Journal: PLoS ONE

Article Title: Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

doi: 10.1371/journal.pone.0125387

Figure Lengend Snippet: Cells were fixed, TRAP stained, and visualized by light microscopy. (A) Representative 100x images of female (top) and male (bottom) mouse bone marrow cells defined by CD11b surface expression driven to form osteoclasts (OCs) with M-CSF (25ng/mL) and RANKL (50 ng/mL). Female OCs (black arrow) were magnified 3x and inset (top). TRAP positive cells with three or more nuclei (OCs) from (B) female and (C) male mice were enumerated from three random images at days 3 (top) and 5 (bottom) of RANKL treatment. Osteoclast size from female (B, right) and male (C, right) was measured by pixels per osteoclast using Adobe PhotoShop CS. Data are expressed as means ± SE compared to Mk2 +/+ controls (* P ≤0.05, ** P ≤0.01, *** P ≤0.001).

Article Snippet: Membranes were incubated in Cell Signaling primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and GAPDH (14C10, rabbit mAb) at a 1:1000 dilution in 5% BSA in TBS-T overnight at 4°C.

Techniques: Staining, Light Microscopy, Expressing

Journal: PLoS ONE

Article Title: Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

doi: 10.1371/journal.pone.0125387

Figure Lengend Snippet: (A) Representative flow cytometry of bone marrow from male and female Mk2 +/+ and Mk2 -/- mice. (B) Enumeration of Gr-1 - CD11b lo cells as a percent of the hematopoietic stem cell population (HSC, top) and CD115+ as a percent of Gr-1 - CD11b lo cells (bottom). (C) Enumeration of CD11b surface expression in female (left) and male (right) mouse bone marrow cells. Data are expressed as means ± SE compared to Mk2 +/+ controls (* P ≤0.05).

Article Snippet: Membranes were incubated in Cell Signaling primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and GAPDH (14C10, rabbit mAb) at a 1:1000 dilution in 5% BSA in TBS-T overnight at 4°C.

Techniques: Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

doi: 10.1371/journal.pone.0125387

Figure Lengend Snippet: (A) Representative western blot of pre-osteoclasts from male mice primed with M-CSF (10 ng/mL) for two days and stimulated with RANKL (100 ng/mL) for the indicated minutes (min). Densiometric analysis of (B) p38 (left) and (C) p-p38 (right) as a percent of GAPDH. Data are expressed as means ± SE compared to Mk2 +/+ controls (* P ≤0.05).

Article Snippet: Membranes were incubated in Cell Signaling primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and GAPDH (14C10, rabbit mAb) at a 1:1000 dilution in 5% BSA in TBS-T overnight at 4°C.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

doi: 10.1371/journal.pone.0125387

Figure Lengend Snippet: (A) Representative confocal images of male dOCP lo cells treated with M-CSF (control) or M-CSF and RANKL for 30 minutes. A 3x magnified portion of p-p38 was inset (middle,left). (B) Pearson’s r correlation coefficients for NFATc1 and DAPI colocalization. (C) Pearson’s r correlation coefficients for p-p38 and DAPI colocalization. (D) Pearson’s r correlation coefficients for NFATc1 and p-p38 colocalization. Data are expressed as means ± SE compared to Mk2 +/+ controls (* P ≤0.05).

Article Snippet: Membranes were incubated in Cell Signaling primary antibodies: p-MK2 (Thr334, 27B7, rabbit mAb), p-p38 (Thr180/Tyr182, rabbit polyclonal), p38 (rabbit polyclonal), and GAPDH (14C10, rabbit mAb) at a 1:1000 dilution in 5% BSA in TBS-T overnight at 4°C.

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Post-Transcriptional Inflammatory Response to Intracellular Bacterial c-di-AMP

doi: 10.3389/fimmu.2019.03050

Figure Lengend Snippet: P38 inhibition leads to the reduction in the expression of ARE-containing mRNAs and induction of TTP. (A) Upper panel multiplex ELISA for the indicated cytokines. BMDM were treated with 5 μM SB203580 or DMSO during the treatment with c-di-AMP for 4 h. Cell medium was used for multiplex ELISA. Lower panel the BMDM were lysed, total RNA was prepared, and the mRNA levels of the indicated cytokines were determined. Individual values of at least three independent experiments are represented as fold of levels in DMSO control treated cells. Means ± SEMs are shown. Asterisk represents P <0.05 of paired t -tests. (B) Similar experiment like (A) but with RAW264.7 cells. (C) RAW264.7 cells were treated with PS alone or PS in the presence of c-di-AMP for the indicated time points. The cells were lysed in SDS loading buffer, and western blots were performed and developed with an antibody specific for phosphorylated p38. β-Actin was used as loading control. (D) Western blot similar to (C) with one PS control at 6 h and at time points as indicated. The blot was developed with anti-TTP antibody and β-actin. (E) RAW264.7 cells were treated for the indicated time points with PS+c-di-AMP or LPS. The cells were lysed in SDS loading buffer, and western blots were performed and developed with antibodies specific for TTP, phosphorylated p38 (P-p38), p38, phosphorylated MK2 (P-MK2), MK2, and β-actin.

Article Snippet: P38 antibody was purchased from Santa Cruz Biotechnology; anti-phospho-p38 (Thr180/Tyr182) was purchased from Millipore/Merck; β-actin, MK2, and P-MK2 (Thr334) antibodies were purchased from Cell Signaling.

Techniques: Inhibition, Expressing, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Control, Western Blot

Journal: Aging (Albany NY)

Article Title: Expression of Rab1A is upregulated in human lung cancer and associated with tumor size and T stage

doi: 10.18632/aging.101087

Figure Lengend Snippet: ( A ) Expression of Rab1A, P-S6K, S6K, P-AKT, AKT, P-ERK, ERK, P-MK2, MK2, P-c-Jun and c-Jun was examined in a panel of lung cancer cell lines by immunoblot. GAPDH served as a loading control. ( B ) Quantitative analysis of Rab1A protein levels in cell lines. The densitometry results for each Rab1A band were normalized to GAPDH, and the ratios between cancer cell lines and BEAS-2B were calculated and results represented in a histogram. Data represent means ± SD of three independent experiments. ( C-F ) Correlation plots of the expression level of Rab1A and P-S6K1 (T389; C ), P-AKT (S473; D ), P-ERK (T202/Y204; E ), P-MK2 (T334; F ) and P-c-Jun (S63; G ). Shown are arbitrary units.

Article Snippet: Reagents were obtained from the following sources: antibody for Rab1A, Proteintech Group (Rosemont, IL); antibodies for P-S6K1(T398), S6K1, P-AKT(S473), AKT, P-ERK(T202/Y204), ERK, P-MK2(T334), MK2 and Tubulin, Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: Expression of Rab1A is upregulated in human lung cancer and associated with tumor size and T stage

doi: 10.18632/aging.101087

Figure Lengend Snippet: ( A ) The effects of Rab1A knockdown was examined in H838, H1975, H650 and A549 cells by immunoblot. Levels of P-S6K1 (T389), S6K, P-AKT (S473) and AKT are shown. β-tubulin was used as a loading control. ( B ) Rab1A was knocked down in H838, H1975, H650 and A549 cells. The relative growth of these cells was analyzed using an MTT assay. Data represent means ± SD of three independent triplicate experiments. si-NC, negative control siRNA; si-Rab1A, Rab1A-specific siRNA.

Article Snippet: Reagents were obtained from the following sources: antibody for Rab1A, Proteintech Group (Rosemont, IL); antibodies for P-S6K1(T398), S6K1, P-AKT(S473), AKT, P-ERK(T202/Y204), ERK, P-MK2(T334), MK2 and Tubulin, Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Western Blot, Control, MTT Assay, Negative Control

Journal: Materials Today Bio

Article Title: Mesenchymal stem cell-derived small extracellular vesicles-loaded GelMA microspheres enhance diabetic wound healing by promoting M2 macrophage polarization through p38 MAPK inhibition

doi: 10.1016/j.mtbio.2025.102423

Figure Lengend Snippet: MSC-sEVs promoted M2 polarization via inhibition of the p38 MAPK pathway. (A) Heatmap depicting cytokine levels in culture supernatants. (B) Heatmap illustrating the relative inflammatory cytokines levels and (C) Immune response-related genes in different treatment groups. (D) GSEA plots indicating enrichment of key pathway. (E) Venn diagram illustrating the overlap of genes across the pathways, with Mapk14 , Nfkb1 , and Fos identified as common regulators of inflammation and immune responses. (F) Bar graph showing the expression levels of key genes (n = 3). (G) Western blot analysis of p-MAPKAPK2, p-p38, and total p38 protein expression. (H–J) Quantification of (H) p38, (I) p-p38, and (J) p-MAPKAPK2 levels, demonstrating that MSC-sEVs treatment significantly reduces p-p38 and p-MAPKAPK2 levels. Data are presented as mean ± SD (n = 3). ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: After blocking with 5 % BSA for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies: CD9 (20597-1-AP, 1:3000, Proteintech, China), CD81 (27855-1-AP, 1:2000, Proteintech, China), Alix (12422-1-AP, 1:10000 Proteintech, China), calnexin (10427-2-AP, Proteintech, China), iNOS (22226-1-AP, 1:1000, Proteintech, China), CD206 (1:1000, 18704-1-AP, Proteintech, China), IL-6 (A0286,1:1000, ABclonal, China), IL-10 (A12255, 1:1000, ABclonal, China), p38 (A5049,1:1000, ABclonal, China), Phospho-p38 MAPK ( PAB43506 -P, 1:1000, Bioswamp, China), Phospho-MAPKAPK2 (AP0588, 1:1000, ABclonal, China) and GAPDH (10494-1-AP, 1:5000, Proteintech, China).

Techniques: Inhibition, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Expression of Rab1A is upregulated in human lung cancer and associated with tumor size and T stage

doi: 10.18632/aging.101087

Figure Lengend Snippet: ( A ) The effects of Rab1A knockdown was examined in H838, H1975, H650 and A549 cells by immunoblot. Levels of P-S6K1 (T389), S6K, P-AKT (S473) and AKT are shown. β-tubulin was used as a loading control. ( B ) Rab1A was knocked down in H838, H1975, H650 and A549 cells. The relative growth of these cells was analyzed using an MTT assay. Data represent means ± SD of three independent triplicate experiments. si-NC, negative control siRNA; si-Rab1A, Rab1A-specific siRNA.

Article Snippet: Reagents were obtained from the following sources: antibody for Rab1A, Proteintech Group (Rosemont, IL); antibodies for P-S6K1(T398), S6K1, P-AKT(S473), AKT, P-ERK(T202/Y204), ERK, P-MK2(T334), MK2 and Tubulin, Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Western Blot, Control, MTT Assay, Negative Control

Journal: Science translational medicine

Article Title: The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress

doi: 10.1126/scitranslmed.abb5445

Figure Lengend Snippet: (A) Heatmap of RPPA results showing significantly upregulated and downregulated markers in Pa01c cells following 16-hour treatment with FIRINOX (5-FU 5μM, SN-38 5μM, oxaliplatin 5μM). Only markers showing significant changes (P<0.05) were illustrated in heatmap. (B) Log2 fold change of the most significantly (> or < 1.5-fold, P<0.001) upregulated and downregulated markers in Pa01c cells following overnight treatment with FIRINOX. (C) Western blots confirming upregulated p-Hsp27 (S82), p-Jun (S73) and p-ERK1/2 (T202/Y204) in Pa01c and MIA Paca-2 cells treated for 16 hours (or overnight) with FIRINOX. (D) Western blot analysis showing phosphorylation of Ser15, Ser78 and Ser82 in Pa01c and MIA PaCa-2 cells following 16-hour treatment with FIRINOX. (E) Western blots showing degree of PARP cleavage (cleaved/full length) in Pa01 cells treated with FIRINOX alone or concomitantly with JNK inhibitor (SP600125 1μM) or MEK inhibitor (trametinib 1μM) for 16 hours, and in Pa01c cells stably expressing two different Hsp27 shRNAs treated without or with FIRINOX. (F) Western blots showing degree of PARP cleavage in scramble control or Hsp27-silenced MIA PaCa-2 cells treated for 16 hours with DMSO vehicle or the individual components of FIRINOX (5-FU 5μM, SN-38 5μM, oxaliplatin 5μM). (G) Flow cytometric quantification of MIA Paca-2 cells as treated (F), with analysis limited to SN-38 treated cells. All quantitative data were presented as mean ± SEM, two-way ANOVA with Tukey’s multiple comparison test; **P<0.01, ****P<0.0001.

Article Snippet: Staining specificity of MK2, p-MK2 (T334) and p-Hsp27 (S82) antibodies were confirmed using isotype control antibody (Cell Signaling Technology # 3990, rabbit) in formalin-fixed paraffin embedded Pa01c cell pellets expressing a scramble sequence or shRNAs against MK2 or Hsp27 with assistance from GI pathologist MBR ( fig. S5A ).

Techniques: Western Blot, Phospho-proteomics, Stable Transfection, Expressing, Control, Comparison

Journal: Science translational medicine

Article Title: The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress

doi: 10.1126/scitranslmed.abb5445

Figure Lengend Snippet: (A, B) Tumor growth curves of (A) Pa01c and (B) MIA PaCa-2 xenografts grown subcutaneously into nude mice and treated with vehicle, ATI-450, FIRINOX, or ATI-450 and FIRINOX when tumors were greater than >50mm3 (N=6-8 tumors per group). (C) Kaplan-Meier survival analysis of C57BL/6J KPC mice treated as indicated till human endpoints (N=12-13 mice per group). (D) Final weights of the pancreata of KPC mice treated in (C) when euthanized at humane endpoints (N=11 per group). (E) Representative H&E, p-MK2 (T334), p-Hsp27 (S82) IHC, Sirius Red and co-IF of pan-cytokeratin plus cleaved caspase-7 (CC7) staining from end-staged pancreata of KPC mice treated as indicated. Quantification was done as below: p-MK2 (T334) by H-score (= 3 x % of strongly staining area + 2 x % of strongly staining area + 1X % of weakly staining area) done manually by GI pathologist MBR in blinded fashion. (F) Representative pictures and quantification of pan-cytokeratin staining of end-staged pancreata of KPC mice treated as indicated. Three different combination-treated pancreata were illustrated to demonstrate variation in histologic changes following FIRINOX and ATI-450 treatment, which all resulted in decreased pan-cytokeratin area. Quantification of p-Hsp27(S82), Sirius red, cleaved caspase-7 and pan-cytokeratin positive areas were quantified using ImageJ software. 6-8 tumors per group, 5-6 random 200X fields per tumor from (C) were analyzed and scored. All quantitative data were presented as mean ± SEM, two-way ANOVA with Welch test; ns=non-significant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (G) Left: Representative p-MK2(T334) IHC images for a PDAC TMA defining strong (3+), moderate (2+) and weak (1+) staining, which was used for collation of H-scores using HALO software. Right: Kaplan-Meier analysis comparing overall survival of PDAC patients undergoing pancreatectomy followed by adjuvant chemotherapy, as stratified into high vs. medium and low p-MK2 H-scores.

Article Snippet: Staining specificity of MK2, p-MK2 (T334) and p-Hsp27 (S82) antibodies were confirmed using isotype control antibody (Cell Signaling Technology # 3990, rabbit) in formalin-fixed paraffin embedded Pa01c cell pellets expressing a scramble sequence or shRNAs against MK2 or Hsp27 with assistance from GI pathologist MBR ( fig. S5A ).

Techniques: Staining, Software, Adjuvant

Journal: Science translational medicine

Article Title: The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress

doi: 10.1126/scitranslmed.abb5445

Figure Lengend Snippet: (A) Western blots showing upregulation of p-Hsp27 and p-MK2 in a panel of PDAC cell lines treated overnight with DMSO, 5-FU 10μM, SN-38 10μM, oxaliplatin 4μM or gemcitabine 10μM. (B) Western blots showing MK2-dependent Hsp27 phosphorylation in scramble control or MK2-silenced Pa01c cells treated overnight with DMSO or SN-38 10μM. (C) Western blots showing PARP cleavage and p-Hsp27 (S82) in scramble control or MK2-silenced MIA PaCa-2 cells treated as in (A). (D) Western blots showing p-Hsp27 (S82) and PARP cleavage of Pa01c and MIA PaCa-2 and treated as indicated overnight. (E) Western blots showing p-Hsp27 at different residues and PARP cleavage of Pa01c and MIA PaCa-2 cells treated with as indicated overnight. (F) Flow cytometric analysis showing Annexin V staining of MIA PaCa-2 cells treated as indicated for 24 hours. (G) Flow cytometric analysis of showing Annexin V staining of different Pa01c variants treated with vehicle or SN-38 4μM for 24 hours. Data were presented as mean ± SEM, P values from two-away ANOVA with Tukey’s multiple comparison test, ns=non-significant, *P<0.05, **P<0.01, ****P<0.0001. (H) Volcano plots of RPPA results following treatment of Pa01c cells with FIRINOX vs. vehicle compared to FIRINOX + ATI-450 vs. vehicle demonstrating selective modulation of p-Hsp27 (S82) by ATI-450.

Article Snippet: Staining specificity of MK2, p-MK2 (T334) and p-Hsp27 (S82) antibodies were confirmed using isotype control antibody (Cell Signaling Technology # 3990, rabbit) in formalin-fixed paraffin embedded Pa01c cell pellets expressing a scramble sequence or shRNAs against MK2 or Hsp27 with assistance from GI pathologist MBR ( fig. S5A ).

Techniques: Western Blot, Phospho-proteomics, Control, Staining, Comparison

Journal: Science translational medicine

Article Title: The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress

doi: 10.1126/scitranslmed.abb5445

Figure Lengend Snippet: (A) Western blots showing p-p38, p-MK2 and p-HSP27 S82 activation in Pa01c and MIA PaCa-2 cells treated overnight with SN-38 alone or concurrently with AS2444697 (IRAK4i) 4μM, HCQ 20μM, KU55933 (ATMi) 20μM, VE821(ATRi) 20μM, or 5Z-7-oxozeaenol (TAKi) 2μM. (B) Western blots of Pa01c and MIA PaCa-2 cells treated with vehicle, SN-38 alone or with TAK1 inhibitor 2uM for 16 hours. (C) Representative immunofluorescence pictures and quantification of Duolink® proximity ligase assay identifying interaction of endogenous p-MK2 and Flag-tagged TAK1 in Pa01c cells treated with DMSO or SN-38 10μM overnight (scale bars=50μM). (D) qPCR showing changes in mRNA expression of TNFA, TNFB, TGFB1, IL1A and IL1B in Pa01c and MIA PaCa-2 cells treated with DMSO or SN-38 10μM for 16 hours. (E) TNFα ELISA of Pa01c cells treated with control or the indicated concentrations of SN-38 overnight. (F) qPCR showing changes in TNFA mRNA expression in Pa01c cells treated as indicated for 16 hours (TAK1i is 5Z-7-oxozeaenol; IKKβi is IMD0354). (G) Western blots showing changes in p-Hsp27(S82) and PARP cleavage in MIA PaCa-2 and Pa01c cells treated overnight as indicated. (H) Western blots showing changes in p-Hsp27(S82) and PARP cleavage in Pa01c and MIA PaCa-2 cells treated overnight as indicated. (I) Flow cytometric analysis showing Annexin V staining of MIA PaCa-2 cells treated as indicated for 36 hours. (J) Immunoblots showing p-Hsp27 and PARP cleavage of Pa01c and MIA PaCa-2 cells stably expressing a scrambled sequence or shRNA against TNFR1 treated with control or SN-38 10μM overnight. All quantitative data were presented as mean±SEM, two-way ANOVA with Tukey’s or Dunnett’s multiple comparison test; *P<0.05, **P<0.01, ****P<0.0001.

Article Snippet: Staining specificity of MK2, p-MK2 (T334) and p-Hsp27 (S82) antibodies were confirmed using isotype control antibody (Cell Signaling Technology # 3990, rabbit) in formalin-fixed paraffin embedded Pa01c cell pellets expressing a scramble sequence or shRNAs against MK2 or Hsp27 with assistance from GI pathologist MBR ( fig. S5A ).

Techniques: Western Blot, Activation Assay, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Control, Staining, Stable Transfection, Sequencing, shRNA, Comparison

Journal: Science translational medicine

Article Title: The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress

doi: 10.1126/scitranslmed.abb5445

Figure Lengend Snippet: (A) Quantification of RPPA results showing fold change of p-AMPKα(T172) and p-ULK1(S757) abundance in Pa01c cells following FIRINOX treatment. (B) Western blots of proteins mediating autophagy in Pa01c and MIA PaCa-2 cells treated overnight with DMSO, gemcitabine 5μM, SN-38 5μM, 5-FU 5μM, oxaliplatin 2μM or FIRINOX (SN-38, oxaliplatin and 5-FU combination). (C) Quantitative flow cytometric-based fluorescence analysis of Pa01c and MIA PaCa-2 cells stably expressing EGFP-LC3 construct treated with DMSO or SN-38 10μM for 24 hours. (D) Western blots showing PARP cleavage (cleaved/full length) and cleaved caspase-7 - in Pa01c and MIA PaCa-2 cells treated as indicated overnight. (E) Flow cytometric quantification of Annexin V staining of MIA PaCa-2 control cells or BECN1 knockout cells treated with control (DMSO) or SN-38 10uM for 24 hours. (F) Western blots of Pa01c and MIA PaCa-2 cells showing markers of autophagy following the indicated treatment overnight. (G) Western blots showing LC3-I/II changes in scramble control or Hsp27-silenced MIA Paca-2 cells treated with DMSO or SN-38 10uM for 16 hours. (H) Western blots showing PARP cleavage and cleaved caspase-7 - in control or BECN1 knockout MIA PaCa-2 cells treated overnight as indicated. All quantitative data were presented as mean ± SEM, two-way ANOVA with Tukey’s or Dunnett’s multiple comparison test; ns=non-significant, **P<0.01, ****P<0.0001.

Article Snippet: Staining specificity of MK2, p-MK2 (T334) and p-Hsp27 (S82) antibodies were confirmed using isotype control antibody (Cell Signaling Technology # 3990, rabbit) in formalin-fixed paraffin embedded Pa01c cell pellets expressing a scramble sequence or shRNAs against MK2 or Hsp27 with assistance from GI pathologist MBR ( fig. S5A ).

Techniques: Western Blot, Fluorescence, Stable Transfection, Expressing, Construct, Staining, Control, Knock-Out, Comparison

Journal: Science translational medicine

Article Title: The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress

doi: 10.1126/scitranslmed.abb5445

Figure Lengend Snippet: (A) Western blots showing activation status of p38, MK2 and Hsp27 across a panel of conventional PDAC and patient-derived cell lines. HPNE cells served as untransformed control. (B) Representative immunohistochemistry (IHC) pictures of human PDAC tumors with adjacent normal or PanIN lesions showing p-MK2 (T334) and p-Hsp27 (S82) staining. Staining intensity of p-MK2 was defined as 0 (absent), 1+ (weak), 2+ (moderate) and 3+ (strong) as demonstrated. Quantification of p-MK2 positive (2+ and 3+) cells was summarized from five different human PDAC samples. For each tumor, five random 200X fields were chosen, and 60-90 neoplastic cells/field were scored. (C) Pictures (400X) of tumoroids of Pa01c and AsPc1 cells stably overexpressing vector or MK2 cDNA grown in Matrigel. (D) Tumor growth curves of Pa01c, MIA PaCa-2 and AsPc-1 cells stably expressing scrambled shRNA, MK2 shRNA or MK2 cDNA (MK2 OE) grown subcutaneously into nude mice (N=4-8 mice per group). (E) Representative H&E, p-p38 and p-MK2 IHC pictures of normal, PanIN and PDAC from KPC mice. (F) Representative H&E images and quantification of serial histopathological changes in the pancreata of KPC mice treated with vehicle or ATI-450 for two weeks. Representative images and quantification of (G) pan-cytokeratin staining, (H) Alcian blue, (I) Trichrome blue and (J) Sirius red staining in the pancreata of KPC mice treated with vehicle or ATI-450 for two weeks. For (F-J), N=7 pancreata per group and in each pancreas random 200X fields were taken and scored using ImageJ software. All quantitative data were presented as mean ± SEM, differences were assessed by two-way ANOVA or unpaired t test with Welch’s correction; ns=non-significant, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Staining specificity of MK2, p-MK2 (T334) and p-Hsp27 (S82) antibodies were confirmed using isotype control antibody (Cell Signaling Technology # 3990, rabbit) in formalin-fixed paraffin embedded Pa01c cell pellets expressing a scramble sequence or shRNAs against MK2 or Hsp27 with assistance from GI pathologist MBR ( fig. S5A ).

Techniques: Western Blot, Activation Assay, Derivative Assay, Control, Immunohistochemistry, Staining, Stable Transfection, Plasmid Preparation, Expressing, shRNA, Software