODE. F ChIP-qPCR estimating MYC binding at SAM68 promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). G qRT-PCR analysis of SAM68 gene expression in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3). H ChIP-qPCR of H3K4me3 deposition at KHDRBS1 ( SAM68 ) promoter in IMEC-WT and M2 cells. Data are mean ± SEM ( n = 3).

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Selection, Microarray, Gene Expression, Expressing, Binding Assay, ChIP-sequencing, ChIP-qPCR, Quantitative RT-PCR

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Kaplan–Meier plots of distant relapse-free survival (DRFS) of BC patients stratified by high or low Sam68 protein expression levels. Patients were categorized according to all molecular subtypes ( n = 211) and Luminal-A ( n = 91), Luminal-B ( n = 61), HER2 + ( n = 27), TNBC ( n = 32), HER2 + + TNBC ( n = 59) BCs. B Box plot representing the distribution of log2 gene expression of KHDRBS1 retrieved from TCGA BRCA gene expression data (RNASeq2GeneNorm). p value was calculated with Wilcoxon rank sum test. The indicated statistics refer to each molecular subtype versus basal subtypes. * p value ≤ 0.05; ** p value ≤ 0.01; **** p value ≤ 0.0001. C ChIP-qPCR estimating MYC and MAX binding at SAM68 promoter in BCSphCs (#4 and #15). Data are mean ± SEM of two independent experiment for each BCSphCs. D Expression of Myc (green color) and Sam68 (red color) on paraffin-embedded sections on parental BC and corresponding PDX tissue. Nuclei were counterstained with Toto-3 (blue color). Scale bar represents 40 µm. E Relative mRNA expression levels of MYC and KHDRBS1 on BCSphCs (#4, #13, and #21) expressing a MycER fusion protein induced by 50 nM of OHT. Data are represented as fold mRNA level changes of OHT-treated cells over vehicle. Data are represented as mean ± SD of three independent experiments. * p value ≤ 0.05; ** p value ≤ 0.01. F Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, and #21) transduced with doxycyclin-inducible non-targeting (nt) and short hairpin Sam68 (shSam68). Data are represented as fold variation of shSam68 over scr. ns not significant; ** p value ≤ 0.01. G Size of tumors generated by orthotopic injection of ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4, #13) in immunocompromised mice (NOD/SCID) at the indicated time points. Data are expressed as mean ± SD ( n = 5 mice per group). ns not significant, *** p value ≤ 0.001.

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Expressing, Gene Expression, ChIP-qPCR, Binding Assay, Transduction, Generated, Injection

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A MYC binding on DNA-damage related genes transcription start sites (TSS) on IMEC-WT and M2 breast cells. B Representative immunofluorescence analysis of Rad51 foci formation in ER+ (MCF7), TNBC (BT549), TNBC BRCA mut (HCC1937) BC established cell lines and BCSphCs (#4) untreated (UT) and after 6 h of 8 Gy single dose γ-irradiation (IR). Nuclei were counterstained by Toto-3 (blue). Scale bar represents 10 µm. C Waterfall plot analysis of doxorubicin (DOX, 200 nM, left panel ), paclitaxel (PTX, 10 nM, middle panel ) and carboplatin (CARB, 100 µM, left panel ) response at 72 h in ER+ and TNBC BC established cell lines and BCSphCs. D Response rate distribution to chemotherapy for ER+ and TNBC BC established cell lines and BCSphCs treated as in ( C ). Middle line shows the median value of response per group, while single points represent the average value of BC cell response to DOX, PTX and CARB. Data are mean of three independent experiments. Statistical analysis was performed by using Kruskal–Wallis test. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01. E Immunoblot analysis of PARP and Sam68 (input) and after immunoprecipitation (IP) with Sam68 antibody in BCSphCs (#15) treated for 4 h with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB). Lamin-B was used as loading control. F Immunoblot analysis of nuclear PAR, PARP, and Sam68 in scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#4) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 4 h. H3 was used as loading control. G Cell proliferation analysis of ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#1, #4, #13, #21) transduced with scramble and short hairpin Sam68 (shSam68) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX) and carboplatin (CARB) for 72 h. Data are represented as fold variation of shSam68 over scramble. Data are mean ± SD of three independent experiments. ns not significant; * p value ≤ 0.05; ** p value ≤ 0.01. H , I Relative mRNA expression levels of RAD51 (H) and MYC (I) on scramble (scr) and short hairpin Sam68 (shSam68) ER+ (MCF7), TNBC (BT549), and TNBC BRCA mut (HCC1937) BC cell lines and BCSphCs (#12 and #13) treated with vehicle, doxorubicin (DOX), paclitaxel (PTX), and carboplatin (CARB) for 24 h. Data are represented as fold mRNA level changes of treated scr and shSam68 cells over vehicle. Data are represented as mean ± SD of three independent experiments. Ns not significant, * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001.

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Binding Assay, Immunofluorescence, Irradiation, Western Blot, Immunoprecipitation, Control, Transduction, Expressing

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Schematic model of DNA-repair signaling pathways mediating the resistance of BC stem-like cells to chemotherapy. B Workflow of purification of sphere cells from serially transplanted BC PDX and their use for in vitro and in vivo drug toxicity testing. C Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and BO2. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). D Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #21) treated with vehicle, olaparib, BO2, cisplatin and olaparib plus BO2 and olaparib plus cisplatin and BO2. Arrows indicate the beginning and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #13, and #21) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. E Immunoblot analysis of Rad51 in BCSphCs (#15) treated with dinaciclib for 24 h at the indicated concentration. Β-actin was used as loading control. F Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs (#4, #13, #15, and #21) treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 2). * p value ≤ 0.05; *** p value ≤ 0.001. G Representative images ( left panel ) and quantification of area ( right panel ) of BC sphere cells (#21), transduced with scramble (scr) and short hairpin Sam68 (shSam68) lentiviral vectors, treated with vehicle and dinaciclib for 6 days. Data are represented as mean ± SEM ( n = 3). Ns not significant, ** p value ≤ 0.01; *** p value ≤ 0.001. Scale bar represents 100 µm. H Size of tumors generated by orthotopic injection of scramble (scr) and short hairpin Sam68 (shSam68) BCSphCs treated with vehicle (veh) and dinaciclib (din). Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated by the injection of BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). **** p value ≤ 0.0001. I Cell viability percentage of BCSphCs (#4, #13, #14, #15, #21) treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). J Synergy plot representing the combination index (CI), computed in CompuSyn by using Chou-Talalay method, for each olaparib and dinaciclib dose pair, calculated from cell viability data of BCSphCs (#13). K Size of tumors generated by orthotopic injection of BCSphCs treated with vehicle, olaparib, dinaciclib and olaparib plus dinaciclib. Arrows indicate the start and the end of treatment. Data are expressed as mean of tumors generated with BCSphCs (#4, #7, #13) ± SEM ( n = 5 mice per group). *** p value ≤ 0.001.

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Protein-Protein interactions, Purification, In Vitro, In Vivo, Generated, Injection, Western Blot, Concentration Assay, Control, Transduction

Journal: Oncogene

Article Title: Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51

doi: 10.1038/s41388-022-02239-4

Figure Lengend Snippet: A Cell viability percentage of scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cell line treated with vehicle and dinaciclib (10 nM) for 6 days. Data are represented as mean ± SEM ( n = 4). * p value ≤ 0.05; ** p value ≤ 0.01; *** p value ≤ 0.001. B Relative mRNA expression levels of RAD51 and MYC on scramble (scr) and short hairpin Sam68 (shSam68) ER+ R (MCF7) BC cells treated with vehicle and dinaciclib for 6 days. Data are represented as fold mRNA level changes of treated scr and shSam68 over vehicle ( n = 3). C Cell viability percentage in ER+ R (MCF7) BC cells treated with vehicle, olaparib and dinaciclib, alone or in combination, at the indicated concentrations for 6 days. Data are represented as mean ± SD ( n = 3). D Kaplan–Meier plots of relapse-free survival (RFS) probability of BC patients of all molecular subtypes stratified by high or low MYC , KHDRBS1 , and RAD51 expression levels. E Schematic model showing the persistence of a BC stem-like population, characterized by high expression levels of MYC, SAM68 , and RAD51 , following standard anticancer therapies.

Article Snippet: For immunohistochemistry analysis, 5 μm-thick paraffin-embedded sections of BC tissues, their normal counterpart and tumor xenografts were heated in a retrieval solution (pH 6.0) for antigen unmasking, permeabilized with PBS plus 0.1% Triton X-100 (TBS) for 10 min on ice and exposed overnight at 4 °C to Sam68 antibody (C-20, rabbit IgG, Santacruz Biotechnology), CD44 (156-3C11, mouse IgG2a, Cell Signaling Technology), γH2AX (Ser139, mouse IgG 1 , JBW301, Merk-Millipore), Rad51 (D4B10 rabbit IgG, Cell Signaling Technology) and Myc (rabbit polyclonal, Cell Signaling Technology).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Checkpoint Kinase 2 Inhibition Can Reverse Tamoxifen Resistance in ER-Positive Breast Cancer

doi: 10.3390/ijms232012290

Figure Lengend Snippet: Knockdown of ATM and CHK2 could reduce BQ expression. ( A ) The effect of ATM knockdown on pATM (Ser1981) and BQ expression. ( B ) The effect of CHK2 knockdown on p-CHK2 (Thr68) BQ expression. ( C ) The effect of ATR knockdown on p-ATR (Ser1989) BQ expression. ( D ) The effect of CHK1 knockdown on p-CHK1 (Ser345) and BQ expression. LCC2 and AK-47 cells were transfected with 20 nM of the siRNA. Cells were harvested 72 h post-transfection. Western blot was employed to detect the expression of candidate proteins. GAPDH was used as the loading control.

Article Snippet: The slides were rinsed with TBST twice, followed by incubation with primary monoclonal anti-p-CHK2 (Thr68) antibody (p-CHK2; 1:50; 2197; Cell Signaling Technology, Danvers, MA, USA) and BQ323636.1 antibody (1:50; D-12, Veritech Ltd., Hong Kong) at 4 °C overnight.

Techniques: Knockdown, Expressing, Transfection, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Checkpoint Kinase 2 Inhibition Can Reverse Tamoxifen Resistance in ER-Positive Breast Cancer

doi: 10.3390/ijms232012290

Figure Lengend Snippet: Knockdown of ATM and CHK2 could reduce TAM resistance. The effect of ATM knockdown on TAM response in ( A ) LCC2 and ( B ) AK-47. The effect of CHK2 knockdown on TAM response in ( C ) LCC2 and ( D ) AK-47. LCC2 and AK-47 are TAM-resistant cell lines. The cells were incubated with 20 nM of the siRNA and 4 µM of TAM for 96 h. Cell viability was determined by MTT assay. Results are shown as mean ± SD from six independent experiments. Student’s t -test was employed to compare the statistical significance between DMSO and TAM groups: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The slides were rinsed with TBST twice, followed by incubation with primary monoclonal anti-p-CHK2 (Thr68) antibody (p-CHK2; 1:50; 2197; Cell Signaling Technology, Danvers, MA, USA) and BQ323636.1 antibody (1:50; D-12, Veritech Ltd., Hong Kong) at 4 °C overnight.

Techniques: Knockdown, Incubation, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Checkpoint Kinase 2 Inhibition Can Reverse Tamoxifen Resistance in ER-Positive Breast Cancer

doi: 10.3390/ijms232012290

Figure Lengend Snippet: Targeting CHK2 by CCT241533 could reverse TAM resistance. The long-term effect of KU-55933 on TAM response in ( A ) LCC2 and ( B ) AK-47. The cells were treated with 10 nM of CCT241533 and 4 µM of TAM for 14 days. A clonogenic assay was employed to determine cell viability. Student’s t -test was employed to compare the statistical significance between DMSO and TAM groups. ( C ) The effect of CCT241533 on BQ expression. LCC2 and AK-47 were treated with 10 nM of CCT241533 for 72 h. Western blot was employed to determine BQ expression. GAPDH was used as the loading control. ( D ) The knockdown efficiency of shRNA against CHK2. LCC2 cells were stably transfected to establish cell lines with stable CHK2 knockdown. Two independent stable clones were selected. Western blot was employed to detect the expression of BQ, p-CHK2 (Thr68) and CHK2. GAPDH was used as the loading control. ( E ) Knockdown of CHK2 could abolish the effect of CCT241533 on reversing TAM resistance. LCC2 and AK-47 were treated with 10 nM of CCT241533 and 4 µM of TAM for 96 h. Cell viability was determined by MTT assay. One-way ANOVA with Bonferroni’s post-test was employed to compare the statistical significance with indicated groups. ( F ) CCT241533 could reduce TAM resistance in female nude mice. LCC2 cells were used for establishing xenografts. The mice were treated with saline, TAM (0.5 mg/Kg) and CCT241533 (1 mg/Kg, 2.5 mg/Kg and 5 mg/Kg) via subcutaneous injection. The mice were treated twice per week for six weeks. The tumour growth curve was plotted. Two-way ANOVA with Turkey post-test was employed to compare the statistical significance with the saline group: ** p < 0.01 and *** p < 0.001.

Article Snippet: The slides were rinsed with TBST twice, followed by incubation with primary monoclonal anti-p-CHK2 (Thr68) antibody (p-CHK2; 1:50; 2197; Cell Signaling Technology, Danvers, MA, USA) and BQ323636.1 antibody (1:50; D-12, Veritech Ltd., Hong Kong) at 4 °C overnight.

Techniques: Clonogenic Assay, Expressing, Western Blot, Control, Knockdown, shRNA, Stable Transfection, Transfection, Clone Assay, MTT Assay, Saline, Injection

Journal: International Journal of Molecular Sciences

Article Title: Checkpoint Kinase 2 Inhibition Can Reverse Tamoxifen Resistance in ER-Positive Breast Cancer

doi: 10.3390/ijms232012290

Figure Lengend Snippet: CHK2 could phosphorylate BQ to enhance its protein stability. ( A ) CHK2 could interact with BQ. LCC2 cells were lysed and immunoprecipitated with anti-CHK2. The immunoprecipitant was analysed by Western blot to detect the presence of endogenous BQ and CHK2. ( B ) The treatment of CCT241533 could reduce the degree of BQ phosphorylation. LCC2 cells were transfected with pcDNA3.1-His-BQ, and the cells were treated with 10 nM of CCT241533 for 48 h. The cells were immunoprecipitated with anti-His antibody. The immunoprecipitant was analysed with anti-His to confirm the presence of BQ in the immunoprecipitant and anti-phos-(Ser/Thr) antibody to determine the degree of phosphorylation on BQ protein. ( C ) The addition of CCT241533 could reduce the relative level of p-BQ in TAM-treated LCC2 cells. LCC2 cells were transfected with pcDNA3.1-His-BQ, and the cells were treated with 4 µM of TAM and 10 nM of CCT241533 for 48 h. The cells were immunoprecipitated with anti-His antibody. The immunoprecipitant was analysed with protein A-HRP and anti-phos-(Ser/Thr) antibody to determine the degree of phosphorylation on BQ protein. ( D ) The effect of TAM and CCT241533 on poly-ubiquitination of BQ. LCC2 cells were transfected with pcDNA3.1-His-BQ, and the cells were treated with 5 µM of MG132 together with 4 µM of TAM or 10 nM of CCT241533 for 24 h. The cells were immunoprecipitated with anti-His antibody. The immunoprecipitant was analysed with anti-His to confirm the presence of BQ in the immunoprecipitant and anti-Ub antibody to determine the degree of poly-ubiquitination on BQ protein.

Article Snippet: The slides were rinsed with TBST twice, followed by incubation with primary monoclonal anti-p-CHK2 (Thr68) antibody (p-CHK2; 1:50; 2197; Cell Signaling Technology, Danvers, MA, USA) and BQ323636.1 antibody (1:50; D-12, Veritech Ltd., Hong Kong) at 4 °C overnight.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Transfection, Ubiquitin Proteomics

Journal: International Journal of Molecular Sciences

Article Title: Checkpoint Kinase 2 Inhibition Can Reverse Tamoxifen Resistance in ER-Positive Breast Cancer

doi: 10.3390/ijms232012290

Figure Lengend Snippet: The clinical significance of p-CHK2 in breast cancer. ( A ) The expression of p-CHK2 and BQ in primary breast tumours on tissue microarray (TMA). Immunohistochemistry was performed on TMA to determine the expression of p-CHK2 and BQ in the tissues. The expression of nuclear p-CHK2 and nuclear BQ were scored. ( B ) Nuclear p-CHK2 was positively correlated with nuclear BQ. Chi-square test was performed. ( C ) Nuclear expression of p-CHK2 in the tumours with the high nuclear BQ expression group was significantly higher than that in the low nuclear BQ expression group. Mann–Whitney U test was employed to determine statistical significance. ( D ) Nuclear p-CHK2 was positively correlated with TAM resistance in ER + ve cases. Chi-square test was performed. ER + ve patients with high nuclear expression of p-CHK2 had poorer outcomes for ( E ) overall and ( F ) disease-specific survival. Log-rank test was employed to determine statistical significance.

Article Snippet: The slides were rinsed with TBST twice, followed by incubation with primary monoclonal anti-p-CHK2 (Thr68) antibody (p-CHK2; 1:50; 2197; Cell Signaling Technology, Danvers, MA, USA) and BQ323636.1 antibody (1:50; D-12, Veritech Ltd., Hong Kong) at 4 °C overnight.

Techniques: Expressing, Microarray, Immunohistochemistry, MANN-WHITNEY

Journal:

Article Title: Activated microglia in cortex of mouse models of mucopolysaccharidoses I and IIIB

doi: 10.1073/pnas.252784899

Figure Lengend Snippet: Histochemical and immunohistochemical characterization of microglia in cortex. (A) G. simplicifolia isolectin IB4-positive microglia apposed to neuron; MPS IIIB, 7 months (×1,750). (B) MOMA-2-positive microglia apposed to neuron; MPS IIIB, 3 months (×1,150). (C and D) MOMA-2-positive cells seen at low power; MPS IIIB 3 months and MPS I, 14 months, respectively (×140). (E–G) Confocal images of sections stained with antibody against Lamp-1 (green), MOMA-2 (red), and merged; MPS IIIB, 6 months (×350). (H–J) Fluorescent images using antibody against ganglioside GM3 (green), MOMA-2 (red), and merged; MPS IIIB, 6 months (×260). (K–M) Images of sections stained with antibody against CD68/macrosialin, MPS IIIB, MPS I, and control, respectively, 3 months (×65). (N and O) Images of sections stained with antibody against IFN-γ; MPS IIIB, and control, respectively, 3 months (×250). (P and Q) Images of sections stained with antibody against IFN-γ receptor; MPS IIIB and control, respectively, 7 months (×250).

Article Snippet: A rat monoclonal antibody against mouse macrophages/monocytes (MOMA-2) and a rat monoclonal antibody against mouse CD68 (macrosialin) were purchased from Serotec.

Techniques: Immunohistochemical staining, Staining

Journal:

Article Title: Activated microglia in cortex of mouse models of mucopolysaccharidoses I and IIIB

doi: 10.1073/pnas.252784899

Figure Lengend Snippet: Transcripts increased in cortex of mouse models of MPS IIIB and MPS I, determined by microarray analysis

Article Snippet: A rat monoclonal antibody against mouse macrophages/monocytes (MOMA-2) and a rat monoclonal antibody against mouse CD68 (macrosialin) were purchased from Serotec.

Techniques: Microarray, Binding Assay

Journal:

Article Title: Activated microglia in cortex of mouse models of mucopolysaccharidoses I and IIIB

doi: 10.1073/pnas.252784899

Figure Lengend Snippet: Increase in transcripts in cortex of mice at different ages determined by Northern blot analysis

Article Snippet: A rat monoclonal antibody against mouse macrophages/monocytes (MOMA-2) and a rat monoclonal antibody against mouse CD68 (macrosialin) were purchased from Serotec.

Techniques: Northern Blot

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: FCER1G and macrophage markers exerted a synergistic effect in predicting ccRCC survival. The Kaplan–Meier survival curve shows the combination of FCER1G and macrophage marker CD163 ( A ) or CD68 ( B ) better predicts patient survival in TCGA-KIRC. ccRCC, clear cell renal cell carcinoma; FCER1G, Fc fragment of IgE receptor Ig; TCGA-KIRC, The Cancer Genome Atlas-kidney renal clear cell carcinoma

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques: Marker

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: The expression characteristics of FCER1G and CD68 in retrospectively collected ccRCC samples. A Thumbnail of TMA-30, showing the expression patterns of CD68 and FCER1G in tumor tissue and normal tissue adjacent to tumor. B The representative samples and violin plot reveal the high expression of CD68 and FCER1G in tumor tissue compared with NATs. C The representative samples and scatter diagram show the correlation of CD68 and FCER1G in ccRCC samples. ccRCC, clear cell renal cell carcinoma; FCER1G, Fc fragment of IgE receptor Ig; NATs, normal tissues adjacent to tumor

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques: Expressing

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: Receiver operating characteristic (ROC) curve based on the CD68 and FCER1G IHC-score. Receiver operating characteristic (ROC) curve for the evaluation of 5-year overall survival based on the CD68 and FCER1G IHC-score of patients with ccRCC from the training set (TMA-30). ccRCC, clear cell renal cell carcinoma; FCER1G, Fc fragment of IgE receptor Ig; IHC, immunohistochemistry; TMA, tissue microarray

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques: Immunohistochemistry, Microarray

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: Correlation between CD68 expression and clinical characteristics of patients with ccRCC in TMA-2020 NO.1-8 ( n = 321)

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques: Expressing

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: Prognostic value of FCER1G and CD68 in retrospectively collected ccRCC samples. A The Kaplan–Meier survival curve shows that high expression of FCER1G was related to poor overall survival and progression-free survival in patients from the validation set (TMA-2020). B The Kaplan–Meier survival curve shows that high expression of CD68 was related to poor overall survival and progression-free survival in patients from the validation set (TMA-2020). ccRCC, clear cell renal cell carcinoma; FCER1G, Fc fragment of IgE receptor Ig; IHC, immunohistochemistry; TMA, tissue microarray

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques: Expressing, Biomarker Discovery, Immunohistochemistry, Microarray

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of patient characteristics with overall survival

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques:

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of patient characteristics with progression-free survival

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques:

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: Combining CD68 and FCER1G expression resulted in better prognostic stratification in patients with ccRCC. The Kaplan–Meier survival curve reveals that combining CD68 and FCER1G expression more accurately determines the overall survival ( A ) and progression-free survival ( B ) in patients with ccRCC from the validation set (TMA-2020). C Immunohistochemistry results of representative samples show an inferior overall survival in patients with high expression of both CD68 and FCER1G. ccRCC, clear cell renal cell carcinoma; FCER1G, Fc fragment of IgE receptor Ig

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques: Expressing, Biomarker Discovery, Immunohistochemistry

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: Concordance index analysis

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques: Biomarker Discovery

Journal: BMC Cancer

Article Title: FCER1G positively relates to macrophage infiltration in clear cell renal cell carcinoma and contributes to unfavorable prognosis by regulating tumor immunity

doi: 10.1186/s12885-022-09251-7

Figure Lengend Snippet: Integration of CD68 and FCER1G expression into TNM staging exhibited high accuracy in assessing prognoses. The Kaplan–Meier survival curve shows that patients with high expression of both CD68 and FCER1G (double high) were associated with worse overall survival ( A ) and progression-free survival ( B ) compared with the remaining patients (non-double high) from the same TNM stage group. ccRCC, clear cell renal cell carcinoma; FCER1G, Fc fragment of IgE receptor Ig

Article Snippet: The slides were subsequently incubated overnight with antibodies against CD68 (#76437s, rabbit anti-human monoclonal, 1:400; Cell Signaling Technology) and FCER1G (ab151986, rabbit anti-human polyclonal, 1:400; Abcam) at 4°C in an incubator.

Techniques: Expressing

Journal: iScience

Article Title: Multi-omics approach reveals posttranscriptionally regulated genes are essential for human pluripotent stem cells

doi: 10.1016/j.isci.2022.104289

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CPSF6 (1:100) , Proteintech , Cat# 15489-1-AP RRID: AB_10694140.

Techniques: Recombinant, Mass Spectrometry, Sequencing, Modification, Protease Inhibitor, Multiplex sample analysis, Multiplex Assay, Fractionation, Cell Culture, Bicinchoninic Acid Protein Assay, Lysis, Cloning, Gene Expression, SYBR Green Assay, Transfection, Ligation, Microarray, TaqMan Assay, Software, Cell Analysis

Journal: Physiological Genomics

Article Title: Microarray analysis of aging-associated immune system alterations in the rostral ventrolateral medulla of F344 rats

doi: 10.1152/physiolgenomics.00131.2016

Figure Lengend Snippet: List of immune system genes used for quantitative PCR validation of microarray analysis and their TaqMan assay IDs

Article Snippet: Actb, Hprt1, Ldha , and Rplp1 genes were used as endogenous controls and the geometric average of their Ct values was used to calculate each gene’s ΔCt value ( 1 ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Functional Category Gene Symbol TaqMan Assay ID Complement system C1qa Rn01519903_m1 C1qc(C1qg) Rn01516757_m1 Cd93(C1qr1) Rn00584525_g1 C3 Rn00584525_g1 C4a Rn00566466_m1 Cfd Rn00709527_m1 Cfh Rn01535436_g1 Vwf Rn00590326_m1 Klkb1 Rn01488161_m1 Microglial cells Cd14 Rn00572656_g1 Cd68 Rn01495634_g1 Tlr2 Rn02133647_s1 Cx3cr1 Rn02134446_s1 Trem2 Rn01512170_m1 Fcrl2 Rn01455191_m1 B2m Rn00560865_m1 Endogenous controls Actb Rn00667869_m1 Hprt1 Rn01527840_m1 Ldha Rn00820751_g1 Rplp1 Rn03467157_gH Open in a separate window List of immune system genes used for quantitative PCR validation of microarray analysis and their TaqMan assay IDs Microarray Data Analysis Hybridization signal intensities from microarrays were extracted with Agilent Feature Extraction Software, version 9.5.1.1. (Agilent Technologies) (Gene Expression Omnibus accession number {"type":"entrez-geo","attrs":{"text":"GSE90956","term_id":"90956"}} GSE90956 ).

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Microarray, TaqMan Assay

Journal: Translational Oncology

Article Title: Disulfidptosis-related gene expression reflects the prognosis of drug-resistant cancer patients and inhibition of MYH9 reverses sorafenib resistance

doi: 10.1016/j.tranon.2024.102091

Figure Lengend Snippet: Association of disulfidptosis gene expression with sorafenib resistance in LIHC. A: GSE109211 Analysis of differences between Sorafenib-treated responder and non-responder in the dataset, volcano plots based on P < 0.05, |log2FC|≥1. B: Wayne plot of the intersection of sorafenib resistance differential genes from the GSE109211 dataset and disulfidptosis-related genes related to sorafenib drug sensitivity ( P < 0.05) from the GDSC database. C: AUC correlation plot of MYH9 with sorafenib in liver cancer cell lines from the GDSC database. D: AUC correlation plot of MYL6 with sorafenib in liver cancer cell lines from the GDSC database. E: AUC correlation plot of SLC7A11 with sorafenib in liver cancer cell lines from the GDSC database. F: Expression levels of MYH9, MYL6 and SLC7A11 in sorafenib-resistant (non-responder) and sensitive groups (responder) in the GSE109211 dataset. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.

Article Snippet: After blocking with 5 % skim milk in TBST for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against MYL6 (1:1000, 68,142–1-Ig, Proteintech, USA), MYH9 (1:1000, 11,128–1-AP, Proteintech, USA), and GAPDH (1:1000, sc-365,062, Santa Cruz Biotechnology, USA).

Techniques: Gene Expression, Expressing

Journal: Translational Oncology

Article Title: Disulfidptosis-related gene expression reflects the prognosis of drug-resistant cancer patients and inhibition of MYH9 reverses sorafenib resistance

doi: 10.1016/j.tranon.2024.102091

Figure Lengend Snippet: Validation of MYH9 associated with sorafenib resistance in hepatocellular carcinoma. A: Western Blot detected the expression of MYH9, MYL6 and SLC7A11 in liver cancer cell lines lysate (3 cases randomly selected in each group). B: Western Blot detected the expression of MYH9 and SLC7A11 in liver cancer cell lines lysate (3 cases randomly selected in each group). C: Workflow of in vivo drug sensitivity assay. D: Samples of HCCLM3R, HCCLM3R+Bl, HepG2R, and HepG2R+Bl cells (1 × 10^6) were inoculated into nude mice subcutaneously. E: Quantitative analysis of tumor volume of xenografts. Tumor volume was compared at indicated time points, and tumor weight was measured at the endpoint. F: Gross photographs of mouse liver orthotopic tumors and lung metastases, as well as H&E staining. Immunohistochemical staining of Ki67 and PCNA in mouse liver orthotopic tumors. * p < 0.05, **p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: After blocking with 5 % skim milk in TBST for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against MYL6 (1:1000, 68,142–1-Ig, Proteintech, USA), MYH9 (1:1000, 11,128–1-AP, Proteintech, USA), and GAPDH (1:1000, sc-365,062, Santa Cruz Biotechnology, USA).

Techniques: Biomarker Discovery, Western Blot, Expressing, In Vivo, Sensitive Assay, Staining, Immunohistochemical staining

Journal: Translational Oncology

Article Title: Disulfidptosis-related gene expression reflects the prognosis of drug-resistant cancer patients and inhibition of MYH9 reverses sorafenib resistance

doi: 10.1016/j.tranon.2024.102091

Figure Lengend Snippet: Tissue chips immunohistochemical analysis. A and F: MYH9 (A) and MYL6 (F) tissue chips immunohistochemical staining. B and G: Representative pictures of MYH9 (B) and MYL6 (G) expressions in HCC and adjacent normal tissues by immunohistochemical. C and H: The statistical results of the difference of MYH9 (C) and MYL6 (H) expressions in HCC and adjacent normal tissues were analyzed by paired t -test, n = 80. D and I: The samples were divided into groups of sorafenib-resistance and sorafenib sensitivity according to whether or not they relapsed after treatment with sorafenib. Representative images of MYH9 (D) and MYL6 (I) expressions in the sorafenib-resistance and sorafenib sensitivity groups by immunohistochemical. E and J: The statistical results of the difference between MYH9 (E) and MYL6 (J) expressions in the sorafenib-resistance and sorafenib-sensitivity groups. n = 80. K: KM survival curve according to whether or not they are resistant to sorafenib based on tissue microarray clinical information. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.

Article Snippet: After blocking with 5 % skim milk in TBST for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against MYL6 (1:1000, 68,142–1-Ig, Proteintech, USA), MYH9 (1:1000, 11,128–1-AP, Proteintech, USA), and GAPDH (1:1000, sc-365,062, Santa Cruz Biotechnology, USA).

Techniques: Immunohistochemical staining, Staining, Microarray