Journal: Journal of Neuropathology & Experimental Neurology

Article Title: Activation of the Heat Shock Response in Familial Amyloidotic Polyneuropathy

doi: 10.1097/nen.0b013e31816fd648

Figure Lengend Snippet: FIGURE 1. Transthyretin (TTR) deposition induces heat shock transcription factor 1 (HSF 1) activation and increases heat shock proteins (Hsp) expression in situ. (A) Human salivary glands from normal control individuals (n = 3) and from human familial amyloidotic polyneuropathy (FAP) patients (n = 5) were immunostained for HSF 1. Greater expression and translocation to the nucleus (arrow) were observed in FAP patient samples. Scale bar = 50 Km (upper panels). Salivary glands from 6-month-old transgenic mice without TTR deposition (TTRj; n = 4) and with TTR deposition (TTR+; n = 5) were analyzed for HSF 1 expression. Only mice with TTR deposits had HSF 1 upregulation and translocation to the nucleus (arrow). Scale bar = 100 Km (bottom panels). (B) Greater expression of Hsp70 in skin from FAP patients (n = 7) compared with controls (n = 3; upper panels). Greater Hsp70 expression in SG samples from TTR positive transgenic (TTR+; n = 7) compared with TTR negative transgenic (TTRj; n = 7) mice. Scale bar = 50 Km (bottom panels). Graphs demonstrate semiquantitative analysis of Hsp70 expression shown as the average of percentage of occupied area with substrate color T standard deviation. *, p G 0.001. (C) Heat shock protein 27 expression in the peripheral nerve is upregulated in advanced stages of FAP. Normal control (n = 5) and FAP 0 (n = 5) have similarly low Hsp27 expression, whereas FAP 1 (n = 6), FAP 2 (n = 4), and FAP 3 (n = 2) have significantly greater expression. Scale bar = 30 Km. The graph demonstrates semiquantitative analysis of Hsp27 expression shown as the percentage of occupied area with substrate color T standard deviation. *, p G 0.02, †, p G 0.01. (D) Increased Hsp in FAP tissues is associated with extracellular TTR deposition. Human FAP skin samples show extracellular TTR deposition, whereas Hsp70 is detected inside gland epithelial cells. Normal human skin does not show TTR, and Hsp70 is undetectable. Arrows with small closed arrowheads point to TTR, and arrows with larger open arrowheads indicate Hsp70 (upper panels). Human FAP peripheral nerve has extracellular TTR deposits (smaller, closed arrowheads; green); Hsp27 expression is in the axons (larger, open arrowheads; red; bottom panels). Scale bar = 20 Km.

Article Snippet: Incubation with hsp70 antibody (Stressgen), diluted 1:200, or with hsp27 (Santa Cruz Biotechnology), diluted 1:100, was performed, followed by incubation with the corresponding secondary Alexa Fluor antibodies (Molecular Probes).

Techniques: Activation Assay, Expressing, In Situ, Control, Translocation Assay, Transgenic Assay, Standard Deviation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Biological Significance of Decreased HSP27 in Human Atherosclerosis

doi: 10.1161/01.atv.0000220108.97208.67

Figure Lengend Snippet: Figure 1. HSP27 proteolysis by plasmin in vitro and ex vivo. A, Recombinant HSP27 was incubated with 125 nmol plasmin for increas- ing periods of time (15 to 60 minutes) with or without inhibitors (VFK and aprotinin). B, Incubation of mammary endartery-released HSP27 with plasmin (125 mmol/L) for 18 hours led to its degradation and aggregation in the conditioned medium, and this effect is pre- vented in the presence of the plasmin inhibitor, aprotinin. C, Incubation of recombinant HSP27 with atherosclerotic plaques, led to HSP27 proteolysis in the conditioned medium. This effect is partially prevented by aprotinin, a serine-protease inhibitor. D, Coincuba- tion of mammary endarteries (M), known to release HSP27, with atherosclerotic plaque samples (P), as a source of proteases, led to HSP27 proteolysis in the conditioned medium. The empty dashed arrow indicates the band corresponding to HSP27 degradation prod- uct and the plain arrow shows the aggregated forms of HSP27.

Article Snippet: Human VSMCs were grown to 50% confluence in 12-well plates and transfected with a mixture composed of 2.5 mol/L CaCl2, 25 nmol small interfering RNA (siRNA) (human HSP27 siRNA sc-29350; Santa Cruz Biotechnology) and calcium phosphate buffer (280 mmol/L NaCl, 1.5 mmol/L Na2HPO4, 12 mmol/L glucose, 10 mmol/L KCl, 50 mmol/L Hepes, pH7) in 10% FCS complete medium.

Techniques: In Vitro, Ex Vivo, Recombinant, Incubation, Protease Inhibitor

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Biological Significance of Decreased HSP27 in Human Atherosclerosis

doi: 10.1161/01.atv.0000220108.97208.67

Figure Lengend Snippet: Figure 3. HSP27 levels modulate plasmin-induced VSMC anoikis. A, Human VSMCs were transfected with calcium phos- phate alone (control of transfection, CT) or with HSP27-siRNA (25 to 50 nM). HSP27 (upper panel) and HSP70 (lower panel) levels were analyzed by Western-blot analysis. Ccontrol cells. B, Immunofluorescence detection of HSP27 in VSMCs trans- fected or not with HSP27-siRNA (25 nM) for 24 hours and cul- tured for additional 48 hours. C, Representative microphoto- graphs of control (C) human VSMCs or treated with plasmin (Pn) for 18 hour in nontransfected cells (upper panel) or in cells transfected with 25 nmol HSP27-siRNA (lower panel).

Article Snippet: Human VSMCs were grown to 50% confluence in 12-well plates and transfected with a mixture composed of 2.5 mol/L CaCl2, 25 nmol small interfering RNA (siRNA) (human HSP27 siRNA sc-29350; Santa Cruz Biotechnology) and calcium phosphate buffer (280 mmol/L NaCl, 1.5 mmol/L Na2HPO4, 12 mmol/L glucose, 10 mmol/L KCl, 50 mmol/L Hepes, pH7) in 10% FCS complete medium.

Techniques: Transfection, Control, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Biological Significance of Decreased HSP27 in Human Atherosclerosis

doi: 10.1161/01.atv.0000220108.97208.67

Figure Lengend Snippet: Figure 4. Effect of HSP27 on apoptosis in vitro. A, Human VSMCs were incubated with plasmin (Pn) for 18 hours in VSMCs transfected or not with HSP27-siRNA (25 nmol). Viability was assessed by the MTT test, which quantifies the remaining adherent cells. Untreated control cells (C) represent 100% of viable adherent cells and the results are expressed as a per- centage of control (mean of 3 separate experiments SD, *P0.05 vs control; †P0.05 vs Pn). B, Quantification of DNA fragmentation in human VSMCs incubated with plasmin (Pn) for 18 hours transfected or not with HSP27-siRNA (25 nmol). Results are expressed in A405 nm 103 (meanSD, *P0.05 vs control; †P0.05 vs Pn). C, Detection of apoptosis in mammary arteries incubated with plasmin (Pn) for 24 hours. Apostain- positive nuclei were absent in control (C) mammary endarteries.

Article Snippet: Human VSMCs were grown to 50% confluence in 12-well plates and transfected with a mixture composed of 2.5 mol/L CaCl2, 25 nmol small interfering RNA (siRNA) (human HSP27 siRNA sc-29350; Santa Cruz Biotechnology) and calcium phosphate buffer (280 mmol/L NaCl, 1.5 mmol/L Na2HPO4, 12 mmol/L glucose, 10 mmol/L KCl, 50 mmol/L Hepes, pH7) in 10% FCS complete medium.

Techniques: In Vitro, Incubation, Transfection, Control

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: Stability results for HSP27: effects of immediate and delayed processing of blood samples with storage at 4℃

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: Clinical Proteomics

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: Stability results for HSP27: effects of repeated freeze-thaw cycles with storage of serum/plasma samples at -80℃

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: Clinical Proteomics

Journal: Annals of Laboratory Medicine

Article Title: In vitro Stability of Heat Shock Protein 27 in Serum and Plasma Under Different Pre-analytical Conditions: Implications for Large-Scale Clinical Studies

doi: 10.3343/alm.2016.36.4.353

Figure Lengend Snippet: In vitro stability of HSP27 under different pre-analytical conditions: (A) relative analyte stability in serum samples and (B) plasma samples stored for 2-6 hr at 4℃ with immediate and delayed sample processing; and (C) the effect of repeated freeze-thaw cycles on HSP27 serum and plasma concentrations. Graphs show relative analyte recoveries at distinct time points (each dot represents the mean analyte concentrations relative to the baseline values of 10 healthy individuals; whiskers indicate standard deviation).

Article Snippet: Circulating HSP27 concentrations were measured by using the Human Total HSP27 DuoSet IC ELISA (Catalog No. DYC1580, R&D Systems, Minneapolis, MN, USA).

Techniques: In Vitro, Clinical Proteomics, Standard Deviation

Journal: Journal of Animal Science

Article Title: Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period

doi: 10.1093/jas/skac129

Figure Lengend Snippet: Primer sequences used to assess transcript abundance in porcine endometrial tissue

Article Snippet: Membranes were then incubated at 4 °C overnight with primary antibodies specific for HSP90AA1 (1:1,000; 8165; Cell Signaling Technology, Danvers, MA), HSP90AB1 (1:1,000; 7411; Cell Signaling Technology, Danvers, MA), HSPA6 (1:1,000; NBP1-85949; Novus Biologicals, Centennial, CO), HSPA1A (1:1,000; NB110-96427; Novus Biologicals, Centennial, CO), HSPB1 (1:1,000; NBP2-25149; Novus Biologicals, Centennial, CO), and HSPD1 (1:1,000; NBP2-32973; Novus Biologicals, Centennial, CO) diluted in 5% milk in TBST.

Techniques: Sequencing

Journal: Journal of Animal Science

Article Title: Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period

doi: 10.1093/jas/skac129

Figure Lengend Snippet: Effect of pregnancy and/or thermal treatment on endometrial transcript abundance of heat shock proteins. Endometrium from cyclic and pregnant gilts maintained under thermal neutral or heat stress conditions were assessed for changes in specific HSP transcript abundance using qPCR. No effect of pregnancy status or thermal treatment on (A) HSP90AA1 and (B) HSP90AB1 was observed. (C) HSPA1A was elevated in the pregnant endometrium irrespective of thermal treatment. (D) HSPA6 was increased in the pregnant endometrium under HS when compared with the other treatment groups. (E) Pregnancy status reduced HSPD1 transcript abundance whereas HS had no effect. Neither pregnancy status nor thermal treatment influenced (F) HSPB1 transcript abundance, but (G) HSPB8 was elevated in the pregnant endometrium under HS conditions. Normalized transcript abundances are reported as fold change. Different lowercase letters indicate statistical significance between treatment groups; P ≤ 0.05.

Article Snippet: Membranes were then incubated at 4 °C overnight with primary antibodies specific for HSP90AA1 (1:1,000; 8165; Cell Signaling Technology, Danvers, MA), HSP90AB1 (1:1,000; 7411; Cell Signaling Technology, Danvers, MA), HSPA6 (1:1,000; NBP1-85949; Novus Biologicals, Centennial, CO), HSPA1A (1:1,000; NB110-96427; Novus Biologicals, Centennial, CO), HSPB1 (1:1,000; NBP2-25149; Novus Biologicals, Centennial, CO), and HSPD1 (1:1,000; NBP2-32973; Novus Biologicals, Centennial, CO) diluted in 5% milk in TBST.

Techniques:

Journal: Journal of Animal Science

Article Title: Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period

doi: 10.1093/jas/skac129

Figure Lengend Snippet: Effect of altrenogest and/or thermal treatment on endometrial transcript abundance of heat shock proteins. Endometrium from pregnant gilts either supplemented with altrenogest or not (postinsemination), and maintained under thermal neutral or heat stress conditions during the peri-implantation period were assessed for changes in specific HSP transcript abundance using qPCR. Neither ALT supplementation nor thermal treatment demonstrated an effect on (A) HSP90AA1 transcript abundance. (B) HSP90AB1 abundance was reduced as a result of ALT supplementation under TN conditions; an effect reversed by HS in spite of ALT supplementation. HS also increased (C) HSPA1A and (D) HSPA6 transcript abundance, in spite of ALT supplementation. No effects of either treatment were observed on (E) HSPD1 , (F) HSPB1 , and (G) HSPB8 transcript abundances. Normalized transcript abundances are reported as fold change. Different lowercase letters indicate statistical significance between treatment groups; P ≤ 0.05.

Article Snippet: Membranes were then incubated at 4 °C overnight with primary antibodies specific for HSP90AA1 (1:1,000; 8165; Cell Signaling Technology, Danvers, MA), HSP90AB1 (1:1,000; 7411; Cell Signaling Technology, Danvers, MA), HSPA6 (1:1,000; NBP1-85949; Novus Biologicals, Centennial, CO), HSPA1A (1:1,000; NB110-96427; Novus Biologicals, Centennial, CO), HSPB1 (1:1,000; NBP2-25149; Novus Biologicals, Centennial, CO), and HSPD1 (1:1,000; NBP2-32973; Novus Biologicals, Centennial, CO) diluted in 5% milk in TBST.

Techniques:

Journal: Journal of Animal Science

Article Title: Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period

doi: 10.1093/jas/skac129

Figure Lengend Snippet: Assessment of heat shock protein abundance in the endometrial samples by Western blotting. Representative figure of immunoblotting of endometrial protein lysates for each treatment group using antibodies directed toward HSP90AA1, HSP90AB1, HSPA1A, HSPA6, HSPD1, and HSPB1. Ponceau S stained blots were imaged to ascertain uniform loading and bands thus obtained were utilized for loading control normalization of specific target proteins. The samples from different treatment groups were uniformly distributed across two gels and pooled samples were used on both gels to allow normalization and unbiased comparisons of band densities across both gels.

Article Snippet: Membranes were then incubated at 4 °C overnight with primary antibodies specific for HSP90AA1 (1:1,000; 8165; Cell Signaling Technology, Danvers, MA), HSP90AB1 (1:1,000; 7411; Cell Signaling Technology, Danvers, MA), HSPA6 (1:1,000; NBP1-85949; Novus Biologicals, Centennial, CO), HSPA1A (1:1,000; NB110-96427; Novus Biologicals, Centennial, CO), HSPB1 (1:1,000; NBP2-25149; Novus Biologicals, Centennial, CO), and HSPD1 (1:1,000; NBP2-32973; Novus Biologicals, Centennial, CO) diluted in 5% milk in TBST.

Techniques: Quantitative Proteomics, Western Blot, Staining, Control

Journal: Journal of Animal Science

Article Title: Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period

doi: 10.1093/jas/skac129

Figure Lengend Snippet: Effect of pregnancy and/or thermal treatment on endometrial protein abundance of heat shock proteins. Endometrium from cyclic and pregnant gilts maintained under thermal neutral or heat stress conditions were assessed for changes in HSP protein abundance using densitometric analysis of immunoblots. Neither pregnancy status nor thermal treatment affected (A) HSP90AA1 or (B) HSP90AB1 abundance. (C) HSPA1A protein abundance was decreased in the pregnant endometrium (in spite of HS), but no effect was observed on (D) HSPA6 protein abundance. Pregnancy status and thermal treatment had no effect on (E) HSPD1 abundance, but HS increased (F) HSPB1 protein abundance in the cyclic gilts. Different lowercase letters indicate statistical significance between treatment groups; P ≤ 0.05.

Article Snippet: Membranes were then incubated at 4 °C overnight with primary antibodies specific for HSP90AA1 (1:1,000; 8165; Cell Signaling Technology, Danvers, MA), HSP90AB1 (1:1,000; 7411; Cell Signaling Technology, Danvers, MA), HSPA6 (1:1,000; NBP1-85949; Novus Biologicals, Centennial, CO), HSPA1A (1:1,000; NB110-96427; Novus Biologicals, Centennial, CO), HSPB1 (1:1,000; NBP2-25149; Novus Biologicals, Centennial, CO), and HSPD1 (1:1,000; NBP2-32973; Novus Biologicals, Centennial, CO) diluted in 5% milk in TBST.

Techniques: Quantitative Proteomics, Western Blot

Journal: Journal of Animal Science

Article Title: Porcine endometrial heat shock proteins are differentially influenced by pregnancy status, heat stress, and altrenogest supplementation during the peri-implantation period

doi: 10.1093/jas/skac129

Figure Lengend Snippet: Effect of altrenogest and/or thermal treatment on endometrial protein abundance of heat shock proteins. Endometrium from pregnant gilts either supplemented with ALT (postinsemination) or not and maintained under thermal neutral or heat stress conditions during the peri-implantation period were assessed for changes in specific HSP protein abundance using densitomentric analysis of immunoblots. ALT supplementation decreased (A) HSP90AA1 protein abundance irrespective of thermal treatment, with a tendency to decrease (B) HSP90AB1 abundance. Neither ALT supplementation nor HS influenced (C) HSPA1A abundance, but ALT reduced (D) HSPA6 protein abundance in the gilts maintained under TN conditions. (E) HSPD1 and (F) HSPB1 protein abundance were unchanged by either treatment. Different lowercase letters indicate statistical significance between treatment groups; P ≤ 0.05.

Article Snippet: Membranes were then incubated at 4 °C overnight with primary antibodies specific for HSP90AA1 (1:1,000; 8165; Cell Signaling Technology, Danvers, MA), HSP90AB1 (1:1,000; 7411; Cell Signaling Technology, Danvers, MA), HSPA6 (1:1,000; NBP1-85949; Novus Biologicals, Centennial, CO), HSPA1A (1:1,000; NB110-96427; Novus Biologicals, Centennial, CO), HSPB1 (1:1,000; NBP2-25149; Novus Biologicals, Centennial, CO), and HSPD1 (1:1,000; NBP2-32973; Novus Biologicals, Centennial, CO) diluted in 5% milk in TBST.

Techniques: Quantitative Proteomics, Western Blot

Journal: International journal of oncology

Article Title: P53-dependent radiosensitizing effects of Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin on human oral squamous cell carcinoma cell lines.

doi: 10.3892/ijo.29.5.1111

Figure Lengend Snippet: Figure 1. Expression of Hsp90 proteins in OSCC tissues and OSCC cell lines. A, immunohistochemical staining of Hsp90 in oral squamous cell carcinoma. Positive staining is seen in the cytoplasm of cancer cells (original magnification x100). B, Western blot analysis of Hsp27, Hsp70 and Hsp90 protein expression in normal keratinocyte cell line HaCaT and oral squamous cell carcinoma cell lines.

Article Snippet: Primary antibodies against Hsp27, Hsp70, Hsp90 and actin were obtained from Santa Cruz Biotech, while antibodies against Akt 1, Raf 1 and p38/MAPK were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: Journal of Clinical Investigation

Article Title: p90 ribosomal S6 kinase 2 promotes invasion and metastasis of human head and neck squamous cell carcinoma cells

doi: 10.1172/jci40582

Figure Lengend Snippet: Figure 5 Targeting RSK2 by shRNA leads to decreased phosphorylation levels of multiple prometastatic protein factors, including tyrosine kinases, c-MET and FAK, and RSK2 phosphorylation substrates, CREB and Hsp27. (A) Stable knockdown of RSK2 did not affect phosphorylation and activation levels of ERK, AKT, and STAT3. Immunoblotting was performed using cell lysates obtained from M4e-pLKO.1 or M4e-pLKO.1- RSK2 shRNA cells. (B) Immunoblotting results demonstrate that targeted downregulation of RSK2 resulted in decreased tyrosine phosphorylation and activation of prometastatic tyrosine kinases c-MET in M4e cells and FAK in 212LN and 37B cells. β-actin was detected as a loading control. (C) RSK2 directly phosphorylated Hsp27 at S78 and S82 but not S15. Purified rHsp27 WT and individual S15A, S78A, or S82A mutant proteins were incubated with active rRSK2 in an in vitro kinase assay. Phosphorylation at Ser15, Ser78, and Ser82 in rHsp27 was detected by specific antibodies phospho-Hsp27 pS15, pS78, and pS82. (D) RNAi-mediated knockdown of RSK2 resulted in reduction in phosphorylation levels of CREB in RSK2-expressing M4e, 212LN, and 37B HNSCC cells and decreased Hsp27 S78 phosphorylation in M4e and 212LN cells but not 37B cells. (E) RNAi-mediated knockdown of RSK2 resulted in reduction in the phosphorylation level of Hsp27 S82 in M4e and 212LN cells but not 37B cells.

Article Snippet: RSK1, RSK2, and phospho-Hsp27 (pS15) antibodies were from Santa Cruz Biotechnology; specific antibodies against phospho-RSK (S380), p44/42 ERK, phospho-p44/42 ERK (T202/T204), AKT, phosphoAKT (S473), STAT3, phospho-STAT3 (Y705), MET, phospho-MET (Y1234/ Y1235), CREB, phospho-CREB (S133), Hsp27, phospho-Hsp27 (S15, S78, and S82), Myc, and FAK were from CST; phospho-FAK (Y397) was from Invitrogen; antibodies against GST and β-actin were from Sigma-Aldrich.

Techniques: shRNA, Phospho-proteomics, Knockdown, Activation Assay, Western Blot, Control, Purification, Mutagenesis, Incubation, In Vitro, Kinase Assay, Expressing

Journal: Journal of Clinical Investigation

Article Title: p90 ribosomal S6 kinase 2 promotes invasion and metastasis of human head and neck squamous cell carcinoma cells

doi: 10.1172/jci40582

Figure Lengend Snippet: Figure 6 RSK2 promotes HNSCC cell invasion through phosphorylation and activation of the downstream substrate CREB. (A) RNAi-mediated stable knockdown of CREB and Hsp27 in M4e cells. (B) Knock- down of CREB and Hsp27 significantly reduced the invasive ability of metastatic M4e cells. Relative invasion was normal- ized to the invasion of M4e-pLKO.1 cells (mean ± SD; *P = 0.01–0.05). (C) Stable knockdown of CREB and Hsp27 did not significantly affect the proliferation rate of M4e cells (mean ± SD). (D) Expression of RSK2 promoted invasive ability of Tu212 cells, whereas targeted downregulation of CREB significantly attenuated RSK2- dependent invasion. Relative invasion was normalized to the invasion of control Tu212 cells (mean ± SD; **P < 0.01). (E and F) Stable expression of the phospho-mimetic CREB S133D mutant but not the phospho- deficient S133A mutant, further significant- ly enhanced 686LN (E) and Tu212 (F) cell invasion, compared with CREB WT. Rela- tive invasion was normalized to the inva- sion of control cells harboring an empty vector (mean ± SD; *P = 0.01–0.05).

Article Snippet: RSK1, RSK2, and phospho-Hsp27 (pS15) antibodies were from Santa Cruz Biotechnology; specific antibodies against phospho-RSK (S380), p44/42 ERK, phospho-p44/42 ERK (T202/T204), AKT, phosphoAKT (S473), STAT3, phospho-STAT3 (Y705), MET, phospho-MET (Y1234/ Y1235), CREB, phospho-CREB (S133), Hsp27, phospho-Hsp27 (S15, S78, and S82), Myc, and FAK were from CST; phospho-FAK (Y397) was from Invitrogen; antibodies against GST and β-actin were from Sigma-Aldrich.

Techniques: Phospho-proteomics, Activation Assay, Knockdown, Expressing, Control, Mutagenesis, Plasmid Preparation

Journal: Journal of Clinical Investigation

Article Title: p90 ribosomal S6 kinase 2 promotes invasion and metastasis of human head and neck squamous cell carcinoma cells

doi: 10.1172/jci40582

Figure Lengend Snippet: Figure 7 RSK2 promotes stabilization of actin filaments in HNSCC cells through phosphorylation and activation of Hsp27. (A) RNAi-mediated knockdown of Hsp27 significantly attenuated Tu212 cell invasion conferred by exogenous expression of RSK2. Relative invasion was normalized to the invasion of control Tu212 cells (mean ± SD; **P < 0.01). (B) Stable expression of the phospho-mimetic Hsp27 S78D/S82D double mutant, but not the phospho-deficient Hsp27 S78A/S82A mutant or the Hsp27 S78D and Hsp27 S82D single mutants, led to further significantly enhanced invasion of poorly invasive Tu212 and 686LN cells, compared with Hsp27 WT. Relative invasion was normalized to the invasion of control cells harboring an empty vector (mean ± SD; *P = 0.01–0.05; **P < 0.01). (C) Stable expression of Hsp27 S78D/S82D mutant, but not WT or S78A/ S82A mutant, rescued the cell invasion attenuated by stable knockdown of RSK2 in M4e cells. Relative invasion was normalized to the invasion of M4e-pLKO.1 cells (mean ± SD; *P = 0.01–0.05). (D) Actin immunofluorescent staining shows that RNAi-mediated stable knockdown of RSK2 resulted in disruption of actin filaments in 212LN and M4e cells, whereas stable expression of the Hsp27 phospho-mimetic mutant S78D/S82D, but not WT or the phospho-deficient S78A/S82A mutant, rescued the formation of actin filaments. Cells were fixed and stained with phalloidin conjugated with Alexa Fluor 555. The integrity of actin filaments was analyzed by confocal microscopy. Original magnification, ×1,000.

Article Snippet: RSK1, RSK2, and phospho-Hsp27 (pS15) antibodies were from Santa Cruz Biotechnology; specific antibodies against phospho-RSK (S380), p44/42 ERK, phospho-p44/42 ERK (T202/T204), AKT, phosphoAKT (S473), STAT3, phospho-STAT3 (Y705), MET, phospho-MET (Y1234/ Y1235), CREB, phospho-CREB (S133), Hsp27, phospho-Hsp27 (S15, S78, and S82), Myc, and FAK were from CST; phospho-FAK (Y397) was from Invitrogen; antibodies against GST and β-actin were from Sigma-Aldrich.

Techniques: Phospho-proteomics, Activation Assay, Knockdown, Expressing, Control, Mutagenesis, Plasmid Preparation, Staining, Disruption, Confocal Microscopy

Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: Paracrine HSP27 promotes OSCC chemoresistance through TAMs. A Schematic presentation of the analyzed tumor models, Vehicle or HSP27-shRNA OSCC cells were orthotopically injected into BALB/c nude mice. B - C Tumor pictures ( B ) and growth analysis ( C ) over 21 days show the mean tumor volume at the indicated time points following implantation, statistical significance was determined by 2-way ANOVA. Volume of individual tumors at day 21 is shown in the box plot; statistical significance was determined by Welch t-test. D - E HSP27 mRNA ( D ), protein ( E ) levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by RT-qPCR and Western blotting in the whole-cell lysates. F Apoptosis relative gene BAX, BIM, BAD and Caspase3 mRNA levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by RT-qPCR. G Apoptosis relative gene BAX, BIM, BAD and Caspase3 mRNA levels in CAL27 cells without or with rhHSP27 (2 μg/mL) induced TAMs-CM, detected by RT-qPCR. H Relative cell apoptosis level in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by Flow Cytometry. I Relative cell apoptosis level in CAL27 cells without or with rhHSP27 (2μg/mL) induced TAMs-CM, detected by Flow Cytometry. J Analysis of ( H ), determined with relative ratio of apoptosis in total per 10 4 cells. K Apoptosis relative BAX, BIM, BAD and Cleaved-caspase3 protein levels in CAL27 cells with NC-siRNA or HSP27-siRNA induced TAMs-CM, detected by Western blotting in the whole-cell lysates. L Analysis of ( I ), determined with relative ratio of apoptosis in total per 10 . 4 cells. M Apoptosis relative BAX, BIM, BAD and Cleaved-caspase3 protein levels in CAL27 cells without or with rhHSP27 (2 μg/mL) induced TAMs-CM, detected by Western blotting in the whole-cell lysates. N – O Analysis of ( K ) and ( M ). T-tests and two-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

Techniques: shRNA, Injection, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: Paracrine HSP27 promotes OSCC invasion, migration, and EMT through TAMs. A GSEA of transcriptome data cells shows enrichment of a gene signature associated with increased metastasis in HNSCC. B The correlation between the expression of HSP27 and the metastasis pathway enrichment score. C The EMT pathway enrichment score of HSP27 and TLR4. D Representative invasive (upper) and migratory (lower) images of the transwell assays using CAL27 cells (upper chamber) cocultured with TAMs (lower chamber) and preincubated in the presence of rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM). E Representative images of the wound-healing assays using CAL27 cells under the stimulation of TAMs-CM from TAMs preincubated with rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM). F – H statistical analysis of cell invasion ( F ), migration ( G ), and stretch ( H ). I Representative immunofluorescence staining images showing the colocation of HSP27 and TLR4 on the cell membranes of the RAW 264.7 cells incubated with rhHSP27 (2 µg/mL) in the TCM of the CAL27 cells. Nuclei were counterstained with DAPI. J - L N-cadherin, Vimentin mRNA and N-cadherin protein levels in the CAL27 cells under stimulation with the TAMs-CM from the TAMs incubated in the presence of rhHSP27 (2 μg/mL) or rhHSP27 (2 μg/mL) with TAK-242 (1 µM), detected by RT-qPCR and Western blotting. One-way ANOVA. Means ± SD of at least three separate experiments. ns, no significance. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

Techniques: Migration, Expressing, Immunofluorescence, Staining, Incubation, Quantitative RT-PCR, Western Blot

Journal: Cell Biology and Toxicology

Article Title: HSP27/IL-6 axis promotes OSCC chemoresistance, invasion and migration by orchestrating macrophages via a positive feedback loop

doi: 10.1007/s10565-024-09983-1

Figure Lengend Snippet: HSP27/IL-6 establishes a positive feedback loop between OSCC cells and M2 TAMs. A - B iNOS, IL-12, TNF-α, Arg-1, IL-10, and IL-6 mRNA levels in RAW264.7cells under stimulation with the TCM of the CAL27 cells pretreated without or with HSP27–siRNA, detected by RT-qPCR. C - D iNOS, IL-12, Arg-1, CD163, and IL-6 mRNA levels in RAW264.7cells pretreated without or with rhHSP27 (2 μg/mL) in the presence of CAL27-CM for 12 h, detected by RT-qPCR. E – F IL-12, Arg-1, and IL-6 mRNA and IL-6 protein levels in RAW264.7cells pretreated without or with TAK-242 (1 µM) under the stimulation of rhHSP27 (2 μg/mL) in the presence of CAL27-CM for 12 h, detected by RT-qPCR ( E ) and ELISA ( F ). G - H IL-6 mRNA, protein levels in THP-1 cells with NC-siRNA or HSP27-siRNA induced TCM, detected by RT-qPCR ( G ) and ELISA ( H ). I - J IL-6 mRNA, protein levels in THP-1 cells without or with rhHSP27 (2 μg/mL) stimulated, detected by RT-qPCR ( I ) and ELISA ( J ). K - L IL-6 mRNA, protein levels in RAW264.7 cells with NC-siRNA or HSP27-siRNA induced TCM, detected by RT-qPCR ( K ) and ELISA ( L ). M – N ), IL-6 mRNA, protein levels in Raw264.7 cells without or with rhHSP27 (2 μg/mL) stimulated, detected by RT-qPCR ( M ) and ELISA ( N ). O - P HSP27 mRNA levels in CAL27 cells without or with IL-6 (100 ng/mL) treated ( O ), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated ( P ), detected by RT-qPCR. ( Q - S ), HSP27protein levels in CAL27 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates ( Q ) and ELISA ( R - S ). T - U HSP27 mRNA levels in SCC9 cells without or with IL-6 (100 ng/mL) treated ( T ), with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated ( U ), detected by RT-qPCR. V-X HSP27 protein levels in SCC9 cells without or with IL-6 (100 ng/mL) treated, with TAMs-CM and TAMs-CM with the IL-6R antagonist tocilizumab (TOC, 5 μg/mL) treated, detected by Western blotting in the whole-cell lysates ( V ) and ELISA ( W - X ). T-tests and one-way ANOVA. Means ± SD of at least three separate experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: In specific experiments, cells will be treated with rhHSP27 from R&D Systems (1580-HS), recombinant human protein IL-6 (rhIL-6) from Peprotech (AF-200–06–20), and the IL-6 receptor inhibitor tocilizumab (anti-IL-6R) from Selleck (A2012).

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