Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: TaqMan ® probes used for PCR.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques:

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: Fold change of YAP and its regulators in A431 cell line. Data presented as fold change with geometric standard deviations. Data presented as fold change (geometric mean) with geometric standard deviations.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques:

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: Detailed mRNA expression of studied Hippo pathway regulators in primary epidermal keratinocytes under the influence of UVB. Data presented as fold change (geometric mean) with geometric standard deviations.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques: Expressing

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: Detailed mRNA expression of studied Hippo pathway regulators in primary epidermal keratinocytes under the influence of narrowband UVB. Data presented as fold change (geometric mean) with geometric standard deviations.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques: Expressing

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: mRNA expression of studied Hippo pathway regulators: LATS1 ( A ), LATS2 ( B ), JNK1 ( C ), JNK2 ( D ), MAP4K4 ( E ), ABL1 ( F ) in primary epidermal keratinocytes (3 repetitions) with preserved (dark grey) and silenced (tilted) expression of YAP under the influence of increasing doses of UVB. Data presented as fold change (columns) with geometric standard deviations (bars). Asterisks above columns indicate level of statistical significance (ns—not significant, * p < 0.05, ** p < 0.001) in comparison to controls.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques: Expressing, Comparison

Journal: Biomedicines

Article Title: Ultraviolet B Exposure Does Not Influence the Expression of YAP mRNA in Human Epidermal Keratinocytes—Preliminary Study

doi: 10.3390/biomedicines13030596

Figure Lengend Snippet: mRNA expression of studied Hippo pathway regulators: LATS1 ( A ), LATS2 ( B ), JNK1 ( C ), JNK2 ( D ), MAP4K4 ( E ), ABL1 ( F ) in primary epidermal keratinocytes (2 repetitions) with preserved (dark grey) and silenced (tilted) expression of YAP under the influence of increasing doses of narrowband UVB. Data presented as fold change (columns) with geometric standard deviations (bars). Asterisks above columns indicate level of statistical significance (ns—not significant, * p < 0.05, ** p < 0.001) in comparison to controls.

Article Snippet: MAP4K4 , Hs01101394_m1.

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Phosphatidylserine-Based Liposomes Encapsulating DMX-5804 Protect Against Doxorubicin-Induced Cardiotoxicity

doi: 10.64898/2026.02.12.705423

Figure Lengend Snippet: Comparative analysis of MAP4K4 inhibitors reveals differential effects on H9c2 cell viability in the presence of doxorubicin. (A) Effect of MAP4K4 Inhibitors on the viability of H9c2 Cells. (B) Schematic illustrating viability studies conducted on H9c2 cells treated with doxorubicin and MAP4K4 inhibitors. (C) Viability of H9c2 Cells Treated with Doxorubicin and DMX-5804. (D) Viability of H9c2 cells treated with doxorubicin and GNE-495, (E) Viability of H9c2 cells treated with doxorubicin and MAP4K4-IN3, (F) Viability of H9c2 cells treated with doxorubicin and PF-06260933. Statistical analysis was performed using two-way ANOVA to assess the effects of treatment and concentration, followed by Sidak’s multiple-comparisons post hoc test to compare groups at matched concentrations. Data are presented as mean ± stdev, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used: MAP4K4 (rabbit IgG, Proteintech catalog no. 55247-1-AP, 1:1000 dilution), phospho-MAP4K4 (Ser629) (rabbit IgG, Bioss, catalog no. BS-5491R, 1:2000 dilution), cJUN (rabbit IgG, catalog no. 1:1000 dilution), phospho-cJUN (Ser73) (rabbit IgG, Cell Signaling Technology catalog no. 3270, 1:1000), and beta-actin, HRP (rabbit IgG, Invitrogen, catalog no. PA1-183-HRP, 1:1000) dilution.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Phosphatidylserine-Based Liposomes Encapsulating DMX-5804 Protect Against Doxorubicin-Induced Cardiotoxicity

doi: 10.64898/2026.02.12.705423

Figure Lengend Snippet: Effects of DMX-5804 on doxorubicin-induced gene expression, drug uptake, and protein signaling in H9c2 cardiomyoblasts. (A) qPCR analysis of H9c2 cells treated with doxorubicin, DMX-5804, or both for 4 hours. (B) qPCR analysis of H9c2 cells treated with doxorubicin, DMX-5804, or both for 24 hours. RT-qPCR data are shown as mean ± SD from n = 3 independent biological replicates, each measured in duplicate technical replicates. (C) Uptake of 10 μM doxorubicin into H9c2 cells co-treated with 10 μM MAP4K4 inhibitors (DMX-5804, GNE-495, MAP4K4-IN3, and PF-06260933) after 24 hours. Data represent mean ± standard deviation from six technical replicates per condition from a single experiment; statistical comparisons were performed using one-way ANOVA. (D) Uptake of 10 μM doxorubicin into H9c2 cells co treated with increasing concentrations of DMX-5804 (5 μM, 10 μM, and 20 μM) after 24 hours. Data represent mean ± standard deviation from six technical replicates per condition from a single experiment; statistical comparisons were performed using one-way ANOVA followed by Dunnett’s multiple comparison’s post-test. (E) Western blot analysis of H9c2 cells treated with doxorubicin, DMX-5804, or both for 24 hours and 4 hours. Membranes were probed for β-actin as a loading control to verify equal protein loading across lanes. Blots shown are representative of at least three independent experiments.

Article Snippet: The following primary antibodies were used: MAP4K4 (rabbit IgG, Proteintech catalog no. 55247-1-AP, 1:1000 dilution), phospho-MAP4K4 (Ser629) (rabbit IgG, Bioss, catalog no. BS-5491R, 1:2000 dilution), cJUN (rabbit IgG, catalog no. 1:1000 dilution), phospho-cJUN (Ser73) (rabbit IgG, Cell Signaling Technology catalog no. 3270, 1:1000), and beta-actin, HRP (rabbit IgG, Invitrogen, catalog no. PA1-183-HRP, 1:1000) dilution.

Techniques: Gene Expression, Quantitative RT-PCR, Standard Deviation, Western Blot, Control

Journal: bioRxiv

Article Title: Phosphatidylserine-Based Liposomes Encapsulating DMX-5804 Protect Against Doxorubicin-Induced Cardiotoxicity

doi: 10.64898/2026.02.12.705423

Figure Lengend Snippet: Effects of MAP4K4 inhibition on doxorubicin uptake, cell viability, and protein signaling in breast cancer cells. (A) Effect of MAP4K4 inhibitors on the cell viability of MDA-MB-231 cells. (B) Uptake of doxorubicin into MDA-MB-231 cells co-treated with MAP4K4 inhibitors after 24 hours. (C) Viability of MDA-MB-231 cells treated with doxorubicin and DMX-5804. (D) Viability of 4T1 cells treated with Doxorubicin and DMX-5804. (E) Western blot analysis of MDA-MB-231 cells treated with doxorubicin, DMX-5804, or both for 24 hours. Data are presented as mean ± stdev, n=4. Statistical analysis was performed using two-way ANOVA to assess the effects of treatment and concentration, followed by Sidak’s multiple-comparisons post hoc test to compare groups at matched concentrations. Data are presented as mean ± stdev, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: The following primary antibodies were used: MAP4K4 (rabbit IgG, Proteintech catalog no. 55247-1-AP, 1:1000 dilution), phospho-MAP4K4 (Ser629) (rabbit IgG, Bioss, catalog no. BS-5491R, 1:2000 dilution), cJUN (rabbit IgG, catalog no. 1:1000 dilution), phospho-cJUN (Ser73) (rabbit IgG, Cell Signaling Technology catalog no. 3270, 1:1000), and beta-actin, HRP (rabbit IgG, Invitrogen, catalog no. PA1-183-HRP, 1:1000) dilution.

Techniques: Inhibition, Western Blot, Concentration Assay

Journal: Cell death & disease

Article Title: HGK-sestrin 2 signaling-mediated autophagy contributes to antitumor efficacy of Tanshinone IIA in human osteosarcoma cells.

doi: 10.1038/s41419-018-1016-9

Figure Lengend Snippet: Fig. 6 HGK acted an upstream regulator that mediated initiation of the JNK MAP kinase and SESN2-dependent autophagy following TIIA treatment. a Western-assisted analysis of HGK after TIIA treatment as indicated for 24 h in 143B and MG63 cells. b 143B cells were pretreated with GNE-495 (8 nM, 1 h) followed by TIIA treatment as indicated for 24 h. Total lysates were immunoblotted for LC3B, HGK, and p-SAPK/JNK expression. c, d shRNA HGK was stably transfected into 143B (c) and MG63 cells (d). Following treatment with TIIA (20 μM) for indicated time intervals, total lysates were immunoblotted for LC3B, HGK, SESN2, p-SAPK/JNK, JNK1, p-c-Jun, and total c-Jun expression. β-actin served as loading control. e, f Representative images of colonies of 143B-HGKKD (shHGK) and 143B-mock (nonsense) cells in a soft agar colony formation assay in the absence or presence of various concentrations of TIIA were captured using a microscope. Scale bar: 500 μm (e). Results were expressed as average number of colonies counted (in six microfields) (f). g, h 143B cells were transiently transfected with the AP-1 luciferase reporter construct (g) or SESN2 promoter luciferase reporter construct (h). After 24 h, the cells were treated with various concentrations of TIIA for another 12 h and the relative luciferase activity was measured and presented as relative AP-1 activity or relative SESN2 promoter activity. The results were expressed as the means ± SD from three independent experiments (n ≥3, *P < 0.05 compared with untreated control)

Article Snippet: For knockdown of SESN2, BECN1, and HGK, pGFP-VRS plasmid encoding shRNA against human SESN2 (TG301755), BECN1 (TG314484), and HGK (TG320615) were purchased from OriGene (Rockville, MD).

Techniques: Western Blot, Expressing, shRNA, Stable Transfection, Transfection, Control, Soft Agar Assay, Microscopy, Luciferase, Construct, Activity Assay

Journal: Nature cell biology

Article Title: Heat stress activates YAP/TAZ to induce the heat shock transcriptome

doi: 10.1038/s41556-020-00602-9

Figure Lengend Snippet: (a) HEK293A cells were cultured under high density (upper panel) or in the absence of serum (lower panel), and were subjected to heat shock for the indicated times. YAP phosphorylation was detected by the phos-tag gel. (b) Heat shock does not affect MST1 phosphorylation. HEK293A cells were transiently transfected with GST-MST1. 24 h after transfection, cells were subjected to heat shock for the indicated times. Glutathione Sepharose 4B beads (GE Healthcare) were used to purify GTS-MST1. Phosphorylation of the purified GST-MST1 was analyzed by Western blot with pMST1 (Thr183) antibody. (c) YAP dephosphorylation time course in MST1-rescued or MAP4K4-rescued MM8KO cells upon heat shock. Plasmids for HA-YAP and FLAG-MST1 or FLAG-MAP4K4 were transiently co-transfected into HEK293A MM8 KO cells. 24 h after transfection, cells were subcultured to new plate and reached a medium confluence the next day, treated with serum starvation for 2 h, then subjected to heat shock for indicated durations. YAP phosphorylation was detected by the phos-tag gel. (d) Heat shock does not affect the LATS1-MOB1 interaction. HEK293A cells were transiently co-transfected with FLAG-LATS1 and 3×HA-MOB1. 24 h after transfection, cells were subjected to heat shock for the indicated times. FLAG antibodies were used for immunoprecipitation and the co-precipitated proteins were detected by Western blot. The uppermost panel were normalized against FLAG-LATS1 protein levels. Immunoblotting in panels a-d has been performed two times with similar results. Source data are available online.

Article Snippet: FLAG (M2) (F1804, 1:2,000), FLAG-HRP (M2) (A8592, 1:5,000) and GST (2H3-D10) (SAB4200237, 1:4,000) were purchased from Sigma, MAP4K4 (A301-502A, 1:2,000) and MAP4K7 (A310-985A, 1:1,000) were purchased from Bethyl Laboratories, HSP90α (610418, 1:2,000) was purchased from BD Biosciences, HSP70 (10995-1-AP, 1:4,000), ITCH (20920-1-AP, 1:2,000), SIAH2 (12651-1-AP, 1:2,000) and AKT (10176-2-AP, 1:4,000) was purchased from Proteintech, SRC (JF0947) (ET1702-03, 1:5,000) was purchased from HuaAn Biotechnology Co., Ltd and HSP25 (ADI-SPA-801-D, 1:4,000) was purchased from Enzo Life Sciences.

Techniques: Cell Culture, Phospho-proteomics, Transfection, Purification, Western Blot, De-Phosphorylation Assay, Immunoprecipitation

Journal: Nature cell biology

Article Title: Heat stress activates YAP/TAZ to induce the heat shock transcriptome

doi: 10.1038/s41556-020-00602-9

Figure Lengend Snippet: (a) Deletion of MAP4Ks, but not MST, slightly delays heat shock-induced YAP dephosphorylation. HEK393A WT, MST1/2 DKO, and MAP4K4/6/7 TKO cells under medial confluence were pretreated with 2-DG (left panels) or sorbitol (right panels) and then subjected to heat shock. The phosphorylation of YAP and LATS1 were analyzed by Western blot. (b) MAP4K4/6/7 knockout delays YAP nuclear localization. Cells were stained for YAP/TAZ for immunofluorescent microscopy. Representative pictures from three independent samples are shown. Scale bars,10 μm. (c) Quantification of YAP/TAZ nuclear and cytosolic localization. Data are mean ± s.d.; n = 3 biologically independent samples. Two-way ANOVA test. (d) MST1/2 knockout delays recovery after heat shock. HEK293A WT, MST1/2 DKO, and MAP4K4/6/7 TKO cells were pretreated with 2-DG, then subjected to heat shock for 1 h followed by recovery at 37°C for the indicated durations. (e) Heat shock does not affect MST1 and MAP4K4 kinase activity. Endogenous MST1 (left panel) or MAP4K4 (right panel) immunoprecipitated from heat shocked HEK293A cells was assayed using GST-LATS2 as a substrate. LATS2 phosphorylation was determined with pLATS (Thr1079) antibody. (f) Heat shock increases LATS and MAP4K4 interaction. HEK293A cells were co-transfected with FALG-MAP4K4 and HA-LATS1. 24 h after transfection, cells were subjected to heat shock. HA (left panel) and FLAG (right panel) antibodies were used for immunoprecipitation and the co-precipitated proteins were detected by Western blot. (g) Heat shock increases LATS and MST1 interaction. Experiments were similar to panel f except cells were co-transfected with FLAG-MST1 and HA-LATS1 (left panel) or 3×HA-LATS2 (right panel). (h) YAP re-phosphorylation time course in MST1-rescued or MAP4K4-rescued MM8KO cells after shifting back to 37°C. Plasmids for HA-YAP and FLAG-MST1 or FLAG-MAP4K4 were co-transfected into HEK293A MM8KO cells. One day after transfection, cells were subcultured to new plates and reached medial confluence the next day, serum starved for 2 h, then subjected to heat shock at 43°C for 1 h followed by recovery at 37°C for the indicated durations. Immunoblotting in panels a and d-h has been performed two times with similar results. Source data are available online.

Article Snippet: FLAG (M2) (F1804, 1:2,000), FLAG-HRP (M2) (A8592, 1:5,000) and GST (2H3-D10) (SAB4200237, 1:4,000) were purchased from Sigma, MAP4K4 (A301-502A, 1:2,000) and MAP4K7 (A310-985A, 1:1,000) were purchased from Bethyl Laboratories, HSP90α (610418, 1:2,000) was purchased from BD Biosciences, HSP70 (10995-1-AP, 1:4,000), ITCH (20920-1-AP, 1:2,000), SIAH2 (12651-1-AP, 1:2,000) and AKT (10176-2-AP, 1:4,000) was purchased from Proteintech, SRC (JF0947) (ET1702-03, 1:5,000) was purchased from HuaAn Biotechnology Co., Ltd and HSP25 (ADI-SPA-801-D, 1:4,000) was purchased from Enzo Life Sciences.

Techniques: De-Phosphorylation Assay, Phospho-proteomics, Western Blot, Knock-Out, Staining, Microscopy, Activity Assay, Immunoprecipitation, Transfection

Journal: Nature cell biology

Article Title: Heat stress activates YAP/TAZ to induce the heat shock transcriptome

doi: 10.1038/s41556-020-00602-9

Figure Lengend Snippet: (a) Heat shock-induced YAP dephosphorylation is not altered in HSF-1 KO cells. HEK293A WT and HSF-1 KO cells (generated by CRISPR) were subjected to heat shock and cell lysates were analyzed by Western blot. Two independent HSF-1 KO clones are shown. (b) HSP70-1/2 deletion moderately delays YAP dephosphorylation induced by heat shock. HEK293A WT and HSP70-1/2 DKO cells were subjected to heat shock and phos-tag gel was used to detect YAP phosphorylation. Two independent HSP70 KO clones are shown. (c) Deletion of HSP90α/β compromises dephosphorylation of LATS1 and YAP by heat shock. HEK293A WT and HSP90α/β DKO cells were subjected to heat shock and cell lysates were analysed by Western blot. Two independent HSP90α/β DKO clones are shown. LE denotes long exposure of the Western blot. (d) Knockdown of HSP90α/β compromises the heat shock-induced dephosphorylation of YAP and LATS1. HEK293A cells were transfected with control siRNA or siRNAs for HSP90α and HSP90β. Two independent siRNAs were used. (e) The knockdown efficiency of HSP90α/β was confirmed by quantitative Real-Time PCR. Data are presented as mean ± s.d.; n = 3 biologically independent samples. Two-way ANOVA test. (f) HSP90α/β knockdown delays the heat shock-induced LATS inactivation. Endogenous LATS1 was immunoprecipitated from heat shocked-high density HEK293A cells with HSP90α/β knockdown or overexpression. In vitro kinase assays were performed using recombinant GST-YAP as the substrate. Phosphorylation of GST-YAP was determined by immunoblotting with the pYAP (Ser127) antibody. (g) Heat shock increases the interaction between HSP90α and LATS1 or MAP4K4. The plasmids for FLAG-MAP4K4, HA-LATS1, MYC-HSP90AA1 (left panel) or MYC-HSP90AB1 (right panel) were transiently co-transfected into HEK293A cells. One day after transfection, cells were subjected to heat shock for the indicated times. MYC antibodies were used for immunoprecipition and the associated FLAG-MAP4K4 and HA-LATS1 were detected by Western blot. Immunoblotting in panels a-d, f and g has been performed two times with similar results. Source data are available online.

Article Snippet: FLAG (M2) (F1804, 1:2,000), FLAG-HRP (M2) (A8592, 1:5,000) and GST (2H3-D10) (SAB4200237, 1:4,000) were purchased from Sigma, MAP4K4 (A301-502A, 1:2,000) and MAP4K7 (A310-985A, 1:1,000) were purchased from Bethyl Laboratories, HSP90α (610418, 1:2,000) was purchased from BD Biosciences, HSP70 (10995-1-AP, 1:4,000), ITCH (20920-1-AP, 1:2,000), SIAH2 (12651-1-AP, 1:2,000) and AKT (10176-2-AP, 1:4,000) was purchased from Proteintech, SRC (JF0947) (ET1702-03, 1:5,000) was purchased from HuaAn Biotechnology Co., Ltd and HSP25 (ADI-SPA-801-D, 1:4,000) was purchased from Enzo Life Sciences.

Techniques: De-Phosphorylation Assay, Generated, CRISPR, Western Blot, Clone Assay, Phospho-proteomics, Knockdown, Transfection, Control, Real-time Polymerase Chain Reaction, Immunoprecipitation, Over Expression, In Vitro, Recombinant

Journal: Life Science Alliance

Article Title: MAP4K4 regulates forces at cell–cell and cell–matrix adhesions to promote collective cell migration

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: (A) Representative immunoblotting of MAP4K4 and actin using lysates of A431 control cells (sgNT), or A431 cells KO for MAP4K4 with two independent sgRNA (M4K4_sg1, M4K4_sg2). (B) Mean velocity of A431 clusters control or KO for MAP4K4 , tracked over 5 h of migration. (C) Mean velocity of A431 clusters treated with DMSO or GNE-495 at different doses (0.1, 0.5, or 1.0 μM), over 5 h of treatment. Number of clusters analyzed (sgNT: 34, M4K4_sg1: 48, M4K4_sg2: 35, DMSO: 22, GNE 0.1 μM: 34, GNE 0.5 μM: 26, GNE 1.0 μM: 33), from three independent experiments. (D, E) z-scan projection of representative confocal images of F-actin stained A431 clusters, showing the differences in the actin cytoskeleton organization and in the morphology of clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2) or (E) clusters treated with DMSO or GNE-495 at 1.0 μM for 24 h. Arrows represent the actin arches at protrusion bases and arrowheads indicate retraction fibers. (F) Protrusion area of control/ MAP4K4 KO cells or DMSO/GNE-495–treated cells with indicated doses. At least five clusters per experiment, three protrusions per cluster from three independent experiments were analyzed. (G) Circularity of control/ MAP4K4 KO cell clusters, or clusters treated with DMSO or GNE-495 at indicated doses. At least 25 clusters from three independent experiments were analyzed. (H, I) Mean velocity extension (H) or retraction (I) events at the periphery of the clusters before or after treatment with DMSO or GNE-495 at 1.0 μM, over 5 h of treatment. Number of clusters analyzed (DMSO: 28, GNE 0.1 μM: 26, GNE 0.5 μM: 26, GNE 1.0 μM: 28) from three independent experiments. All the data are presented as mean ± s.d. and tested by Kruskal–Wallis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Western Blot, Control, Migration, Staining

Journal: Life Science Alliance

Article Title: MAP4K4 regulates forces at cell–cell and cell–matrix adhesions to promote collective cell migration

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: (A) Representative confocal images of pERM staining, at the substrate z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (B) Quantification of mean intensity of pERM from clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (C, D) Number and (D) length of retraction fibers of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (E) Representative confocal images of pERM staining at the cell–cell junction z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (F) Quantification of mean intensity of pERM of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (G) Representative immunoblotting of moesin and actin from lysates of A431 cells control (Rosa_sg) or KO for MSN using two independent sgRNA sequences ( MSN _sg1, MSN _sg2). (H) Representative confocal images of control or MSN KO clusters stained for zyxin. (I) Number of zyxin-positive focal adhesions in control or MSN KO clusters. (J) Representative confocal z-scan projection of p120 stained cells, control or KO for MSN , showing cell–cell junction morphology. (K) Tortuosity index of cell–cell junction on control or MSN KO cells. At least three junctions of five different clusters per experiment, from three independent experiments were analyzed. All the data are presented as mean ± s.d. and tested by Mann–Whitney test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Staining, Western Blot, Control, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: MAP4K4 regulates forces at cell–cell and cell–matrix adhesions to promote collective cell migration

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: (A, B, C) z-scan projection of representative confocal images of β-catenin stained A431 clusters, stably expressing (A) eGFP–MAP4K4_WT, (B) eGFP–MAP4K4 kinase dead (MAP4K4 D153N ), or (C) deleted for the CNH domain (MAP4K4 ΔCNH ). Line scan indicates the colocalization between β-catenin and MAP4K4. (D) Immunobloting of MAP4K4 or actin for lysates of A431 cells controls (non-infected or sgNT), KO for MAP4K4 (sg_1 or sg_2) alone or expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. (E) Number of zyxin-positive focal adhesions for A431 clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2) and stably expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. (F) Cell–cell junction tortuosity index for A431 clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2), and stably expressing eGFP–MAP4K4 WT, KD, or ΔCNH, resistant to sg_2. Data on (E, F) are represented as mean ± s.d. and tested by Kruskal–Wallis (ns, nonsignificant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Staining, Stable Transfection, Expressing, Western Blot, Infection, Control

Journal: Molecular Oncology

Article Title: MAP 4K4 is a novel MAPK / ERK pathway regulator required for lung adenocarcinoma maintenance

doi: 10.1002/1878-0261.12055

Figure Lengend Snippet: MAPK / ERK 1/2 is a downstream signaling mediator of MAP4K4 in lung adenocarcinoma cells. (A) The whole‐cell lysates of different lung adenocarcinoma cell lines, including two KRAS ‐mutant cell lines, A549 and H23; one KRAS and EGFR wild‐type cell line, H1793; three EGFR ‐mutant cell lines, H1650, H1975, and H3255; and one lung bronchus cell line, BEAS ‐2B, were used for IB with indicated antibodies. (B) MAP 4K4‐knockdown cell lines (sh‐M 1 and sh‐M 2) or sh RNA control cell lines (sh‐C) were generated with two different lentiviral‐based sh RNA targeting MAP 4K4 or scrambled sh RNA in H23, H1975, and H1650 cell lines. The whole‐cell lysates were used for IB with indicated antibodies. To detect GTP ‐bound RAS , the cell lysates were incubated with RAF ‐1 RBD agarose. The bound proteins were then resolved by SDS / PAGE and blotted with anti‐ RAS antibody. (C) MAP 4K4‐overexpressing cell lines ( HA ‐M) and control cell lines ( HA ‐C) were established by transfecting pc DNA 3.1‐ HA ‐ MAP 4K4 or pc DNA 3.1‐ HA into A549 or H3255 cell lines followed by G418 selection. The whole‐cell lysates were prepared for IB or subjected to RAS activation assay. (D–F) Constitutively active ERK 2 (act ERK 2) or vector was transfected into MAP 4K4‐knockdown cell lines (sh‐M 1) with Polyjet In Vitro DNA Transfection Reagent. Data in column charts were shown as means ± SD ; ** and # denote a statistically significant difference ( P < 0.01) and no statistically significant difference ( P > 0.05), respectively, compared with sh RNA control cell lines (sh‐C). (D) Left panel: representative pictures of soft agar assay. Right panel: quantification of soft agar assay. (E) Left panel: representative pictures of in vitro cell invasion assay. Right panel: quantification of in vitro cell invasion assay. (F) The whole‐cell lysates of different cell lines were used for IB with indicated antibodies. (G) H1975‐sh‐control (sh‐C) and H1975‐sh‐ MAP 4K4 (sh‐M 1 and sh‐M 2) cells were treated with 3 μ m of erlotinib for 6 and 24 h. IB was performed with indicated antibodies.

Article Snippet: The sections were incubated overnight with MAP4K4 antibody (Biorbyt, San Francisco, CA, USA) in a humidified chamber at room temperature.

Techniques: Mutagenesis, Knockdown, Control, Generated, Incubation, SDS Page, Selection, Activation Assay, Plasmid Preparation, Transfection, In Vitro, Soft Agar Assay, Invasion Assay

Journal: Life Science Alliance

Article Title: MAP4K4 regulates forces at cell–cell and cell–matrix adhesions to promote collective cell migration

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: (A) Representative immunoblotting of MAP4K4 and actin using lysates of A431 control cells (sgNT), or A431 cells KO for MAP4K4 with two independent sgRNA (M4K4_sg1, M4K4_sg2). (B) Mean velocity of A431 clusters control or KO for MAP4K4 , tracked over 5 h of migration. (C) Mean velocity of A431 clusters treated with DMSO or GNE-495 at different doses (0.1, 0.5, or 1.0 μM), over 5 h of treatment. Number of clusters analyzed (sgNT: 34, M4K4_sg1: 48, M4K4_sg2: 35, DMSO: 22, GNE 0.1 μM: 34, GNE 0.5 μM: 26, GNE 1.0 μM: 33), from three independent experiments. (D, E) z-scan projection of representative confocal images of F-actin stained A431 clusters, showing the differences in the actin cytoskeleton organization and in the morphology of clusters control (sgNT) or KO for MAP4K4 (M4K4_sg2) or (E) clusters treated with DMSO or GNE-495 at 1.0 μM for 24 h. Arrows represent the actin arches at protrusion bases and arrowheads indicate retraction fibers. (F) Protrusion area of control/ MAP4K4 KO cells or DMSO/GNE-495–treated cells with indicated doses. At least five clusters per experiment, three protrusions per cluster from three independent experiments were analyzed. (G) Circularity of control/ MAP4K4 KO cell clusters, or clusters treated with DMSO or GNE-495 at indicated doses. At least 25 clusters from three independent experiments were analyzed. (H, I) Mean velocity extension (H) or retraction (I) events at the periphery of the clusters before or after treatment with DMSO or GNE-495 at 1.0 μM, over 5 h of treatment. Number of clusters analyzed (DMSO: 28, GNE 0.1 μM: 26, GNE 0.5 μM: 26, GNE 1.0 μM: 28) from three independent experiments. All the data are presented as mean ± s.d. and tested by Kruskal–Wallis (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Western Blot, Control, Migration, Staining

Journal: Life Science Alliance

Article Title: MAP4K4 regulates forces at cell–cell and cell–matrix adhesions to promote collective cell migration

doi: 10.26508/lsa.202302196

Figure Lengend Snippet: (A) Representative confocal images of pERM staining, at the substrate z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (B) Quantification of mean intensity of pERM from clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (C, D) Number and (D) length of retraction fibers of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (E) Representative confocal images of pERM staining at the cell–cell junction z focal plane, from clusters treated with DMSO or GNE-495 at 1.0 μM. (F) Quantification of mean intensity of pERM of clusters treated with DMSO or GNE-495 at 1.0 μM. At least 25 clusters from three independent experiments were analyzed. (G) Representative immunoblotting of moesin and actin from lysates of A431 cells control (Rosa_sg) or KO for MSN using two independent sgRNA sequences ( MSN _sg1, MSN _sg2). (H) Representative confocal images of control or MSN KO clusters stained for zyxin. (I) Number of zyxin-positive focal adhesions in control or MSN KO clusters. (J) Representative confocal z-scan projection of p120 stained cells, control or KO for MSN , showing cell–cell junction morphology. (K) Tortuosity index of cell–cell junction on control or MSN KO cells. At least three junctions of five different clusters per experiment, from three independent experiments were analyzed. All the data are presented as mean ± s.d. and tested by Mann–Whitney test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: MAP4K4 KO cells were generated using the pLenti.Cas9-blast (#52962; Addgene) construct and the following sgRNA constructs: MAP4K4_sg1 (#76263; Addgene) and MAP4K4_sg2 (#76264; Addgene).

Techniques: Staining, Western Blot, Control, MANN-WHITNEY