Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. MEFs harboring TetO-OKSM and M2rtTA cassettes were transfected with siRNA targeting different canonical NuRD components (indicated in the illustration), 2 and 4 days after reprogramming initiation following DOX administration. Reprogramming was then evaluated by AP staining at day 8. B. Reprograming efficiency following siRNA treatments was evaluated using AP staining, after 8 days of reprogramming (n=3, two-sided Student’s t-test p values are indicated). C. Cell growth curves of MEFs treated with siRNA for the indicated NuRD components (two-sided Student’s t-test p values are indicated). Knockdown (KD) of Gatad2a, unlike KD of Chd4, Mbd3 and Hdac2, does not severely inhibit cell proliferation. D. Representative images of cells treated with siRNA targeting Mbd3 or Gatad2a and exposed to BrdU in order to evaluate proliferation. E. Quantitative evaluation of BrdU incorporation test, which shows normal proliferation in siScramble and siGatad2a, unlike in cells treated with siMbd3 (n=8, two-sided Student’s t-test p values are indicated Student’s t-test). F . Knockdown for different canonical NuRD components does not show a significant elevation in cell death or apoptosis. Viability and apoptosis induction were measured using FACS following Annexin-PI staining. G. Reprogramming efficiency following siRNA treatments targeting different NuRD components, at different time points. KD was performed at two distinct cycles: the early one (Regimen 1, marked in black) started one day prior to DOX induction, and the second one (Regimen 2, marked in grey) started one day post-DOX induction. H. iPSC reprogramming efficiency following different siRNA treatments. was evaluated at day 8. (n=3 per each condition, two-sided Student’s t-test p values are indicated). Gatad2a siRNA improves reprogramming whether administrated prior or post DOX administration, unlike siRNA targeting Mbd3 or Chd4, in which only in Regimen #2 iPSC colony formation efficiency was increased.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Transfection, Staining, Knockdown, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A . Scheme demonstrating strategy for generating secondary isogenic Gatad2a WT and KO lines, and comparing their reprogramming efficiency side by side. 2i/LIF-KSR conditions were introduced at day 3.5 during the 8-day course. More detailed information is provided in Supplementary regarding different systems used herein. B . Targeting scheme of Gatad2a locus to generate knockouts by CRISPR/Cas9. C . Western blot validation of Gatad2a knockout in iPSC harboring M2rtTA and TetO-OKSM cassettes in comparison to its parental isogenic line. D . Representative images of Gatad2a -/- iPSC derived E13.5 chimera. Red arrow highlights mCherry+ chimera which originates from mCherry labeled iPSCs that were microinjected. E. Bulk iPSC reprogramming as in F , but experiment was terminated after 6 days and iPSC colony formation was evaluated by Alkaline Phosphatase staining (AP+). F . Representative flow cytometry measurements of ΔPEOct4-GFP reactivation dynamics in polyclonal/bulk Gatad2a-WT and Gatad2a-KO isogenic cell lines. Throughout the course of the reprogramming experiment the cells were not passaged to avoid any biases. Reprogramming of secondary MEFs seeded as single cells. G. Representative summaries of single-cell iPSC reprogramming efficiency experiment. Secondary isogenic Gatad2a WT and KO reprogrammable MEFs carrying constitutively expressed mCherry-NLS and naïve pluripotency specific ΔPE-Oct4-GFP reporter were sorted and seeded as single-cell per well. Reprogramming was initiated by DOX administration according to panel A . Reprogramming efficiency was assessed after 8 days based on the number of wells in which mCherry+ cells formed an ΔPE-Oct4-GFP positive colony. Throughout the course of the reprogramming experiment the cells were not passaged to avoid any biases. H. The indicated secondary Gatad2a +/+ and Gatad2a -/- somatic cell types were isolated and subjected to single cell reprogramming and evaluation of iPSC efficiency following 8 days of DOX. Reprogramming efficiency was assessed after 8 days based on the number of wells in which mCherry+ cells formed an ΔPE-Oct4-GFP positive colony. In summary, these results indicate that complete inhibition of Gatad2a (also known as P66a), a NuRD specific subunit, does not compromise somatic cell proliferation as previously seen upon complete Mbd3 protein elimination, and yet disrupts Mbd3/NuRD repressive activity on the pluripotent circuitry and yields 90-100% highly-efficient reprogramming within 8 days as similarly observed previously in Mbd3 hypomorphic Mbd3 flox/- donor somatic cells ( ; ).

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: CRISPR, Western Blot, Biomarker Discovery, Knock-Out, Comparison, Derivative Assay, Labeling, Staining, Flow Cytometry, Isolation, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Three different clonal sets of isogenic secondary OSKM reprogrammable cells/sets were generated: (i) MEFs were reprogrammed to iPSC following viral infection of FUW-M2rtTA and FUW-OKSM. mCherry constitutive marker and ΔPE-Oct4-GFP markers were then introduced to the iPSC. (ii) R26-M2rtTA +/- m. Col1a-OKSM +/- ES were derived from E3.5 embryos following mating of mice. (iii) ES Kh2 m.Col1a-TetO-2XFlag-Mbd3 were injected to blastocysts, and MEF were harvested at E12.5. MEF were then reprogrammed following viral infection of FUW-OKSM. All described cell lines were then subjected to Gatad2a-KO using CRISPR/Cas9, followed by an injection of both the KO and its isogenic WT to blastocysts. Chimeric fibroblasts were separated from the donor cells by Puromycin selection. B . Summary of CRISPR/Cas9 strategy to generate Gatad2a null cells. sgRNA targeted sequence is indicated. C . Correct targeting efficiency by the strategy described in B to generate mouse Gatad2a KO PSCs as determined both by Western blot analysis and PCR sequencing. D . Representative Western blot analysis for sub cloned lines following targeting Gatad2a with sgRNA. Blue arrows indicate examples of KO clonal lines. E. Transcriptome landscape (RNA-seq), alongside ATAC-seq and H3K27Ac ChIP-seq, of fibroblasts (FSP-1 and Thy1) and pluripotent (Nanog and Sall4) related genes. IGV Data range is indicated at the top right corner of the signal.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Generated, Infection, Marker, Derivative Assay, Injection, CRISPR, Selection, Sequencing, Western Blot, Clone Assay, RNA Sequencing, ChIP-sequencing

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Mass spectrometry analysis of NuRD complex components binding efficiency to Mbd3 in Gatad2a-KO and its isogenic Gatad2a-WT line, in MEF cells during reprogramming by OSKM. Only putative NuRD components are presented and can be seen in an equal strength at both platforms, except for Chd4, which does not bind Mbd3 in Gatad2aKO. Flag-Tagged Mbd3 was used to establish a platform for studying Mbd3-binding proteins, by correct targeting of TetO-Mbd3-Flag into the M. Col1a locus. Isogenic Gatad2a-KO were generated from this line with CRISPR/Cas9 and used for the IP and MS analysis indicated above (See ). B. Flag-Mbd3 CoIP in Gatad2a-WT and Gatad2a-KO cells. Experiments were conducted both in MEF cells and ESs. C. Cells during reprogramming were treated with siRNA targeting Gatad2a, and pellets collected after four days. CoIP of Chd4 shows that siGatad2a prevents Mbd3 binding to Gatad2a but also to Chd4. D. Gatad2a-KO MEF with overexpression of Gatad2a (transgenic recovery; abbreviated as Gatad2a-Tg) or Mock were subjected to Chd4-CoIP. Gatad2aoverexpression recovers the binding of Chd4 to Mbd3 and does not affect its binding to Mta2. E. The coiled coil region of Mbd3 is highly conserved between different organisms, and different proteins in the MBD family. The highlighted amino acids are crucial for Gatad2a binding to Mbd2 or Mbd3. F. A scheme of Flag tagged Mbd3, and mutant forms of Mbd3: lacking the Coiled coil region (#CCR-Mbd3) or the methyl-binding domain (#MBD-Mbd3). G. WT-Mbd3 and both mutants were over-expressed in 293T cells and were subjected to Flag-Mbd3 CoIP to examine their protein interactions. While the deletion of MBD prevents the binding of Klf4, only the deletion of the coiled coil region abolished the binding to Chd4 and Gatad2a. H-I. Mbd3 fl/- secondary MEF, harboring #PE-Oct4-GFP reporter, were transfected with two different forms of Flag tagged WTMbd3 and ΔCCR-Mbd3. The cells were then subjected to reprogramming. While WT-Mbd3 significantly reduced reprogramming efficiency ( p Value<0.0001, two-sided Student’s t-test, n=3), ΔCCR-Mbd3 expression was not able to inhibit deterministic reprogramming in the cells, consistent with its inability to interact and recruit Chd4 to the assembled complex.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Mass Spectrometry, Binding Assay, Generated, CRISPR, Over Expression, Transgenic Assay, Mutagenesis, Transfection, Expressing

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. A scheme of Gatad2a constructs and mutants used. Gatad2a-WT, Gatad2a-Truncated (lacking the CCR2 of Gatad2a) and HA-tagged-CCR1-Gatad2a construct. B. Flag-Mbd3 and different Gatad2a constructs were cotransfected in 293T cells. Flag-Mbd3 binds both forms of Gatad2a (WT and truncated), as shown by Flag-Mbd3 CoIP. C. Dot blot analysis to validate protein expression following HA-Gatad2aCCR1 peptide overexpression. D. Overexpression of Flag-Mbd3 with HA-Gatad2a-CCR1 or control (HA-GFP) in 293T cells, followed by Co-IP for anti-Flag-Mbd3. Over-expression of Gatad2a-CCR1 reduces the binding of Mbd3 to endogenous Gatad2a and Chd4, comparing to the control specimen. Further, CCR can cause a reduction in Mbd3 binding to endogenous Gatad2a and Chd4, without changing other NuRD components (such as Hdac2) binding. E. Overexpression of STEMCCA-OKSM vector, HA-MBD (methyl-binding domain of Mbd3) or HA-GFP in 293T cells, was followed by Co-IP with anti-HA. The results demonstrate that MBD domain can bind to pluripotency factors such as Oct4 and to the NuRD component Hdac2 but cannot bind Gatad2a or Chd4. F. Summarizing scheme for three different approaches for neutralizing Mbd3-Gatad2a-Chd4 axis: (i) by deleting the CCR domain of Mbd3 (ii) KO or KD of Gatad2a protein and (iii) overexpression of an exogenous Gatad2a-CCR competitive peptide that interferes with Gatad2aCCR interaction with Mbd3.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Construct, Dot Blot, Expressing, Over Expression, Control, Co-Immunoprecipitation Assay, Binding Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Rosa26-M2rtTA Col1a:TetO-2XFlag-Mbd3 ES cells were subjected to different differentiation protocols, and cells from 5 distinct states (naïve ESC, EpiLC, EBs, MEF, 4-day OSKM Reprogramming) were subsequently subjected to CoIP with anti- Flag-Mbd3. Lysates were then analyzed by western blot, and reacted with different antibodies against different NuRD components, Pluripotency factors, and other epigenetic proteins. While some of the proteins show constitutive binding to Mbd3 throughout all differentiation states (Mta2, Prmt5)- other proteins show differential binding (Oct4, Klf4, Cdk2ap1) and the latter is not correlated with the proteins level in the cell. B. Rosa26-M2rtTA Col1a:TetO-2XFlag-Mbd3 ES cells were either maintained in ground state naïve conditions or in priming conditions (Fgf2/Activin A). Lysates were subjected to Co-IP with anti-Flag-Mbd3 and examined by Western blot. Oct4 protein expression is significantly reduced after priming, but its binding to Mbd3 can be detected only in the primed pluripotent state. C. Mbd3 expression in ES cells treated with growth media containing different small molecules, after 72 hours of treatment. Unlike other treatments, PKCi Go6983 (5 μM) treatment resulted in a radical decrease in Mbd3 protein expression. D. PKCi Go6983 effect on Mbd3 level is seen after approximately 48 hours, in different concentration ranging from 0.5 to 10 μM. E. WT EpiSCs reversion efficiency to naïve ESCs in different conditions. Anova test P values are indicated. (one representative experiment out of 3 performed is shown). F. Isogenic WT and Gatad2a KO ESCs were expanded on feeder free gelatin coated plates in N2B27 LIF only or LIF/PKCi conditions. Phase images and Oct4-GFP signal maintenance are shown after 8 passages (P8). Oct4-GFP. G. ES cells treated with naïve ground state condition were treated with shRNA targeting Ubc9 or Scramble. Cells were lysed and subsequent CoIP of Chd4 shows a decrease in Gatad2a binding to the protein. H. Cells induced in naïve ground state 2i/LIF conditions and subsequent shRNA targeting either for Ubc9 or scramble negative control. The cells were lysed and fractioned – Cytoplasm, Nucleoplasm and Chromatin fractions, proteins were analyzed by western blot. The NuRD components Mbd3 and Gatad2a, but not Mta2, can be seen mainly in the chromatin fraction, and their expression is significantly reduced following shUbc9 treatment.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Western Blot, Binding Assay, Co-Immunoprecipitation Assay, Expressing, Concentration Assay, shRNA, Negative Control

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Western blot showing Mbd3 protein levels in ESCs expanded in the indicated growth conditions. Mbd3 levels were decreased following PKCi (Go6983) treatment in ES cells maintained in different conditions (FBS and KSR based growth media, supplemented with 2i). B. Reduction in Mbd3 protein expression is maintained following long term PKCi (Go6983) treatment, as examined in different concentrations (0.5-5 μM). Media was exchanged ever 48h. C. Western blot analysis for different NuRD components in ESC cells with and without Go6983. Analysis shows that other NuRD components expression level is not affected from PKCi (Go6983) treatment. D. as in A, but a different PKCi was used, termed GFX which does not inhibit atypical PKC pathway. E. Mbd3 protein levels following shRNA treatment of mouse V6.5 ESCs for atypical PKCzeta isoform. F. Mbd3 levels in MEFs following treatment with Go6983 are not affected. G. RT-PCR analysis for Mbd3 transcript abundance 24 and 48h following PKCi Go6983 treatment, do not show significant changes and reduction in Mbd3 transcript level. This suggests that the depletion in Mbd3 protein levels shown in A-C is post-translational. H. Go6983 causes mild depletion in Mbd3 in MEFs following 3 days of OSKM expression. I . Gatad2a +/+ WT MEFs carrying ΔPE -Oct4-GFP reporter and constitutive mCherry markers (secondary system i) were subjected to iPSC reprogramming protocols in in and iPSC efficiency was quantified at day 8. In the last two conditions included in the panel, the MEFs were pre-treated with control and Ubc9 shRNA following with Neomycin selection , and then subjected to DOX mediated iPSC reprogramming. Anova P values are indicated. J. A Schematic representation of Gatad2a known domains and confirmed SUMOylation sites . Importantly, harnessing prediction tools for identification of possible SUMO consensus sites results in 2 additional sites inside the coiled coil regions (Chi-squared test p value<0.05). K. 2-d08 specific small molecule inhibitor for SUMOylation was applied during co-IP experiments for Chd4 at the indicated increasing concentrations known to deplete global SUMOylation levels. Cells were lysed the CoIP of Mbd3 shows a decrease in Gatad2a binding to Chd4, as similarly seen with shRNA depletion of Ubc9 . L. As in with immunoblot for SUMO2/3 on the same exact gel series.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Control, Selection, Co-Immunoprecipitation Assay, Binding Assay

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 4 Effects of SPD on NF-kB signaling pathway. (A-C) Western blot and semi-quantitative analysis of p-IkBa, IkBa, p-p65, and p65. (D) Immunofluorescence to observe NF-kB P65 nuclear translocation. We stained the nuclei using DAPI (blue) and labeled NF-kB p65 using anti-NF-kB p65 rabbit fluorescent antibody (green), while labeling the cytoskeleton using YF®594-Phalloidin. Significant NF-kB p65 nuclear translocation was observed in the IL-1b group. pretreatment with SPD reversed IL-1b-induced nuclear translocation, whereas no significant changes were observed when SPD was applied alone. *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the IL-1b group.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Western Blot, Translocation Assay, Staining, Labeling, Control

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 5 The protective effect of the SPD depends on the AhR. (A-C) Western blot and qRT-PCR analysis for AhR knockdown. (D-G) Expression of IL-6, TNF- a and PGE2 in cell suspensions by ELISA and NO by Griess reaction. (H-P) Western blot and semi-quantitative analysis of COX-2, iNOS, MMP-3, MMP-13, Collagen-II, ADAMTS-5 and p-IkBa, IkBa, p-p65 and p65. *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the IL-1b group; ※p < 0.05, ※※p < 0.01 vs. the IL-1b+SPD group.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Western Blot, Quantitative RT-PCR, Knockdown, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 6 SPD ameliorates IL-1b-induced chondrocyte pyroptosis by inhibiting the NLRP3/caspase-1/GSDMD pathway. (A-E) Western blot measuring the differences in the expression levels of cellular pyroptosis-related proteins in each group. To elucidate whether SPD ameliorates chondrocyte pyroptosis by inhibiting AhR/NF-kB, NF-kB p65 overexpression plasmid was used to characterize the effect of the AhR/NF-kB pathway. (F-J) The inflammation-associated cellular pyroptosis proteins NLRP3, caspase-1, and GSDMD were significantly increased in the IL-1b group compared with the CG group, whereas downregulation of cellular pyroptosis was observed in the SPD group. Administration of P65 overexpression plasmid (OE group) blocked the therapeutic effect of SPD. (K-L) Flow cytometry showing chondrocytes labeled with caspase-1 fluorescence inhibitor probe FLICA 660-YVAD-FMK. IL-1b induces chondrocyte pyroptosis. SPD significantly alleviated IL-1b-induced chondrocyte death. (M) Transmission electron microscopy (TEM) images of chondrocytes from different treatment groups, such as cytoplasmic edema, swelling and rupture of cell membrane, nuclear consolidation, and organelle cavitation were more obvious in the IL-1b group; cell morphology of the SPD pretreatment group was significantly improved. Black arrows indicate cell membrane swelling; white arrows indicate organelle cavitation (Low magnification: ×2.0k, high magnification: ×10.0k). *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the IL-1b group; ※p < 0.05, ※※p < 0.01 vs. the IL- 1b+SPD group.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation, Flow Cytometry, Labeling, Transmission Assay, Electron Microscopy, Membrane, Control

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 7 SPD attenuates the progression of OA in a rat model. (A-C) A representative image of H&E, saffron O-solid green and toluidine blue staining of cartilage sections from different groups. (D) Articular cartilage Pritzker score in different groups of rats. (E) The modified Mankin score for different groups of cartilage. (F) The cartilage OARSI scores of different groups. (G-L) Immunohistochemical staining and percentage of positive cells for IL-6, NF-kB p65, Caspase-1, NLRP3, Gasdermin-D and Collagen II. Scale bar = 20 mm, ×400. *p < 0.05, **p < 0.01 vs. the control group; #p < 0.05, ##p < 0.01 vs. the ACLT group.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Staining, Immunohistochemical staining, Control

Journal: Frontiers in immunology

Article Title: Spermidine attenuates chondrocyte inflammation and cellular pyroptosis through the AhR/NF-κB axis and the NLRP3/caspase-1/GSDMD pathway.

doi: 10.3389/fimmu.2024.1462777

Figure Lengend Snippet: FIGURE 8 Mechanism of SPD inhibition of NF-kB signaling via AhR receptors and attenuation of chondrocyte inflammation and pyroptosis via NLRP3/caspase- 1/GSDMD signaling pathway. A rat OA model was constructed using ACLT. Changes in the levels of inflammatory factors in animal samples were observed by intraperitoneal injection of SPD. We found that the drug entered the cells and bound to AhR targeting to inhibit chondrocyte inflammation and inflammation-associated cellular pyroptosis, thereby ameliorating the pathologic changes in OA. In vitro, SPD inhibited chondrocyte inflammation and inflammation-associated cellular pyroptosis by blocking the activation of AhR/NF-kB and NLRP3/caspase-1/GSDMD signaling pathways in IL-1b-treated chondrocytes.

Article Snippet: After three washes with trisbuffered saline (TBS) containing 0.1% Tween-20 (TBST) for 10 min, overnight incubation was carried out with primary antibodies targeting Phospho-NF-kB p65 (1:1000; CST, #3033), NF-kB p65 (1:1000; CST, #8242), Phospho-IkBa (1:1000; CST, #2859), IkBa (1:1000; CST, #9242), iNOS (1:1000; Abcam, ab178945), COX2 (1:1000; Abcam, ab179800), Collagen II (1:1000; Abcam, ab34712), MMP13 (1:2000; Proteintech, 18165-1-AP), MMP3 (1:1000; Proteintech, 17873-1-AP), AhR (1:1000; Proteintech, 67785-1), ADAMTS-5 (1:1000; Abcam, ab41037), NLRP3 (1:1000; Proteintech, 19771-1-AP), Caspase-1/p20/ p10 (1:4000; Proteintech, 22915-1-AP), GSDMD (1:1000; #AF4012, Affinity Biosciences), and GAPDH (1:20000; Proteintech, 10494-1- AP).

Techniques: Inhibition, Construct, Injection, In Vitro, Blocking Assay, Activation Assay, Protein-Protein interactions

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: Expression levels of XBP1 and XBP1s are positively correlated with resistance to CDK4/6 inhibitors and endocrine therapy in patients with HR+/HER2− metastatic breast cancer. A) Schematic for the study design and analytical workflow. PFS, progression‐free survival; RNA‐seq, RNA sequencing; DEG, differentially expressed gene; BRCA, breast invasive carcinoma. B) Venn diagram showing the shared numbers and overlaps of upregulated genes identified in HR+/HER2− breast cancer tumors from the TCGA and METABRIC datasets, and the treatment‐resistant cohort of patients with HR+/HER2− metastatic breast cancer. ET, endocrine therapy. C) Kaplan–Meier curves of progression‐free survival (PFS) in the validation cohort of patients with high and low levels of XBP1 and TMEM26 expressions based on qRT‐PCR assay. P ‐values are indicated and were calculated using the log‐rank test. D) XBP1 and XBP1s expression levels in primary breast tumors of different molecular subtypes (HR+/HER2− subtype, n = 152; HER2+ subtype, n = 84; Triple‐negative subtype, n = 38). * p < 0.05, *** p < 0.001, **** p < 0.0001. E) Kaplan–Meier curves of PFS in the validation cohort of patients with high and low levels of XBP1s expression. P ‐values are indicated and were calculated using the log‐rank test. F) Immunohistochemistry (IHC) staining for XBP1s and XBP1 in representative tumor samples from patients who were sensitive (PFS > 6 months) or resistant (PFS ≤ 6 months) to combination therapy of CDK4/6 inhibitors and endocrine therapy. Scale bar, 50 µm. G) The immunoreactive score of IHC staining for XBP1s and XBP1 in tumors from the validation cohort of patients. ** p < 0.01. For statistical analysis, the log‐rank test was employed in (C) and (E); one‐way ANOVA with Tukey's post hoc test was utilized for (D); the Mann–Whitney U test was employed in (G). Data are represented as the mean ± standard deviation.

Article Snippet: Immunoprecipitation was carried out using XBP1s (27901, Cell Signaling Technology) and normal mouse IgG (sc‐2025, Santa Cruz) antibodies at 4 °C overnight with rotation.

Techniques: Expressing, RNA Sequencing, Biomarker Discovery, Quantitative RT-PCR, Immunohistochemistry, MANN-WHITNEY, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: XBP1s attenuated the therapeutic efficacy of CDK4/6 inhibitors and endocrine therapy in vivo. A) Schematic diagram illustrating the workflow of the in vivo study. B–H) Mice with cell‐derived xenografts of MCF7/pCDH or MCF7/XBP1s were randomly divided into four treatment groups: vehicle, palbociclib, fulvestrant, or their combination. B) Representative images of IHC staining for XBP1s in MCF7/pCDH and MCF7/XBP1s cell‐derived xenografts from different treatment groups. Scale bar, 50 µm. C) Tumor growth curve plots of MCF7/pCDH tumor in each treatment group after treatment administration. n = 6 per treatment group. Each data point represents the mean tumor volume ± standard deviation. D) Waterfall plot showing the change in tumor volume of individual MCF7/pCDH cell‐derived xenografts at the end of the experiment compared with the baseline tumor volume on day 0 of the treatment. n = 6 per treatment group. E) Percentage of tumors exhibiting different drug responses (PD, SD, R) in MCF7/pCDH cell‐derived xenografts. PD, progression disease; SD, stable disease; R, regression. F) Tumor growth curve plots of MCF7/XBP1s tumor in each treatment group after treatment administration. n = 6 per treatment group. Each data point represents the mean tumor volume ± standard deviation. G) Waterfall plot showing the change in tumor volume of individual MCF7/ XBP1s cell‐derived xenografts at the end of the experiment compared with the baseline tumor volume on day 0 of the treatment. n = 6 per treatment group. H) Percentage of tumors exhibiting different drug responses (PD, SD, R) in MCF7/XBP1s cell‐derived xenografts.

Article Snippet: Immunoprecipitation was carried out using XBP1s (27901, Cell Signaling Technology) and normal mouse IgG (sc‐2025, Santa Cruz) antibodies at 4 °C overnight with rotation.

Techniques: Drug discovery, In Vivo, Derivative Assay, Immunohistochemistry, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: Overexpression of XBP1s confers resistance to fulvestrant and palbociclib and impairs the synergistic effect of palbociclib combined with fulvestrant in vitro. A) The dose‐response curves and half‐maximal inhibitory concentration (IC 50 ) values of palbociclib in MCF7 and T‐47D cells transfected with the control or XBP1s‐overexpressing plasmid after being treated for 96 h. B) Representative images and quantification of colony formation assay in MCF7 and T‐47D cells treated with vehicle or palbociclib (3.5 × 10 −6 m for MCF7 cells, 1 × 10 −6 m for T‐47D cells). ns, not significant; *** p < 0.001. C) Western blotting of the indicated proteins in MCF7 cells with or without XBP1s overexpression after being treated with the indicated concentration of palbociclib. D) Cell cycle distribution of MCF7 cells with or without XBP1s overexpression after being treated with palbociclib (3.5 × 10 −6 m ). E) The dose‐response curves and IC 50 values of fulvestrant in MCF7 and T‐47D cells transfected with the control or XBP1s ‐overexpressing plasmid after being treated for 96 h. F) Representative images and quantification of colony formation assay after treatment with vehicle or fulvestrant in MCF7 (15 × 10 −9 m ) cells with or without XBP1s overexpression. * p < 0.05, *** p < 0.001. G) Representative images and quantification of colony formation assay after treatment with vehicle or fulvestrant in T‐47D cells (120 × 10 −9 m ) with or without XBP1s overexpression. ns, not significant; ** p < 0.01. H) The dose‐response curves and IC 50 values of palbociclib in patient‐derived organoids (PDOs) after being treated for 96 h. ** p < 0.01. I) The dose–response curves and IC 50 values of palbociclib in PDOs after being treated for 96 h. ** p < 0.01. J,K) Relative spheroid area in PDO‐8912 transfected with the control or XBP1s ‐overexpressing plasmid following treatment with palbociclib and fulvestrant for the indicated days. The culture medium was replaced every 3 d. * p < 0.05, ** p < 0.01, *** p < 0.001. For statistical analysis, two‐tailed unpaired Student's t ‐test was utilized for (B), (F), (G), (H), (I), (J), and (K). Data are presented as the mean ± standard deviation.

Article Snippet: Immunoprecipitation was carried out using XBP1s (27901, Cell Signaling Technology) and normal mouse IgG (sc‐2025, Santa Cruz) antibodies at 4 °C overnight with rotation.

Techniques: Over Expression, In Vitro, Concentration Assay, Transfection, Control, Plasmid Preparation, Colony Assay, Western Blot, Derivative Assay, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: Epigenetic and pharmacological inhibition of XBP1s enhances the therapeutic sensitivity of fulvestrant and palbociclib in HR+/HER2− breast cancer cells. A) The dose–response curves and IC 50 values of palbociclib in MCF7 cells with or without XBP1 knockdown after being treated for 96 h. B) The dose–response curves and IC 50 values of fulvestrant in MCF7 cells with or without XBP1 knockdown after being treated for 96 h. C) Schematic illustrating the mechanism through which 4µ8C and MKC8866 hinder the synthesis of XBP1s. D) The mRNA levels of XBP1s downstream target genes were assessed in MCF7 cells by the qRT‐PCR assay following 6 h of treatment with the vehicle control (dark red bars), 0.5 × 10 −6 m of thapsigargin (Tg) alone (orange bars), or 0.5 × 10 −6 m of Tg combined with increasing concentrations of 4µ8C (light yellow bars), from 1.25 to 40 × 10 −6 m (twofold). E) The mRNA levels of XBP1s downstream target genes were assessed in MCF7 cells by the qRT‐PCR assay following 24 h of treatment with the vehicle control (dark red bars), 0.5 × 10 −6 m of thapsigargin (Tg) alone (orange bars), or 0.5 × 10 −6 m of Tg combined with increasing concentrations of MKC8866 (light yellow bars), from 0.125 to 4 × 10 −6 m (twofold). F,G) Cell proliferation analysis and cell cycle distribution for MCF7 cells treated with or without 4µ8C and MKC8866. * p < 0.05, ** p < 0.01. H) The dose–response curves and IC 50 values of palbociclib in MCF7 cells transfected with or without 4µ8C (left) and MKC8866 (right) after being treated for 96 h. I) The dose–response curves and IC 50 values of fulvestrant in MCF7 cells transfected with or without 4µ8C (left) and MKC8866 (right) after being treated for 96 h. J) Dose–response matrix (relative cell viability) of 4µ8C combined with palbociclib (left) or fulvestrant (right) in MCF7 cells based on the CCK8 assay. K) Combination index (CI) values for the antiproliferation effects of 4µ8C combined with palbociclib or fulvestrant in MCF7 cells. CI values were calculated based on the dose–response matrix in (J). For statistical analysis, two‐way ANOVA with Bonferroni's method correction was applied in (F). Data are presented as the mean ± standard deviation.

Article Snippet: Immunoprecipitation was carried out using XBP1s (27901, Cell Signaling Technology) and normal mouse IgG (sc‐2025, Santa Cruz) antibodies at 4 °C overnight with rotation.

Techniques: Inhibition, Knockdown, Quantitative RT-PCR, Control, Transfection, CCK-8 Assay, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: XBP1s overexpression promotes cell proliferation and cell cycle progression by regulating the G1/S transition in HR+/HER2− breast cancer cells. A) Enrichment plots for hallmarks of the E2F target, estrogen response, and the MYC target generated using gene set enrichment analysis (GSEA) based on the results of RNA sequencing of MCF7/pCDH and MCF7/XBP1s cells. FDR, false discovery rate. NES, normalized enrichment score. B) Representative images and quantification of EdU‐positive MCF7 and T‐47D cells with or without XBP1s overexpression. Scale bar, 50 µm. ** p < 0.01, *** p < 0.001. C) Images of tumors from the MCF7/pCDH group and MCF7/XBP1s group at the end of the experiments. D) IHC staining for XBP1s in tumor slides from the MCF7/pCDH and MCF7/XBP1s groups. Scale bar, 50 µm. E) Tumor growth curves of cell‐derived xenografts of MCF7/pCDH and MCF7/XBP1s cells. Tumor volumes were recorded every 4 d starting from day 12 after the injection of cells. n = 6 per group. * p < 0.05, ** p < 0.01. F–I) MCF7/pCDH and MCF7/XBP1s cells were synchronized to the G1/S phase by double thymidine blockage, then released to a fresh medium and collected for the indicated time. F) Cell cycle distribution was measured using flow cytometry. G) Representative time‐lapse images of MCF7 cells, with or without XBP1s overexpression, were presented at regular 3‐h intervals after synchronization. Scale bar, 25 µm. H) Quantification of the cell cycle duration based on time‐lapse live‐cell imaging. ** p < 0.01. I) The levels of proteins related to cell cycle determination were detected by western blotting in MCF7 cells after synchronization and subsequent release for the indicated duration. J) Western blotting for the indicated proteins related to G1 to S phase transition in MCF7 and T‐47D cells with or without XBP1s overexpression. For statistical analysis, two‐tailed unpaired Student's t ‐test was used for (B) and (H); two‐way ANOVA with Bonferroni's method correction was applied in (E). Data are presented as the mean ± standard deviation.

Article Snippet: Immunoprecipitation was carried out using XBP1s (27901, Cell Signaling Technology) and normal mouse IgG (sc‐2025, Santa Cruz) antibodies at 4 °C overnight with rotation.

Techniques: Over Expression, Generated, RNA Sequencing, Immunohistochemistry, Derivative Assay, Injection, Flow Cytometry, Live Cell Imaging, Western Blot, Sublimation, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: SND1 is a direct target of XBP1s and is responsible for the effects of XBP1s on cell proliferation, G1/S transition, and resistance to therapy. A) The Venn diagram shows the intersection of hallmark gene sets identified by GSEA based on the RNA‐seq data. B) KEGG pathway enrichment analysis of ChIP‐seq data obtained from MCF7 cells. C) Scatterplot showing the correlation between the expression of XBP1s and SND1 in tumors obtained from patients with HR+/HER2− early breast cancer ( n = 152). The correlation was assessed using Pearson's correlation coefficient. D,E) MCF7 and T‐47D cells were transfected with the control or XBP1s ‐overexpressing plasmid. The mRNA expression of SND1 was detected using qRT‐PCR assay (D), and the protein level of SND1 was measured via western blotting assay (E). ** p < 0.01, ### p < 0.001. F) ChIP‐qPCR of predicted XBP1s binding sites on SND1 promoters in MCF7 and T‐47D cells. ** p < 0.01, ### p < 0.001. G) The upper panel shows the XBP1s binding motif; the lower panel shows a schematic diagram of dual‐luciferase reporter vectors containing wild‐type or mutant XBP1s binding sites on the SND1 promoter. H) The activity of wild‐type (pGL3–SND1–WT) and mutant (pGL3–SND1–MUT) SND1 promoter in MCF7 cells, as indicated, was measured by dual‐luciferase reporter assay. * p < 0.05, *** p < 0.001, **** p < 0.0001. I) The protein level of SND1 was detected using western blotting in MCF7 and T‐47D cells treated with the vehicle control, 0.5 × 10 −6 m of Tg alone, or 0.5 × 10 −6 m of Tg combined with increasing concentrations of 4µ8C. J–M) Cells were treated with shNC, SND1‐sh2, or SND1‐sh3 after being stably transfected with the control or XBP1s ‐overexpressing plasmid. The cell proliferation analysis (J), cell cycle distribution (K), dose–response curves and IC 50 values of palbociclib (L) and fulvestrant (M) in MCF7 and T‐47D cells. * p < 0.05, ** p < 0.01. For statistical analysis, two‐tailed unpaired Student's t ‐test was used for (D) and (F); one‐way ANOVA with Turkey's post hoc test was utilized for (H); two‐way ANOVA with Turkey's post hoc test was applied in (J). Data are presented as the mean ± standard deviation.

Article Snippet: Immunoprecipitation was carried out using XBP1s (27901, Cell Signaling Technology) and normal mouse IgG (sc‐2025, Santa Cruz) antibodies at 4 °C overnight with rotation.

Techniques: RNA Sequencing, ChIP-sequencing, Expressing, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, ChIP-qPCR, Binding Assay, Luciferase, Mutagenesis, Activity Assay, Reporter Assay, Stable Transfection, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: SND1 facilitates E2F1‐mediated transcriptional activation of target genes in HR+/HER2− breast cancer cells. A) Whole‐cell extracts of MCF7 and T‐47D cells were subjected to co‐immunoprecipitation (co‐IP) using anti‐SND1 antibodies, and the interaction between E2F1 and SND1 was identified via western blotting. B) Whole‐cell extracts of MCF7 and T‐47D cells were subjected to co‐IP using anti‐E2F1 antibodies, and the interaction between E2F1 and SND1 was identified by western blotting. C) Immunofluorescence (IF) staining assays exhibited the subcellular localization of SND1 (yellow) and E2F1 (green) in MCF7 and T‐47D cell lines. Nuclei were stained with DAPI (blue). D) The mRNA levels of E2F1 downstream target genes in MCF7 and T‐47D cells after transfection with the control or SND1 ‐overexpressing plasmid were determined by qRT‐PCR assay. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. E) Scatterplot showing the correlation between the expression of XBP1s and E2F1 , CCNE1 , CCNA2 , and RRM2 in tumors obtained from patients with HR+/HER2− early breast cancer ( n = 152). The correlation was assessed using Pearson's correlation coefficient. F,G) After being treated with or without 0.1 × 10 −6 of palbociclib for 24 h, whole‐cell extracts of MCF7 and T‐47D cells were immunoprecipitated with anti‐SND1 antibodies or anti‐E2F1 antibodies, followed by western blotting was conducted to investigate the interaction between SND1 and E2F1. H) The qRT‐PCR assay was used to detect the mRNA levels of E2F1 downstream target genes in MCF7 and T‐47D cells treated with or without palbociclib following stable transfection with either the control or XBP1s ‐overexpressing plasmid. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. For statistical analysis, two‐tailed unpaired Student's t ‐test was used for (D); one‐way ANOVA with Turkey's post hoc test was utilized for (H). Data are presented as the mean ± standard deviation.

Article Snippet: Immunoprecipitation was carried out using XBP1s (27901, Cell Signaling Technology) and normal mouse IgG (sc‐2025, Santa Cruz) antibodies at 4 °C overnight with rotation.

Techniques: Activation Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Expressing, Stable Transfection, Two Tailed Test, Standard Deviation

Journal: Advanced Science

Article Title: XBP1s Mediates Cross‐resistance to Combination Treatment of CDK4/6 Inhibitors plus Endocrine Therapy in Breast Cancer

doi: 10.1002/advs.202409588

Figure Lengend Snippet: 4µ8C sensitizes HR+/HER2− breast cancer to the combination therapy of fulvestrant and palbociclib in PDO models. A) Dose–response matrix (relative cell viability) and combination index (CI) for the effect of palbociclib (left) or fulvestrant (right) combined with 4µ8C in PDO‐0912. B) Dose–response matrix (relative cell viability) and CI for the effect of palbociclib (left) or fulvestrant (right) combined with 4µ8C in PDO‐3890. C) Acridine orange/propidium iodide (AO/PI) staining was performed on HR+/HER2− patient‐derived organoids PDO‐0912 and PDO‐3890 following treatment, as indicated. The left panel shows the representative images of live/dead organoids 96 h after the indicated treatment; the right panel shows the quantification results of live (green)/dead (red) analysis. PDO‐0912 was subjected to the vehicle, 10 × 10 −6 m 4µ8C, a combination of 5 × 10 −6 m palbociclib and 0.5 × 10 −6 m fulvestrant (Pal+Ful), or their combination with 10 × 10 −6 m 4µ8C (Pal+Ful+4µ8C); PDO‐3890 was subjected to the vehicle, 10 × 10 −6 m 4µ8C, a combination of 14 × 10 −6 m palbociclib and 1 × 10 −6 m fulvestrant (Pal+Ful), or their combination with 10 × 10 −6 m 4µ8C (Pal+Ful+4µ8C). Scale bar, 50 µm. D,E) Following transfection with the control or XBP1s ‐overexpressing plasmid, cell viability assay was performed to determine the organoid growth following treatment with the vehicle and Pal+Ful for the indicated days in PDO‐0912 and PDO‐3890. * p < 0.05, ** p < 0.01. F) Cell viability assay was performed to determine the organoid growth following treatment with the vehicle, 4µ8C, Pal+Ful, or Pal+Ful+4µ8C for the indicated days in PDO‐1553 and PDO‐4576. G–I) Mice with MCF7 cell‐derived xenografts were randomly divided into four treatment groups: vehicle, 4µ8C, Pal+Ful, or Pal+Ful+4µ8C. G) Representative images of IHC staining for XBP1s, SND1, and E2F1 in MCF7 cell‐derived xenografts from different treatment groups. Scale bar, 50 µm. H) Tumor growth curve plots of the MCF7 tumor in each treatment group after treatment administration. n = 6 per treatment group. I) Images of MCF7 cell‐derived xenografts at the end of the experiment. n = 6 per treatment group. For statistical analysis, two‐way ANOVA with Bonferroni's method correction was applied in (D) and (E). Data are presented as the mean ± standard deviation.

Article Snippet: Immunoprecipitation was carried out using XBP1s (27901, Cell Signaling Technology) and normal mouse IgG (sc‐2025, Santa Cruz) antibodies at 4 °C overnight with rotation.

Techniques: Staining, Derivative Assay, Transfection, Control, Plasmid Preparation, Viability Assay, Immunohistochemistry, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: BMP2 secretion from hepatocellular carcinoma cell HepG2 enhances angiogenesis and tumor growth in endothelial cells via activation of the MAPK/p38 signaling pathway

doi: 10.1186/s13287-019-1301-2

Figure Lengend Snippet: Silencing of BMP2 suppresses tumor angiogenesis. a Tumor volume and weight after over-expression or silencing of BMP2. b Representative image of xenograft tumors. c HE staining analysis of pathological changes in response to over-expression or silencing of BMP2 (× 400). d Vessel formation in response to over-expression or silencing of BMP2 observed under a laser confocal microscope (× 400). e VEGF protein level in serum of nude mice injected with cells infected with lentivirus expressing oeBMP2 or shBMP2 measured by ELISA. f Immunohistochemical staining for CD31 (× 400) showing MVD value in response to over-expression or silencing of BMP2. g Cell apoptosis in response to over-expression or silencing of BMP2 detected by TUNEL (× 400). h Positive rate of Caspase-3 and PCNA in response to over-expression or silencing of BMP2 detected by immunohistochemistry (× 400). i Western blot analysis of VEGF, VEGF-C, MMP-2, MMP-9, Vimentin, and E-cadherin proteins in response to over-expression or silencing of BMP2. n = 6. Data were expressed as mean ± standard deviation. p values were obtained by the Mann-Whitney U test. * p < 0.05. HCC, hepatocellular carcinoma; OD, optical density; BMP2, bone morphogenetic proteins 2; HE, hematoxylin-eosin; NC, negative control; VEGF, vascular endothelial growth factor; PCNA, proliferating cell nuclear antigen; MVD, microvessel density; ELISA, enzyme-linked immunosorbent assay; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling

Article Snippet: Nucleus staining was then carried out using hematoxylin (C0105, Beyotime Institute of Biotechnology, Shanghai, China) for 30 s, and diaminobenzidine (DAB) (P0202, Beyotime Institute of Biotechnology, Shanghai, China) was employed for color development.

Techniques: Over Expression, Staining, Microscopy, Injection, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, TUNEL Assay, Immunohistochemistry, Western Blot, Standard Deviation, MANN-WHITNEY, Negative Control, End Labeling

Journal: Stem Cell Research & Therapy

Article Title: BMP2 secretion from hepatocellular carcinoma cell HepG2 enhances angiogenesis and tumor growth in endothelial cells via activation of the MAPK/p38 signaling pathway

doi: 10.1186/s13287-019-1301-2

Figure Lengend Snippet: BMP2 enhances angiogenesis in vivo in HCC through MAPK/p38 signaling pathway activation. The MARK/p38 signaling pathway was inhibited by SB-239063 when BMP2 was over-expressed to identify the functional significance of MARK/p38 signaling pathway activation in the effects of BMP2 on tumor angiogenesis. a Tumor volume and weight of nude mice. b Representative images of xenograft tumors. c HE staining analysis of pathological changes in tumor tissues (× 400). d Angiogenesis in tumor tissues under a laser confocal microscope (× 400). e VEGF expression patterns in the serum of nude mice detected by ELISA. f Immunohistochemical staining for CD31 (× 400) showing MVD value. g Cell apoptosis detected by TUNEL (× 400). h Positive expression of Caspase-3 and PCNA examined by immunohistochemistry (× 400). i Western blot analysis of VEGF, VEGF-C, MMP-2, MMP-9, Vimentin, and E-cadherin protein patterns. n = 6. Data were expressed by mean ± standard deviation. p values were obtained using the Mann-Whitney U test. * p < 0.05; HCC, hepatocellular carcinoma; MAPK, mitogen-activated protein kinase; NC, negative control; VEGF, vascular endothelial growth factor; PCNA, proliferating cell nuclear antigen; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin-eosin; MVD, microvessel density; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling

Article Snippet: Nucleus staining was then carried out using hematoxylin (C0105, Beyotime Institute of Biotechnology, Shanghai, China) for 30 s, and diaminobenzidine (DAB) (P0202, Beyotime Institute of Biotechnology, Shanghai, China) was employed for color development.

Techniques: In Vivo, Activation Assay, Functional Assay, Staining, Microscopy, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, TUNEL Assay, Immunohistochemistry, Western Blot, Standard Deviation, MANN-WHITNEY, Negative Control, End Labeling

Journal: Translational Cancer Research

Article Title: The prognostic value and mechanisms of centromere protein M in patients with lung adenocarcinoma

doi: 10.21037/tcr-22-491

Figure Lengend Snippet: CENPM inhibits the cell apoptosis in LUAD cells. (A) CENPM downregulation increased H1975 cell apoptotic rate, as measured by flow cytometer. (B) CENPM upregulation decreased H292 cell apoptotic rate, as measured by flow cytometer. All data are the results of three independent experiments. Bars indicate the means ± SD. ***, P<0.001; ****, P<0.0001. CENPM, centromere protein M; SD, standard deviation; LUAD, lung adenocarcinoma; NC, negative control; PE, phycoerythrin; FITC-A, fluorescein isothiocyanate-acrylate; oe, overexpression; UL, upper left; UR, upper right; LL, lower left; LR, lower right; SD, standard deviation.

Article Snippet: Annexin V and PI staining were carried out using the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, China).

Techniques: Flow Cytometry, Standard Deviation, Negative Control, Over Expression

Journal: Translational Cancer Research

Article Title: The prognostic value and mechanisms of centromere protein M in patients with lung adenocarcinoma

doi: 10.21037/tcr-22-491

Figure Lengend Snippet: CENPM promotes cell proliferation, causes cell cycle arrest at G0/G1 phase, enhances cell migration and invasion capacity, promotes apoptosis in LUAD cell lines via regulation of AKT1/mTOR pathway. (A) LY294002 treatment inhibited H292 cell proliferation compared with oeCENPM treatment, measured through CCK-8. (B,C) LY294002 treatment decreased H292 cells G0/G1 phase while increased G2/M phase of cell cycle compared with oeCENPM treatment, as measured by flow cytometer. (D,E) LY294002 treatment inhibited H292 cell migration and invasion capacity compared with oeCENPM treatment, as determined through Transwell assay (crystal violet staining). (F) LY294002 treatment promoted H292 cell apoptosis compared with oeCENPM treatment, as measured by flow cytometer. Bars indicate the means ± SD. *, P<0.05; **, P<0.01; ****, P<0.0001. # , P<0.05 compared with the sham group. CENPM, centromere protein M; mTOR, mammalian target of rapamycin; oe, overexpression; CCK-8; cell counting kit-8; ns, not significant; RMS, root mean square; Freq., frequency; cv, coefficient of variation; PE, phycoerythrin; OD, optical density; SD, standard deviation; UL, upper left; UR, upper right; LL, lower left; LR, lower right.

Article Snippet: Annexin V and PI staining were carried out using the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, China).

Techniques: Migration, CCK-8 Assay, Flow Cytometry, Transwell Assay, Staining, Over Expression, Cell Counting, Standard Deviation

Journal: Translational Cancer Research

Article Title: The prognostic value and mechanisms of centromere protein M in patients with lung adenocarcinoma

doi: 10.21037/tcr-22-491

Figure Lengend Snippet: CENPM gene knockdown inhibits cell proliferation and promotes cell apoptosis in vivo . Xenograft nude mouse model was used to investigate the effect of CENPM knockdown on LUAD growth. H1975 cells were transduced with shCENPM and injected subcutaneously into BALB/c nude mice. (A,B) The nude mice were injected with shNC or shCENPM treated cells. (C) Representative images of tumors were observed in shNC (upper panel) or shCENPM (lower panel) group. (D) CENPM downregulation inhibited tumor volume. (E) CENPM downregulation inhibited tumor weight. (F) Representative images of immunohistochemical staining of tumors, and disorganized structure was witnessed in the xenograft of shCENPM group. (G) CENPM downregulation promoted apoptosis in tumors, as measured through increased TUNEL positive cells by immunofluorescence staining. (H) CENPM downregulation inhibited proliferation in tumors, as measured through decreased positivity for Ki67 by immunofluorescence staining. (I) AKT1/mTOR pathway activity were significantly declined in mice injected with shCENPM H1975 cells. Relative protein levels were determined after normalization to GAPDH. Bars indicate the means ± SD. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. CENPM, centromere protein M; LUAD, lung adenocarcinoma; NC, negative control; mTOR, mammalian target of rapamycin; SD, standard deviation.

Article Snippet: Annexin V and PI staining were carried out using the Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, China).

Techniques: Knockdown, In Vivo, Transduction, Injection, Immunohistochemical staining, Staining, TUNEL Assay, Immunofluorescence, Activity Assay, Negative Control, Standard Deviation

Journal: BioMed Research International

Article Title: Accumulation of AGO2 Facilitates Tumorigenesis of Human Hepatocellular Carcinoma

doi: 10.1155/2020/1631843

Figure Lengend Snippet: Clinical analysis of AGO2 expression in hepatocellular carcinoma patients. (a) Representative image of AGO2 IHC staining in noncancerous (epithelium) tissues and HCC tissues. (b) Relative expression levels of AGO2 in noncancerous and HCC tissues were determined by measuring the staining intensity (ratings: 0, 1, 2, and 3). Values represented as the means ± S.D. ( n = 87). Kaplan-Meier survival analysis according to AGO2 expression (c), age (d), gender (e), and tumor size (f) in 90 patients with HCC (log-rank test). Probability of survival of patients: (c) low expression of AGO2 ( n = 26) vs. overexpression of AGO2 ( n = 61) ( P = 0.037); (d) age > 65 ( n = 12) vs. age≦65 ( n = 77) ( P = 0.597); (e) male ( n = 81) vs. female ( n = 9) ( P = 0.625); (f) tumor size > 5 cm ( n = 50) vs. tumor size≦5 cm ( n = 39) ( P = 0.005).

Article Snippet: The pCMV3 vector carrying human AGO2 cDNA was obtained from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Immunohistochemistry, Staining, Over Expression

Journal: BioMed Research International

Article Title: Accumulation of AGO2 Facilitates Tumorigenesis of Human Hepatocellular Carcinoma

doi: 10.1155/2020/1631843

Figure Lengend Snippet: The correlation of AGO2 expression with clinical parameters of HCC patients. CC, correlation coefficient.

Article Snippet: The pCMV3 vector carrying human AGO2 cDNA was obtained from Sino Biological Inc. (Beijing, China).

Techniques: Expressing

Journal: BioMed Research International

Article Title: Accumulation of AGO2 Facilitates Tumorigenesis of Human Hepatocellular Carcinoma

doi: 10.1155/2020/1631843

Figure Lengend Snippet: AGO2 promoted cell proliferation and migration in HCC in vitro. (a) AGO2 protein abundances in different cell lines were detected by western blot analysis; GAPDH was used as a loading control. (b) The ratio of AGO2 to GAPDH in each cell line was measured by densitometric analysis of western blot bands, respectively. (c) Designation of AGO2 protein depletion in SMMC-7721 cells using CRISPR/Cas9 gene editing strategy. (d) AGO2 expression in SMMC-7721 cells was determined by western blotting. (e) Colony formation detected by Giemsa staining for normal and AGO2 −/− SMMC-7721 cells. (f) AGO2 knockout induced inhibition in colony formation as compared to control. (g) Diagram of the cell cycle analysis for normal and AGO2 −/− SMMC-7721 cells based on flow cytometry. (h) The proportions of cells in G1, S, and G2 phases were calculated. (i) Representative images for cell migration assay. (j) Numbers of stained cells in migration assay were counted. Data are normalized and expressed as the fold change relative to control values. Values represented as the means ± S.D. ( n > 3 each group); ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.005.

Article Snippet: The pCMV3 vector carrying human AGO2 cDNA was obtained from Sino Biological Inc. (Beijing, China).

Techniques: Migration, In Vitro, Western Blot, Control, CRISPR, Expressing, Staining, Knock-Out, Inhibition, Cell Cycle Assay, Flow Cytometry, Cell Migration Assay

Journal: BioMed Research International

Article Title: Accumulation of AGO2 Facilitates Tumorigenesis of Human Hepatocellular Carcinoma

doi: 10.1155/2020/1631843

Figure Lengend Snippet: AGO2 enhanced tumor growth and size in HCC in vivo. (a) Control and AGO2 knockout SMMC-7721 cells were subcutaneously injected in the flank of BALB/c nude mice with 2 × 10 6 cells. About 7 days post injection, the tumor size was measured every other day. The tumor size was calculated as (length × width 2 )/2. At the indicated time, all mice were sacrificed and the tumors were photographed. (b) The size of killed nude mice of the indicated groups. (c) The size of tumors isolated from nude mice. (d) The weights of tumors were measured. Values represented as the means ± S.D.

Article Snippet: The pCMV3 vector carrying human AGO2 cDNA was obtained from Sino Biological Inc. (Beijing, China).

Techniques: In Vivo, Control, Knock-Out, Injection, Isolation

Journal: BioMed Research International

Article Title: Accumulation of AGO2 Facilitates Tumorigenesis of Human Hepatocellular Carcinoma

doi: 10.1155/2020/1631843

Figure Lengend Snippet: Bioinformatics analysis of microarray data. (a) The heat map of the pairwise comparison in control and AGO2 knockout SMMC-7721 cells. The colored bar shows the expression levels of genes. (b) Gene Ontology (GO) functional classification of differentially expressed genes (DEGs). The GO terms are shown in the bar chart of biological processes, cellular components, and molecular functions. (c) Heatmaps showed the upregulation and downregulation of DEGs associated with disease. Orange: Z ‐score > 0, blue: Z ‐score < 0, gray: no Z -score value. Z ‐score > 2 indicates that the function is significantly activated, while Z ‐score < ‐2 indicates that the function is significantly inhibited.

Article Snippet: The pCMV3 vector carrying human AGO2 cDNA was obtained from Sino Biological Inc. (Beijing, China).

Techniques: Microarray, Comparison, Control, Knock-Out, Expressing, Functional Assay

Journal: BioMed Research International

Article Title: Accumulation of AGO2 Facilitates Tumorigenesis of Human Hepatocellular Carcinoma

doi: 10.1155/2020/1631843

Figure Lengend Snippet: AGO2 upregulated Survivin, Vimentin, and Snail expression. Western blot results showed that the expression of Survivin (a), Vimentin (b), and Snail (c) were significantly decreased in SMMC-7721 AGO2 −/− cells compared with control cells. GAPDH was used as a loading control. (d) Quantification of protein expression of Survivin (a), Vimentin (b), and Snail (c) that was normalized by GAPDH, respectively. Values represented as the means ± S.D. ( n > 3 each group); ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.005.

Article Snippet: The pCMV3 vector carrying human AGO2 cDNA was obtained from Sino Biological Inc. (Beijing, China).

Techniques: Expressing, Western Blot, Control

Journal: BioMed Research International

Article Title: Accumulation of AGO2 Facilitates Tumorigenesis of Human Hepatocellular Carcinoma

doi: 10.1155/2020/1631843

Figure Lengend Snippet: AGO2 overexpression promoted proliferation and migration of SMMC-7721 and Huh-7 in vitro. (a–f) Huh-7 cells were transiently transfected with pCMV3-AGO2 (AGO2 OE ) and an empty vector pCMV3 (control), respectively. (g–l) SMMC-7721 cells were used for AGO2 overexpression and subsequently analysis. Western blot was used to examine the effect of pCMV3-AGO2 transfection into Huh-7 (a) and SMMC-7721 (g) cells. (b and h) CCK8 assay was performed to detect cell proliferation of AGO2 OE Huh-7 (b) and SMMC-7721 (h) cells. (c and i) Cell migration rates of AGO2 OE Huh-7 (c) and SMMC-7721 (i) were determined by transwell assay. Western blot was performed to detect the effect of AGO2 overexpression on Survivin, Snail, and Vimentin expression in Huh-7 (d–f) and SMMC-7721 (j–l) cells. The ratio of Survivin, Snail, and Vimentin to GAPDH in Huh-7 and SMMC-7721 was measured by densitometric analysis of western blot bands, respectively. Values represented as the means ± S.D. ( n > 3 each group); ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.005.

Article Snippet: The pCMV3 vector carrying human AGO2 cDNA was obtained from Sino Biological Inc. (Beijing, China).

Techniques: Over Expression, Migration, In Vitro, Transfection, Plasmid Preparation, Control, Western Blot, CCK-8 Assay, Transwell Assay, Expressing

Journal: Oncology reports

Article Title: HOXA7 stimulates human hepatocellular carcinoma proliferation through cyclin E1/CDK2.

doi: 10.3892/or.2014.3668

Figure Lengend Snippet: Figure 2. Impact of the knockdown of HOXA7 on the proliferation of HCC cells. (A and B) RT-PCR and western blotting verified the knockdown effect of HOXA7 in the HepG2 and QGY-7703 cell lines. (C and D) Cell proliferation curves of HepG2/si-HOXA7, QGY-7703/si-HOXA7 and negative control cells as detected by MTT assay. (E and F) Colony formation analysis of HepG2/si‑HOXA7 and HepG2/NC cell lines (**P<0.01, HepG2/si-HOXA7 vs. HepG2/NC). (G and H) Colony formation analysis of QGY-7703/si-HOXA7 and QGY-7703/NC cell lines (*P<0.05, QGY-7703/si-HOXA7 vs. QGY-7703/NC).

Article Snippet: Western blotting was carried out using the following antibodies: rabbit anti-HOXA7 antibody (Santa Cruz Biotechnology), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology), rabbit anti-cyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody, and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control, MTT Assay

Journal: Oncology reports

Article Title: HOXA7 stimulates human hepatocellular carcinoma proliferation through cyclin E1/CDK2.

doi: 10.3892/or.2014.3668

Figure Lengend Snippet: Figure 1. Expression of HOXA7 in the HepG2, Hep3B-2, QGY-7703 and QSG‑7701 cell lines. (A) mRNA expression of HOXA7 and GAPDH detected by RT-PCR. (B) Protein expression of HOXA7 and β-actin as detected by western blotting.

Article Snippet: Western blotting was carried out using the following antibodies: rabbit anti-HOXA7 antibody (Santa Cruz Biotechnology), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology), rabbit anti-cyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody, and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncology reports

Article Title: HOXA7 stimulates human hepatocellular carcinoma proliferation through cyclin E1/CDK2.

doi: 10.3892/or.2014.3668

Figure Lengend Snippet: Figure 3. Cell cycle distribution. (A) Cell cycle distribution of HepG2/si‑HOXA7 and HepG2/NC cells as detected by flow cytometry. (B) The percentage of HepG2/si-HOXA7 and HepG2/NC cells in the phases of the cell cycle. (C) Cell cycle distribution of QGY-7703/si-HOXA7 and QGY-7703/NC cells as detected by flow cytometry. (D) The percentage of QGY-7703/si-HOXA7 and QGY-7703/NC cells in the phases of the cell cycle.

Article Snippet: Western blotting was carried out using the following antibodies: rabbit anti-HOXA7 antibody (Santa Cruz Biotechnology), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology), rabbit anti-cyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody, and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Flow Cytometry

Journal: Oncology reports

Article Title: HOXA7 stimulates human hepatocellular carcinoma proliferation through cyclin E1/CDK2.

doi: 10.3892/or.2014.3668

Figure Lengend Snippet: Figure 5. Tumor formation of the HepG2/si-HOXA7, QGY-7703/si-HOXA7 and control cell xenograft tumors in the Balb/c nude mice. (A and B) Tumor volume was measured every 5 days. (C-F) Forty days after tumor cell injection, the mice were sacrificed and the tumor weight was recorded. (G and H) RT-PCR and western blot analyses of the expression of HOXA7 in the representative xenograft tumors.

Article Snippet: Western blotting was carried out using the following antibodies: rabbit anti-HOXA7 antibody (Santa Cruz Biotechnology), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology), rabbit anti-cyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody, and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

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

Journal: Oncology reports

Article Title: HOXA7 stimulates human hepatocellular carcinoma proliferation through cyclin E1/CDK2.

doi: 10.3892/or.2014.3668

Figure Lengend Snippet: Figure 4. Overexpression of HOXA7 promotes QSG‑7701 cell proliferation. (A) RT-PCR and western blotting verified the overexpression of HOXA7 in the QSG-7701 cells. (B) Cell proliferation curve of QSG-7701-HOXA7 and negative control cells as detected by MTT analysis. (C and D) Colony formation analysis of the QSG-7701-HOXA7 and QSG-7701-C cell lines (**P<0.01, QSG-7701‑HOXA7 vs. QSG-7701-C). (E) Cell cycle distribution of QSG‑7701‑HOXA7 and QSG-7701-C cells as detected by flow cytometry. (F) The percentage of QSG-7701-HOXA7 and QSG-7701-C cells in each phase of the cell cycle.

Article Snippet: Western blotting was carried out using the following antibodies: rabbit anti-HOXA7 antibody (Santa Cruz Biotechnology), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology), rabbit anti-cyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody, and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control, Flow Cytometry

Journal: Oncology reports

Article Title: HOXA7 stimulates human hepatocellular carcinoma proliferation through cyclin E1/CDK2.

doi: 10.3892/or.2014.3668

Figure Lengend Snippet: Figure 6. HOXA7 affects the expression of cell cycle-related proteins in the HCC and normal hepatic cells in the xenograft tumors. (A and B) Western blot analyses detected the expression of cyclin D1/CDK4 and cyclin E1 and CDK2 in the HCC cells. (C and D) Western blotting detected the expression of cyclin D1/CDK4 and cyclin E1 and CDK2 in normal hepatic cells and xenograft tumors.

Article Snippet: Western blotting was carried out using the following antibodies: rabbit anti-HOXA7 antibody (Santa Cruz Biotechnology), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology), rabbit anti-cyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody, and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: Oncology reports

Article Title: Cell motility and spreading promoted by CEACAM6 through cyclin D1/CDK4 in human pancreatic carcinoma.

doi: 10.3892/or.2015.4338

Figure Lengend Snippet: Figure 1. The expression level of CEACAM6 protein in pancreatic carcinoma and non-cancerous tissues. (A) Pancreatic carcinoma tissues (magnification, x100). (B) Pancreatic carcinoma tissues (magnification, x200). (C) Pancreatic carcinoma tissues (magnification, x400). (D) Non-cancerous (magnification, x100). (E) Non-cancerous (magnification, x200). (F) Non-cancerous (magnification, x400).

Article Snippet: Western blot analysis was carried out using the following antibodies: rabbit anti-CEACAM6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, uSA), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology, Danvers, CA, uSA), rabbit anticyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Expressing

Journal: Oncology reports

Article Title: Cell motility and spreading promoted by CEACAM6 through cyclin D1/CDK4 in human pancreatic carcinoma.

doi: 10.3892/or.2015.4338

Figure Lengend Snippet: Figure 2. The expressions of CEACAM6 in pancreatic carcinoma cell lines. (A) mRNA expression of CEACAM6 and GAPDH detected by RT-PCR. (B) Protein expression of CEACAM6 and β-actin as detected by western blot analysis.

Article Snippet: Western blot analysis was carried out using the following antibodies: rabbit anti-CEACAM6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, uSA), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology, Danvers, CA, uSA), rabbit anticyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncology reports

Article Title: Cell motility and spreading promoted by CEACAM6 through cyclin D1/CDK4 in human pancreatic carcinoma.

doi: 10.3892/or.2015.4338

Figure Lengend Snippet: Figure 3. The investigation of the biological significance of CEACAM6 overexpression in pancreatic carcinoma cell lines. (A and B) The RNAi effect of CEACAM6 in pancreatic carcinoma cell lines. (C and D) CCK-8 assay of CEACAM6-siRNA on the growth of pancreatic carcinoma cells. (E-H) Colony formation analysis of BxPC-3/si-CEACAM6, BxPC-3/NC and SW1990/si-CEACAM6, SW1990/NC cell lines (*P<0.05, si-CEACAM6 vs. NC).

Article Snippet: Western blot analysis was carried out using the following antibodies: rabbit anti-CEACAM6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, uSA), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology, Danvers, CA, uSA), rabbit anticyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Over Expression, CCK-8 Assay

Journal: Oncology reports

Article Title: Cell motility and spreading promoted by CEACAM6 through cyclin D1/CDK4 in human pancreatic carcinoma.

doi: 10.3892/or.2015.4338

Figure Lengend Snippet: Figure 4. Cell cycle distribution. (A) The percentage of BxPC-3/si-CEACAM6 and BxPC-3/NC cells in the phases of the cell cycle. (B) Cell cycle distribution of BxPC-3/si-CEACAM6 and BxPC-3/NC cells as detected by flow cytometry. (C) The percentage of SW1990/si-CEACAM6 and SW1990/NC cells in the cell cycle. (D) Cell cycle distribution of SW1990/si-CEACAM6 and SW1990/NC cells as detected by flow cytometry.

Article Snippet: Western blot analysis was carried out using the following antibodies: rabbit anti-CEACAM6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, uSA), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology, Danvers, CA, uSA), rabbit anticyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Flow Cytometry

Journal: Oncology reports

Article Title: Cell motility and spreading promoted by CEACAM6 through cyclin D1/CDK4 in human pancreatic carcinoma.

doi: 10.3892/or.2015.4338

Figure Lengend Snippet: Figure 6. Tumor formation in nude mice. (A and B) Tumor formation of the BxPC-3/si-CEACAM6, SW1990/si-CEACAM6 and xenograft tumors in the Balb/c nude mice. (C and D) Tumor volume was measured every 5 days. Forty days after tumor cell injection, the mice were sacrificed and the tumor weight was recorded. (E and F) RT-PCR and western blot analyses of the expression of CEACAM6 in the representative xenograft tumors.

Article Snippet: Western blot analysis was carried out using the following antibodies: rabbit anti-CEACAM6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, uSA), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology, Danvers, CA, uSA), rabbit anticyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Injection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

Journal: Oncology reports

Article Title: Cell motility and spreading promoted by CEACAM6 through cyclin D1/CDK4 in human pancreatic carcinoma.

doi: 10.3892/or.2015.4338

Figure Lengend Snippet: Figure 5. Overexpression of CEACAM6 promotes cells of MIA PaCa-2 proliferation. (A) RT-PCR and western blot analysis verified the overexpression effect of CEACAM6 in MIA PaCa-2. (B) Cell proliferation curve of MIA PaCa-2-CEACAM6 and negative control cells detected by CCK-8 analysis. (C and D) The colony formation analysis of MIA PaCa-2-CEACAM6 and MIA PaCa-2-C cell lines (**P<0.01, MIA PaCa-2-CEACAM6 vs. MIA PaCa-2-C). (E) Cell cycle distribution of MIA PaCa-2-CEACAM6 and MIA PaCa-2-C detected by flow cytometry. (F) Cell cycle distribution percent of MIA PaCa-2-CEACAM6 and MIA PaCa-2-C.

Article Snippet: Western blot analysis was carried out using the following antibodies: rabbit anti-CEACAM6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, uSA), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology, Danvers, CA, uSA), rabbit anticyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Negative Control, CCK-8 Assay, Flow Cytometry

Journal: Oncology reports

Article Title: Cell motility and spreading promoted by CEACAM6 through cyclin D1/CDK4 in human pancreatic carcinoma.

doi: 10.3892/or.2015.4338

Figure Lengend Snippet: Figure 7. CEACAM6 affects the expression of cell cycle-related proteins in pancreatic carcinoma cells and xenograft tumors. (A and B) Western blotting detected the expression of cyclin D1/CDK4 in pancreatic carcinoma cells. (C and D) Western blotting detected the expression of cyclin D1/CDK4 in xenograft tumors.

Article Snippet: Western blot analysis was carried out using the following antibodies: rabbit anti-CEACAM6 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, uSA), rabbit anti-cyclin D1 antibody (Cell Signaling Biotechnology, Danvers, CA, uSA), rabbit anticyclin E1 antibody (Abcam Biotechnology), rabbit anti-CDK4 antibody and rabbit anti-CDK2 antibody (both from Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: The FASEB Journal

Article Title: Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia

doi: 10.1096/fj.201802058rr

Figure Lengend Snippet: Figure 1. ACTN4 expression pattern in placenta. A) IHC staining of ACTN4 in first trimester human placenta villi (a, b, d, e) and decidua (c, f). iEVTs and CTBs were stained by HLA-G and CK7, respectively; AV, anchoring villi; FV, floating villi; CCT, cell column trophoblast; STB, syncytiotrophoblasts. Scale bars, 100 mm. B) Western blotting of ACTN4 in normal and EO-PE placentas, Data are presented as the median + range. *P , 0.05, Mann-Whitney U test. C) IHC staining of ACTN4 in floating villi and BP of normal and EO-PE placentas on serial sections. Cell column trophoblasts (CCTs), iEVTs, and CTBs were stained by HLA-G and CK7, respectively, DS, decidual side. Scale bars: 100 mm (a, b, e, f); 200 mm (c, d, g, h). D) Western blotting of ACTN4 in primary CTB cells isolated from normal and EO-PE placentas. Data are presented as the means 6 SEM. **P , 0.01, ***P , 0.001.

Article Snippet: Adenoviral-carrying shRNAtargeting mouse ACTN4 was purchased from Cyagen Biosciences (Santa Clara, CA, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot, MANN-WHITNEY, Isolation

Journal: The FASEB Journal

Article Title: Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia

doi: 10.1096/fj.201802058rr

Figure Lengend Snippet: Figure 2. ACTN4 regulates trophoblast proliferation. A) Western blotting of ACTN4 in HTR-8/SVneo (HTR8), JAR, and JEG3 cells. B) Interference efficiency of siRNAs targeting ACTN4 in HTR-8/SVneo (HTR8) cells was assessed by Western blotting. NC, negative control C) CCK-8 assay on HTR-8/SVneo cells after 0, 24, 48, and 72 h of si-ACTN4#1 and si-ACTN4#2 transfection. OD, optical density. D) Cell cycle analysis of HTR-8/SVneo cells by flow cytometry after 48 h of si-ACTN4#1 and si-ACTN4#2 transfection. E) EdU (pink) staining on HTR-8/SVneo cells after 48 h of si-ACTN4#1 and si-ACTN4#2 transfection. Nuclei were counterstained with DAPI (blue). Scale bars, 200 mm. NS, nonsignificant. All data are means 6 SEM of 3 independent experiments performed in triplicate. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: Adenoviral-carrying shRNAtargeting mouse ACTN4 was purchased from Cyagen Biosciences (Santa Clara, CA, USA).

Techniques: Western Blot, Negative Control, CCK-8 Assay, Transfection, Cell Cycle Assay, Cytometry, Staining

Journal: The FASEB Journal

Article Title: Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia

doi: 10.1096/fj.201802058rr

Figure Lengend Snippet: Figure 3. ACTN4 regulates the PI3K, AKT, and GSK3b pathway in trophoblasts. A) Western blotting of ACTN4, p-AKTSer473, AKT, p-GSK3b, GSK3b, cyclin D1, and p21 in HTR-8/SVneo cells transfected with si-ACTN4#1 or ACTN4 overexpression (ACTN4-OE) plasmid for 48 h in the presence and absence of LY294002 (20 mM) for 24 h after transfection. B) Densitometry quantification of Western blots. NS, nonsignificant. Data are means 6 SEM of 3 independent experiments performed in triplicate. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: Adenoviral-carrying shRNAtargeting mouse ACTN4 was purchased from Cyagen Biosciences (Santa Clara, CA, USA).

Techniques: Western Blot, Transfection, Over Expression, Plasmid Preparation

Journal: The FASEB Journal

Article Title: Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia

doi: 10.1096/fj.201802058rr

Figure Lengend Snippet: Figure 4. ACTN4 binds to and facilitates AKT translocation onto the plasma membrane in trophoblasts. A) IF staining of AKT (red) and ACTN4 (green) in HTR-8/SVneo (HTR8) and primary human CTB cells. Nuclei were counterstained by DAPI (blue). Scale bars, 200 mm. B) IP of ACTN4 in HTR-8/SVneo cells and primary human CTBs by the use of anti-ACTN4 rabbit pAb or control rabbit IgG. The IP products and input control were then blotted with antibodies against ACTN4 and AKT. C) Western blotting of membranous and cytoplasmic AKT in HTR-8/SVneo cells that were transfected with si-ACTN4#1 or ACTN4 overexpression (ACTN4-OE) plasmid. Na, K-ATPase, and b-actin were applied for loading controls. NC, negative control.

Article Snippet: Adenoviral-carrying shRNAtargeting mouse ACTN4 was purchased from Cyagen Biosciences (Santa Clara, CA, USA).

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Staining, Control, Western Blot, Transfection, Over Expression, Plasmid Preparation, Negative Control

Journal: The FASEB Journal

Article Title: Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia

doi: 10.1096/fj.201802058rr

Figure Lengend Snippet: Figure 5. The ACTN4–b-catenin complex is essential for the regulation of podosome formation by E-cadherin. A) IF staining of ACTN4 (green) and b-catenin (red) in JEG3 cells. Scale bar, 200 mm. B) Co-IP of ACTN4 and b-catenin in JEG3 cells. C) IF staining of ACTN4 (green) and b-catenin (red) in E-cadherin–deficient JEG3 cells; white arrows: podosomes. Scale bars, 100 mm. D) Co-IP of ACTN4 and b-catenin in E-cadherin–deficient JEG3 cells. E) TRITC-phalloidin staining (red) of actin filament in E- cadherin–deficient JEG3 cells in the presence of si-ACTN4#1 or control (si-NC) for 24 h. Con, blank control. Nuclei were counterstained by DAPI (blue). Scale bars, 200 mm. Insets: higher-magnification views of the boxed areas. Scale bars, 100 mm. F) Matrigel invasion assays (upper panel) and transwell migration assays (lower panel) of E-cadherin–deficient JEG3 cells in the presence of si-ACTN4#1 or si-NC. NS, nonsignificant. All data are means 6 SEM of 3 independent experiments performed in triplicate. **P , 0.01, ***P , 0.001, ****P , 0.0001.

Article Snippet: Adenoviral-carrying shRNAtargeting mouse ACTN4 was purchased from Cyagen Biosciences (Santa Clara, CA, USA).

Techniques: Staining, Co-Immunoprecipitation Assay, Control, Migration

Journal: The FASEB Journal

Article Title: Trophoblastic proliferation and invasion regulated by ACTN4 is impaired in early onset preeclampsia

doi: 10.1096/fj.201802058rr

Figure Lengend Snippet: Figure 6. ACTN4-deficient mice have abnormal placentation and PE-like phenotypes. A) Western blotting of ACTN4, p- AKTSer473, AKT, p-GSK3b, and GSK3b in placentas collected from Ad-Ctrl (n = 4)– or Ad-ACTN4 (n = 5)–injected mice on GD 14.5 and Ad-Ctrl (n = 5)– or Ad-ACTN4 (n = 5)–injected mice on GD 18.5. B) Hematoxylin and eosin (H&E) staining (upper panel) and IHC staining of Ki67 (lower panel) on GD-14.5 placentas from Ad-Ctrl (n = 4)– and Ad-ACTN4 (n = 5)–injected mice. The area of labyrinth zone (Lz) and Jz and the Lz:Jz ratio were analyzed. Scale bars, 1000 mm (upper panel) and 200 mm (lower panel). C) SBP was monitored daily in Ad-Ctrl (n = 6) and Ad-ACTN4 (n = 6) groups during GD 6.5 and GD 18.5, as well as in NP female mice treated with Ad-Ctrl (n = 5) and Ad-ACTN4 (n = 5). P, pregnant. D) Placental weight on GD 18.5. E) Fetal weight on GD 18.5 (Ad-Ctrl, n = 73 pups from 5 dams; Ad-ACTN4, n = 69 pups from 5 dams). NS, nonsignificant. Data are expressed as the means 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: Adenoviral-carrying shRNAtargeting mouse ACTN4 was purchased from Cyagen Biosciences (Santa Clara, CA, USA).

Techniques: Western Blot, Injection, Staining, Immunohistochemistry

Journal: Cancer Research Communications

Article Title: Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer

doi: 10.1158/2767-9764.CRC-22-0330

Figure Lengend Snippet: Clinical relevance of MELK in breast cancer and expression of MELK in breast cancer cell lines. A, MELK mRNA levels in HR − HER2 + , HR + HER2 − , HR + HER2 + , and TNBC tumors in the World Consortium IBC dataset, analyzed by multiple comparison using a general linear model. Data are presented as mean ± SD. – represents median, X represents mean. B, IHC images (200× magnification) showing MELK protein expression in normal epithelial tissue and luminal, HER2 + , and TNBC breast tumors. Correlation between MELK mRNA levels (<7 or ≥7) and OS, PFS, and DMFS in patients in the overall cohort ( C ) and among those with TNBC ( D ), determined by the Kaplan–Meier method. MELK mRNA levels ( E ) and protein levels ( F ) in TNBC and non-TNBC cells. Inset, correlation between MELK mRNA and protein levels in TNBC cell lines. In F, α-tubulin was used as a loading control. MELK mRNA levels were determined using quantitative reverse transcriptase PCR.

Article Snippet: Cells (1 × 10 6 ) were transfected with 8 μg of the HA-DDK-pcDNA3 empty vector (OriGene) or the HA-DDK-pcDNA3 vector carrying wild-type (WT) MELK (OriGene) or kinase-dead (D150A; KD) MELK using the Neon Transfection System.

Techniques: Expressing, Comparison, Control, Reverse Transcription

Journal: Cancer Research Communications

Article Title: Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer

doi: 10.1158/2767-9764.CRC-22-0330

Figure Lengend Snippet: Univariate Cox regression model for OS, PFS, and DMFS among patients with breast cancer (World IBC Consortium dataset; n = 314)

Article Snippet: Cells (1 × 10 6 ) were transfected with 8 μg of the HA-DDK-pcDNA3 empty vector (OriGene) or the HA-DDK-pcDNA3 vector carrying wild-type (WT) MELK (OriGene) or kinase-dead (D150A; KD) MELK using the Neon Transfection System.

Techniques:

Journal: Cancer Research Communications

Article Title: Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer

doi: 10.1158/2767-9764.CRC-22-0330

Figure Lengend Snippet: Multicovariate Cox regression model for OS, PFS, and DMFS among patients with breast cancer (World IBC Consortium dataset; n = 314)

Article Snippet: Cells (1 × 10 6 ) were transfected with 8 μg of the HA-DDK-pcDNA3 empty vector (OriGene) or the HA-DDK-pcDNA3 vector carrying wild-type (WT) MELK (OriGene) or kinase-dead (D150A; KD) MELK using the Neon Transfection System.

Techniques:

Journal: Cancer Research Communications

Article Title: Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer

doi: 10.1158/2767-9764.CRC-22-0330

Figure Lengend Snippet: Effect of MELK knockdown or overexpression on proliferation, motility, EMT, and CSC properties in TNBC cells. Effect of MELK knockdown or KO ( A ) or overexpression ( B ) on proliferation and anchorage-independent growth of TNBC cells. HCC70 and BT-549 cells (A, bottom left) were treated with two different siMELK sequences for 48 hours. MELK expression was then determined using Western blot analysis (top), and cell viability was determined using trypan blue exclusion assay (bottom left). In MELK KO MDA-MB-231 cells (A, bottom right), anchorage-independent growth was determined using soft-agar assay at week 3 following incubation. β-Actin was used as a loading control for Western blot analysis. SUM149 and SUM159 cells (B) were transfected with plasmids encoding WT MELK gene or KD MELK gene and cultured for 48 hours. MELK expression was then determined using Western blot analysis (top), and cell growth was determined using anchorage-independent growth assay (bottom). β-Actin was used as a loading control for Western blot analysis. Effect of MELK knockdown ( C ) or overexpression ( D ) on migration and invasion of TNBC cells. Cells were transfected with two different siMELK sequences for 72 hours or plasmids encoding WT MELK gene or KD MELK gene for 48 hours and then assayed for migration and invasion. E, Effect of MELK knockdown on expression of EMT epithelial and mesenchymal markers in TNBC cells. Cells were treated with two different siMELK sequences for 48 hours, and then MELK expression was determined using Western blot analysis. α-Tubulin was used as a loading control. Effect of knockdown or KO ( F ) or overexpression ( G ) of MELK on mammosphere formation in TNBC cells. Cells were transfected with two different siMELK sequences or plasmids encoding WT MELK gene or KD MELK gene and 48 hours later were seeded for mammosphere formation. MELK KO clones were cultured for 7 days and then analyzed for mammosphere formation. H, Effect of MELK knockdown on CD44 + /CD24 − and ALDH1 + subpopulations in TNBC cells. Cells were treated with two different siMELK sequences and 48 hours later subjected to flow cytometry analysis. In A and F, P15 indicates Cas9-p15 control cells; C3, MELK KO C3 clones; and C28, MELK KO C28 clones. In A–D, G, and F, data are presented as mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Cells (1 × 10 6 ) were transfected with 8 μg of the HA-DDK-pcDNA3 empty vector (OriGene) or the HA-DDK-pcDNA3 vector carrying wild-type (WT) MELK (OriGene) or kinase-dead (D150A; KD) MELK using the Neon Transfection System.

Techniques: Knockdown, Over Expression, Expressing, Western Blot, Trypan Blue Exclusion Assay, Soft Agar Assay, Incubation, Control, Transfection, Cell Culture, Growth Assay, Migration, Clone Assay, Flow Cytometry, Standard Deviation

Journal: Cancer Research Communications

Article Title: Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer

doi: 10.1158/2767-9764.CRC-22-0330

Figure Lengend Snippet: Inhibitory effect of MELK-In-17 on anchorage-independent growth, motility, CSC properties, and EMT transition in TNBC cells. A–D, MELK-In-17 inhibited anchorage-independent growth ( A ), suppressed migration and invasion ( B ), reduced mammosphere formation ( C ), and reduced CD44 + /CD24 − and ALDH1 + CSC subpopulations ( D ) in TNBC cells. In A, the anchorage-independent growth was determined using a soft-agar assay at week 3 following treatment. In B, cells were pretreated with MELK-In-17 for 2 hours and then assayed for migration and invasion in the presence of MELK-In-17. In C, cells were seeded for mammosphere formation and the next day treated with MELK-In-17. In D, cells were treated with MELK-In-17 for 48 hours and then subjected to flow cytometry analysis. E, Western blot analysis of the effects of treatment with MELK-In-17 on the expression of EMT markers. α-Tubulin was used as a loading control. Band intensity of proteins is normalized to that of α-Tubulin. In A–C, data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Cells (1 × 10 6 ) were transfected with 8 μg of the HA-DDK-pcDNA3 empty vector (OriGene) or the HA-DDK-pcDNA3 vector carrying wild-type (WT) MELK (OriGene) or kinase-dead (D150A; KD) MELK using the Neon Transfection System.

Techniques: Migration, Soft Agar Assay, Flow Cytometry, Western Blot, Expressing, Control

Journal: Cancer Research Communications

Article Title: Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer

doi: 10.1158/2767-9764.CRC-22-0330

Figure Lengend Snippet: Effect of inhibition of MELK signaling on tumor growth and lung metastasis in TNBC xenograft mouse models. A, MELK expression in Cas9-p15 (P15) and MELK KO MDA-MB-231 (C3 and C28) cells. α-Tubulin was used as a loading control. B–F, MELK KO significantly suppressed lung metastases in an MDA-MB-231 xenograft mouse model. Female athymic nude mice were injected intravenously with luciferase-transfected Cas9-p15 or MELK KO MDA-MB-231-Luc-GFP stable cells. Metastatic tumors were measured weekly for 7 weeks by whole-body luciferase imaging using an IVIS 100 Imaging System. Shown are mouse whole-body luciferase images ( B ), photon counts per area ( C ), lung weight per mouse measured at week 7 following cell inoculation ( D ), mouse OS over a period of 80 days following cell inoculation ( E ), and IHC staining for hematoxylin and eosin (H&E), MELK, proliferation (Ki67), and mesenchymal markers (vimentin and fibronectin; F ) in mice inoculated with Cas9-p15 or MELK KO MDA-MB-231 cells. Images were taken under 200 × magnification. G, MELK-In-17 significantly suppressed tumor growth in a 4T1 xenograft mouse model ( P < 0.05). Murine 4T1 TNBC cells were injected into the mammary fat pads of female BALB/c mice. When tumor size reached 75–150 mm 3 , grouped mice were treated with vehicle or MELK-In-17 at 5 or 10 mg/kg via intraperitoneal injections daily for 25 days. In C–G, data are presented as mean ± SD. *, P < 0.05; **, P < 0.01.

Article Snippet: Cells (1 × 10 6 ) were transfected with 8 μg of the HA-DDK-pcDNA3 empty vector (OriGene) or the HA-DDK-pcDNA3 vector carrying wild-type (WT) MELK (OriGene) or kinase-dead (D150A; KD) MELK using the Neon Transfection System.

Techniques: Inhibition, Expressing, Control, Injection, Luciferase, Transfection, Imaging, Immunohistochemistry

Journal: Cancer Research Communications

Article Title: Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer

doi: 10.1158/2767-9764.CRC-22-0330

Figure Lengend Snippet: Differential gene expression profile, consolidated gene set enrichment, and network analyses. A, Differences in gene expression are shown in volcano plot format, where the X -axis denotes the log 2 -transformed fold-change and the Y -axis denotes the –log 10 -transformed P value. The log 2 fold-change was calculated as Cas9-p15 control cell fold-change/MELK KO MDA-MB-231 cell fold-change, resulting in positive values for genes overexpressed in the WT condition and negative values for genes overexpressed in the KO condition. The nominal P value threshold at 5% is indicated by a dashed blue horizontal line. The 10 most differentially expressed genes by P value are labeled using their gene symbol. B, Differences in the hallmark enrichment pattern are shown in a modified volcano plot format, where the X -axis denotes the normalized enrichment score and the Y -axis denotes the –log 10 -transformed P value. The normalized enrichment score was calculated as Cas9-p15 control cell score/MELK KO MDA-MB-231 cell score, resulting in positive values for hallmarks enriched in the WT condition and negative values for hallmarks enriched in the KO condition, as indicated at the top of the plot. The nominal P value threshold at 5% is indicated by a dashed blue horizontal line. Significant hallmarks are labeled using their gene set name. C, Pathway enrichment profile of the MELK-centered PPI network. In the left part of the plot, significantly enriched pathways, listed along the Y -axis, are represented in dot plot format; the X -axis represents the –log 10 -transformed P value; and the size of the dots reflects the number of pathway genes contained in the network. To the right of the dot plot, overlaps between enriched pathways and three gene sets defined after analyzing the MELK-centered protein–protein interaction network are indicated in heat map format. The gene sets of interest are indicated in the X -axis underneath the heat map, where dark blue denotes the presence of pathway genes and light blue denotes the absence of pathway genes in these gene sets.

Article Snippet: Cells (1 × 10 6 ) were transfected with 8 μg of the HA-DDK-pcDNA3 empty vector (OriGene) or the HA-DDK-pcDNA3 vector carrying wild-type (WT) MELK (OriGene) or kinase-dead (D150A; KD) MELK using the Neon Transfection System.

Techniques: Gene Expression, Transformation Assay, Control, Labeling, Modification