Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Snx14 localizes at ER–LD contacts after OA treatment. (A) Localization of Snx14 in U2OS cells in the absence and presence of OA, respectively. CoIF staining of the cells transiently expressing an untagged Snx14 were performed with α-Snx14 antibody (green), and α-HSP90B1 (ER marker, red) antibodies and LDs were stained with MDH (blue) and imaged by confocal microscopy. Inset of OA-treated cells displays Snx14 accumulating around LDs. Scale bar = 20 µm. Scale bar of insets = 5 µm. (B) Schematic diagram showing Snx14 EGFP-APEX2 fusion at ER–LD contacts. (C) TEM micrographs showing untreated and DAB + H 2 O 2 –treated cells following OA treatment. The dark precipitate with DAB + H 2 O 2 treatment indicates presence of Snx14 EGFP-APEX2 . Scale bar = 0.5 µm. (D) TEM micrograph showing SNX14 EGFP-APEX2 -expressing cell with DAB precipitate at ER–LD contacts. (D′) Image from D showing pseudocolored ER (red), LD (yellow), and green arrows pointing at DAB precipitate specifically at the junction of ER and LD. The blue arrows indicate a region of LD surface that lacks ER wrapping and also lacks DAB precipitate. Purple stars indicate LDs without any detectable ER association (and no DAB precipitate). The orange arrow indicates an ER membrane itself. Scale bar = 0.5 µm. (E) Lower-magnification TEM micrographs of a Snx14 EGFP-APEX2 -expressing cell following OA treatment and stained with DAB, showing precipitate surrounding various LDs entangled with the ER network. Scale bar = 0.5 µm.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Staining, Expressing, Marker, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Snx14 is topologically anchored in the ER and interacts with LDs in trans. (A) LD flotation assay of OA-treated U2OS cells expressing Snx14 EGFP . Lanes indicate postnuclear supernatant (PNS), cytosol, total membrane, and LD float fractions. (B) Schematic diagram of Snx14 fragment constructs tagged with EGFP. Snx14 FL depicts the full-length human Snx14. Snx14 N is the N-terminal fragment from the start that includes PXA and RGS domains. Snx14 PXCN includes the PX domain and C-Nexin domains. Snx14 PX consists of PX and Snx14 CN represents C-Nexin domain. An AH in the C-Nexin domain is identified as depicted in the schematic diagram. Snx14 PXCNΔH indicates the PX and C-Nexin domain from which the AH is deleted. Snx14 FLΔH depicts the full-length Snx14 with AH deletion. (C) Western blot showing distribution of Snx14 FL , Snx14 N , and Snx14 PXCN tagged with 3XFlag among total membrane and LD fractions following OA treatment. (D) U2OS cells transfected with Snx14 FL , Snx14 N , Snx14 PXCN , Snx14 PX , Snx14 CN , Snx14 PXCNΔH , and Snx14 FLΔH , respectively, and treated with OA for 16 h. CoIF staining with α−EGFP (green) and α-HSP90B1 (ER marker, red) and LDs stained with MDH (blue) and imaged by confocal microscopy. Scale bar = 20 µm. Inset scale bar = 5 µm. Cartoons represent the localization of the respective Snx14 fragments with respect to ER and LD.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Expressing, Construct, Western Blot, Transfection, Staining, Marker, Confocal Microscopy

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Loss of Snx14 perturbs LD size and morphology but does not change neutral lipid levels. (A) Confocal micrographs of WT and SNX14 KO cells treated with OA overnight. LDs visualized by MDH (black) and nucleus stained with Syto 85 orange fluorescent stain (blue outline). Images were processed so that LDs were converted to grayscale and inverted. Scale bar = 25 µm. (B) Quantification of average area covered by LDs per cell of representative images from A. Total LD area was derived from more than five fields of view, each consisting of approximately five cells or more of two different sets of experiments (total no. of cells >75; ***, P < 0.0001 unpaired t test with α = 0.05). (C) TEM micrographs of WT and SNX14 KO cells treated with OA overnight to visualize LD distribution and morphology. The top panels are lower magnification (scale bar = 2 µm). The bottom panels are higher magnification (scale bar = 1 µm). (D) Scatter dot plot of cross-sectional areas of LDs in WT and SNX14 KO cells as in C. Total LDs = 896; ***, P < 0.0001, Kolmogorov–Smirnov D test with α = 0.05. (E) Rescue of LD morphology in SNX14 KO cells by readdition of empty vector (EV), Snx14 FL , Snx14 N , Snx14 PXCN , Snx14 PXCNΔH , and Snx14 FLΔH , respectively, all tagged with EGFP. Cells were coIF stained with α-EGFP (green), α-HSP90B1 (ER, red), and LDs stained with MDH (blue) and imaged with confocal microscope. Scale bar = 50 µm. (F) Area covered by LDs in each cell from E analyzed and plotted. Total no. of cells quantified are 45 from two different sets of experiments (***, P < 0.0001, one-way ANOVA with α = 0.05). Line bars indicate mean ± SD.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Staining, Derivative Assay, Plasmid Preparation, Microscopy

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Snx14 functions independently of ER–LD protein, Seipin. (A) Confocal micrographs showing localization of Snx14 in WT and SEIPIN KO SUM159 cells. The cells were transfected with Snx14 EGFP and treated with OA overnight. Labels were Snx14 EGFP (α-EGFP), ER (α-HSP90B1), and LDs (MDH). Scale bar = 20 µm. Inset scale bar = 5 µm. (B) Test of rescue of LD morphology in SEIPIN KO examined by ectopic expression of Snx14 EGFP into SEIPIN KO . Labels were Snx14 EGFP (α-EGFP), ER (α-HSP90B1), and LDs (MDH stained, converted to grayscale, and inverted by ImageJ). Scale bar = 20 µm. (C) Area covered by LDs in each cell from B analyzed and plotted. Total no. of cells quantified is 23 from two different sets of experiments (***, P < 0.0001, one-way ANOVA with α = 0.05). Line bars indicate mean ± SD. (D) Test of rescue of LD morphology in SNX14 KO examined by readdition of Seipin mCherry and comparing to that of WT and SNX14 KO . Blue line depicts cell boundary. Black speckles represent LDs that are MDH-stained, converted to grayscale, and inverted by ImageJ. Scale bar = 20 µm. (E) Area covered by LDs in each cell from D analyzed and plotted. Total no. of cells quantified is 45 from two different sets of experiments (***, P < 0.0001, one-way ANOVA with α = 0.05). Line bars indicate mean ± SD. (F) Confocal micrographs of U2OS cells to examine localization of Seipin mCherry in WT cells, those transfected with Snx14 EGFP , and in SNX14 KO cells. Cells were transfected with Seipin-mCherry and treated with OA overnight. Labels were Snx14 EGFP (α-EGFP), Seipin-mCherry (α-mCherry), and LDs (MDH). Scale bar = 20 µm.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Transfection, Expressing, Staining

Journal: The Journal of Cell Biology

Article Title: Cerebellar ataxia disease–associated Snx14 promotes lipid droplet growth at ER–droplet contacts

doi: 10.1083/jcb.201808133

Figure Lengend Snippet: Snx14 localizes at ACSL3-positive preLDs following OA addition. (A) Confocal micrographs of immunofluorescently stained Snx14 EGFP -expressing cells following OA treatment for t = 0, 1, 2, 4, 8, and 16 h. Labels were Snx14 EGFP (α-EGFP), ER (α-HSP90B1), and LDs (MDH). Scale bar = 2 µm. Line scans show the spatial distribution of Snx14 (green) with respect to ER (red) and LDs (blue). (B) Confocal micrographs of immunofluorescently stained Snx14 EGFP -expressing cells following OA treatment for t = 0, 1, 2, 4, 8, and 16 h. Labels were Snx14 EGFP (α-EGFP), native ACSL3 (α-ACSL3), and LDs (MDH). Scale bar = 2 µm. Line scans show the spatial distribution of Snx14 (green) with respect to ACSL3 (red) and LDs (blue). Scale bar = 2 µm. (C) Quantification of Pearson’s coefficient between Snx14 and ER of n = ∼20 cells depicted in A. (D) Quantification of Pearson’s coefficient between ACSL3 and LDs, and Snx14 and LDs of n = ∼20 cells depicted in B. (E) Quantification of Pearson’s coefficient between Snx14 and ACSL3 of n = ∼20 cells depicted in B. (F) Confocal micrographs examining localization of ACSL3 in WT and SNX14 KO cells treated with OA overnight. Labels were native ACSL3 (α-ACSL3, green) and LDs (MDH, magenta). Scale bar = 20 µm. (G) Confocal micrographs to examine localization of Snx14 in WT cells treated with negative scrambled siRNA (neg Si) or ACSL3-targeted siRNA (ACSL3 Si), respectively. The cells were transfected with Snx14 EGFP along with the respective siRNAs and treated with OA overnight. Labels were Snx14 EGFP (α-EGFP), ER (α-HSP90B1), and LDs (MDH). Scale bar = 20 µm. Inset scale bar = 5 µm.

Article Snippet: The primary antibodies used are mouse anti-Hsp90B1 (1:300; Sigma-Aldrich; AMAb91019), rabbit anti-Snx14 (HPA017639), rabbit anti-EGFP (1:300), rabbit anti-Flag (1:200; Sigma-Aldrich; F7425), mouse anti-ACSL3 (1:200; Novus Biologicals; H00002181-B01P).

Techniques: Staining, Expressing, Transfection

Journal: Journal of Clinical Medicine

Article Title: A New Histology-Based Prognostic Index for Aggressive T-Cell lymphoma: Preliminary Results of the “TCL Urayasu Classification”

doi: 10.3390/jcm13133870

Figure Lengend Snippet: Overall survival in TCL patients with and without prognostic factors—comparison of the Kaplan–Meier survival curves and prognostic factors, positive/negative immunostaining between Group 2 and Group 3 (log-rank test). ( A , B ) show the results of comparisons among 3 groups, and C to F show the results of comparisons between 2 groups. ( A ) GRP94+, CR1+, n = 2; GRP94+, CR−, n = 8; GRP94−, CR+, n = 6; p < 0.01. GRP94-positive patients showed a non-CR treatment response, resulting in a poor prognosis. ( B ) GRP94-positive patients with relapse ( n = 10), poor prognosis with a median OS of 13.5 months ( p < 0.01). GRP94-negative patients without relapse ( n = 6), relatively good prognosis with a median OS of 102 months ( p < 0.01). A total of 10 patients, consisting of 6 patients who showed a non-CR treatment response after the initial therapy and 4 patients who developed relapse within 1 year, were identical to the 10 patients showing positive tumor staining for GRP94. GRP94-positive patients showed a “non-CR” treatment response after the initial therapy or developed relapse within 1 year, resulting in a poor prognosis. In the tumor microenvironment, GRP94 expression is associated with cell survival of the TCL cells and leads to a poor prognosis. In order to overcome various stressful conditions, such as altered cellular metabolism and acidosis, TCL cells survive and lead to a poor prognosis. ( C ) PD-L1-positive patients ( n = 4, median OS, 5.5 months, p < 0.01); PD-L1-negative patients ( n = 12, median OS, 50 months, p < 0.01). PD-L1 positivity allows TCL cells to proliferate by escaping the surveillance mechanism, which results in a poor prognosis. ( D ) AKR1C3-positive patients ( n = 6, median OS, 10 months: p < 0.01); AKR1C3-negative patients ( n = 10, median OS, 62 months; p < 0.01). AKR1C3 expression in TCL cells decreases the intracellular metabolism of HO of the CHOP regimen drugs, attenuating their cytotoxic activity against the TCL cells and making the disease refractory, which results in a poor prognosis. ( E ) TP-positive patients ( n = 3, median OS, 6 months; p < 0.01); TP-negative patients ( n = 13, median OS, 56 months; p < 0.01). TP expression is mainly linked to antiapoptotic and angiogenic activities, leading to a poor prognosis. ( F ) CYP2B6-positive patients ( n = 4, median OS, 13.5 months; p < 0.05); CYP2B6-negative patients ( n = 12, median OS, 46 months; p < 0.01).

Article Snippet: The primary antibodies against the major proteins involved in anticancer drug metabolism included (1) GRP94: Proteintech (Rosemont, IL 60018, USA), clone 1H10B7 (this monoclonal antibody was generated against the N-terminal region of full-length HSP90b1); (2) CYP3A4: Sigma-Aldrich (St. Louis, MO 63103, USA), SAB1400064 (this polyclonal antibody was generated against CYP3A4); (3) AKR1C3: Proteintech, 11194-1-AP (this polyclonal antibody was generated against AKRC3); (4) MDR1 (P-glycoprotein): Proteintech, 22336-1-AP (this polyclonal antibody was generated against MDR1); (5) MRP1 (CD9): Proteintech, 60232-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length MRP1); (6) TGF beta1: Proteintech, 21898-1-AP (this polyclonal antibody was generated against TGF-beta); (7) GRP78: Proteintech, 66574-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length GRP78); (8) glutathione S-transferase kappa1 (GST): Proteintech, 14535-1-AP (this polyclonal antibody was generated against GST1); (9) thymidine phosphorylase: Abcam (Cambridge, UK), ab226917 (this polyclonal antibody was generated against thymidine phosphorylase); (10) MRP4 (ABCC4): SANTA CRUZ BIOTECHNOLOGY (Dallas, TX 75220, USA), SC-376262 (this monoclonal antibody was generated against the N-terminal region of full-length MRP4 (amino acid 1-280)); (11) CYP2B6: LifeSpan BioSciences, Inc. (Seattle, WA 98121, USA), LS-C352084 (this polyclonal antibody was generated against CYP2B6); (12) TNF1 alpha: Sigma-Aldrich, SAB4502982 (this polyclonal antibody was generated against TNF1 alpha); (13) PD-1; (14) PD-L1: Proteintech (Rosemont, IL, USA), 66248-1-IG, mouse IgG1 monoclonal antibody, clone 2B11D11; (15) PD-L2: Proteintech (Rosemont, IL, USA), 18251-1-AP 16, rabbit IgG polyclonal antibody; (16) P53: Cell Signaling Technology, Inc. (3 Trask Lane Danvers, MA 01923, USA), DO-7 mouse monoclonal antibody #48818; (17) c-MYC: Abcam (Kendall Sq Cambridge, MA 02139, USA), Y69 clone ab32072; (18) ENT-1 (equilibrative nucleoside transporter 1): Proteintech (Rosemont, IL, USA), 1337-1-AP rabbit IgG polyclonal antibody; (19) AKR1B1: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit polyclonal antibody HPA052751; (20) AKR1B10: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit monoclonal antibody HPA020280.

Techniques: Comparison, Immunostaining, Staining, Expressing, Activity Assay

Journal: Journal of Clinical Medicine

Article Title: A New Histology-Based Prognostic Index for Aggressive T-Cell lymphoma: Preliminary Results of the “TCL Urayasu Classification”

doi: 10.3390/jcm13133870

Figure Lengend Snippet: HPI (TCL Urayasu classification) Group 3 (the very poor prognosis group) Case: A 33-year-old woman diagnosed with stage IIA ALK-positive ALCL (low-intermediate IPI (IPIe), PIT-Group 1). ALK positivity is generally associated with a good prognosis. However, this patient developed resistance to both CHOP and ESHAP regimens, and died after only about 2 months of treatment. The diagnosis was ALCL. Her ALCL showed positive staining for GRP94 (shown in 8) and 3 (PD-L1, TP, and GRP78, shown in 10, 12, and 20) of the other 6 poor prognostic factors (PD-L1, TP, AKR1C3, P53, PD1, and GRP78). In the tumor microenvironment, expression of PD-L1 on the cell surface blocks the immune checkpoint molecules, allowing the tumor to grow. TP is involved in starvation resistance, angiogenesis, invasion, and metastasis. GRP78 allows the tumors to overcome various stressful conditions, such as hypoxia, hypoglycemia, dysregulation of homeostasis, altered cell metabolism, and acidosis, which results in treatment resistance.

Article Snippet: The primary antibodies against the major proteins involved in anticancer drug metabolism included (1) GRP94: Proteintech (Rosemont, IL 60018, USA), clone 1H10B7 (this monoclonal antibody was generated against the N-terminal region of full-length HSP90b1); (2) CYP3A4: Sigma-Aldrich (St. Louis, MO 63103, USA), SAB1400064 (this polyclonal antibody was generated against CYP3A4); (3) AKR1C3: Proteintech, 11194-1-AP (this polyclonal antibody was generated against AKRC3); (4) MDR1 (P-glycoprotein): Proteintech, 22336-1-AP (this polyclonal antibody was generated against MDR1); (5) MRP1 (CD9): Proteintech, 60232-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length MRP1); (6) TGF beta1: Proteintech, 21898-1-AP (this polyclonal antibody was generated against TGF-beta); (7) GRP78: Proteintech, 66574-1-IG (this monoclonal antibody was generated against the N-terminal region of full-length GRP78); (8) glutathione S-transferase kappa1 (GST): Proteintech, 14535-1-AP (this polyclonal antibody was generated against GST1); (9) thymidine phosphorylase: Abcam (Cambridge, UK), ab226917 (this polyclonal antibody was generated against thymidine phosphorylase); (10) MRP4 (ABCC4): SANTA CRUZ BIOTECHNOLOGY (Dallas, TX 75220, USA), SC-376262 (this monoclonal antibody was generated against the N-terminal region of full-length MRP4 (amino acid 1-280)); (11) CYP2B6: LifeSpan BioSciences, Inc. (Seattle, WA 98121, USA), LS-C352084 (this polyclonal antibody was generated against CYP2B6); (12) TNF1 alpha: Sigma-Aldrich, SAB4502982 (this polyclonal antibody was generated against TNF1 alpha); (13) PD-1; (14) PD-L1: Proteintech (Rosemont, IL, USA), 66248-1-IG, mouse IgG1 monoclonal antibody, clone 2B11D11; (15) PD-L2: Proteintech (Rosemont, IL, USA), 18251-1-AP 16, rabbit IgG polyclonal antibody; (16) P53: Cell Signaling Technology, Inc. (3 Trask Lane Danvers, MA 01923, USA), DO-7 mouse monoclonal antibody #48818; (17) c-MYC: Abcam (Kendall Sq Cambridge, MA 02139, USA), Y69 clone ab32072; (18) ENT-1 (equilibrative nucleoside transporter 1): Proteintech (Rosemont, IL, USA), 1337-1-AP rabbit IgG polyclonal antibody; (19) AKR1B1: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit polyclonal antibody HPA052751; (20) AKR1B10: Sigma-Aldrich (3050 Spruce Street Saint Louis, MO 63103, USA), rabbit monoclonal antibody HPA020280.

Techniques: Biomarker Discovery, Staining, Expressing

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: RT-qPCR primer sequences

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques:

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 is overexpressed in NPC. A : Bioinformatics website http://gepia.cancer-pku.cn/index.html analyzed the expression parttern of HSP90B1 in HNSC; B : RT-qPCR detection of HSP90B1 in NPC tissues and normal nasopharyngeal epithelial samples; C : Western blot detection of HSP90B1 expression pattern in NPC tissues and normal nasopharyngeal epithelial samples; D : IHC staining to assess the expression pattern of HSP90B1 in NPC tissues and normal nasopharyngeal epithelial samples; E : RT-qPCR to assess the expression pattern of HSP90B1 in the NPC cell lines (HNE1, HNE2, CNE1, CNE2, HONE1) and human immortalized nasopharyngeal epithelial cell line (NP69); F : Western blot assessment of HSP90B1 expression in NPC cell lines (HNE1, HNE2, CNE1, CNE2, HONE1) and NP69; G : Prognostic chart of 5 year survival of 42 NPC patients. Data are expressed as mean ± SD (N = 3). p < 0.05 was considered statistically significant

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: Relationship between HSP90B1 and clinicopathological features of NPC patients

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Expressing

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 affects NPC proliferation, glycolysis, and angiogenesis. The siRNA targeting HSP90B1 and pcDNA 3.1 overexpression vector were transfected into HNE1 and CNE2 cells. A : RT-qPCR to detect the expression of HSP90B1 in the cells; B : Western blot to detect the expression of HSP90B1 in the cells; C : Colony formation assay to detect the proliferation of HNE1 and CNE2 cells; D : EdU assay to detect proliferation of HNE1 and CNE2 cells; E : Flow cytometry to detect apoptosis in HNE1 and CNE2 cells; F : Commercial kits to detect ECAR in HNE1 and CNE2 cells; G : Commercial kits to detect glucose consumption in HNE1 and CNE2 cells; H : Commercial kit to detect lactate production in HNE1 and CNE2 cells; I : Western blot to assess GLUT1 and HK2 protein expression in HNE1 and CNE2 cells; J : Tube formation assay to detect angiogenic capacity of HUVECs; K : HUVEC migration capacity assay; L : Western blot to assess the expression of angiogenic proteins Ang-1 and VEGF in HUVECs. Data are expressed as mean ± SD (N = 3). p < 0.05 was considered statistically significant

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Over Expression, Plasmid Preparation, Transfection, Quantitative RT-PCR, Expressing, Western Blot, Colony Assay, EdU Assay, Flow Cytometry, Tube Formation Assay, Migration

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 targets EGFR expression. A : Western blot to detect the effect of knockdown or overexpression of HSP90B1 on the expression of phosphorylated EGFR; B : Biosignature website ( https://www.ebi.ac.uk ) to analyze the binding relationship between HSP90B1 and EGFR; C : Co-IP assay to detect the interaction between HSP90B1 and EGFR; D : RT-qPCR to detect EGFR expression pattern in NPC tissues and normal nasopharyngeal epithelial samples; E : Western blot to detect the expression pattern of EGFR in NPC tissues and normal nasopharyngeal epithelial samples; F : IHC staining to assess the expression pattern of EGFR in NPC tissues and normal nasopharyngeal epithelial samples; G : RT-qPCR to assess the expression pattern of EGFR in NPC cell lines (HNE1, HNE2, CNE1, CNE2, and HONE1) and human immortalized nasopharyngeal epithelial cell line (NP69); H : Western blot to assess EGFR expression in NPC cell lines (HNE1, HNE2, CNE1, CNE2, and HONE1) and NP69. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Expressing, Western Blot, Over Expression, Binding Assay, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Immunohistochemistry

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 by regulating EGFR affects NPC malignancy. HNE1 and CNE2 cells transfected with pcDNA 3.1-HSP90B1 were treated with an EGFR inhibitor (cetuximab). A : Western blot to detect the phosphorylation level of EGFR; B : Colony formation assay to detect proliferation of HNE1 and CNE2 cells; C : EdU assay to detect proliferation of HNE1 and CNE2 cells; D : Flow cytometry assay to detect apoptosis in HNE1 and CNE2 cells; E : Commercial kits to detect ECAR in HNE1 and CNE2 cells; F : Commercial kits to detect glucose depletion in HNE1 and CNE2 cells; G : Commercial kits to detect lactate production in HNE1 and CNE2 cells; H : Western blot assessment of HNE1 and CNE2 cells GLUT1 and HK2 protein expression; I : Tube formation assay to detect angiogenic capacity of HUVECs; J : HUVEC migration capacity assay; K : Western blot to assess the expression of angiogenic proteins Ang-1 and VEGF in HUVECs. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Transfection, Western Blot, Colony Assay, EdU Assay, Flow Cytometry, Expressing, Tube Formation Assay, Migration

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: HSP90B1 activates the PI3K/Akt/mTOR pathway in NPC cells by regulating EGFR. HNE1 and CNE2 cells transfected with pcDNA 3.1-HSP90B1 were treated with an EGFR inhibitor (cetuximab). A – B : Western blot assessed the phosphorylation level of PI3K/Akt/mTOR. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Transfection, Western Blot

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: FKC by inhibiting HSP90B1 limits NPC proliferation, glycolysis, and angiogenesis. FKC-treated HNE1 and CNE2 cells were transfected with pcDNA 3.1-HSP90B1. A : CCK-8 assay to assess the effect of different concentrations of FKC treatment on NP69 cell viability; B : RT-qPCR to detect the expression of HSP90B1; C : Western blot to detect the expression of HSP90B1; D : Colony formation assay to detect HNE1 and CNE2 cell proliferation; E : EdU assay to detect HNE1 and CNE2 cell proliferation; F : Flow cytometry to detect HNE1 and CNE2 cell apoptosis; G : Commercial kits to detect ECAR in HNE1 and CNE2 cells; H : Commercial kits to detect glucose consumption in HNE1 and CNE2 cells; I : Commercial kits to detect HNE1 and lactate production in CNE2 cells; J : Western blot assessment of GLUT1 and HK2 protein expression in HNE1 and CNE2 cells; K : Tube formation assay to detect the angiogenic capacity of HUVECs; L : Migration capacity assay of HUVECs; M : Western blot assessment of angiogenic proteins Ang-1 and VEGF in HUVEC. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Transfection, CCK-8 Assay, Quantitative RT-PCR, Expressing, Western Blot, Colony Assay, EdU Assay, Flow Cytometry, Tube Formation Assay, Migration

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: FKC by inhibiting HSP90B1 affects the PI3K/Akt/mTOR pathway in NPC cells. pcDNA 3.1-HSP90B1 was transfected into FKC-treated HNE1 and CNE2 cells. A – B : Western blot detected phosphorylation levels of PI3K/Akt/mTOR. Data are presented as mean ± SD (N = 3). P < 0.05 was considered statistically significant

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Transfection, Western Blot

Journal: Cancer Cell International

Article Title: Flavokawain C inhibits glucose metabolism and tumor angiogenesis in nasopharyngeal carcinoma by targeting the HSP90B1/STAT3/HK2 signaling axis

doi: 10.1186/s12935-024-03314-4

Figure Lengend Snippet: FKC by modulating the HSP90B1/EGFR axis affects NPC tumor growth and metastasis. A : Tumor images; B : Tumor volume; C : Tumor weight; D : Western blot assessment of relevant protein expression in tumors; E : Noninvasive bioluminescence imaging of luciferase-expressing intrahepatic HNE1 cell xenografts showing FKC inhibition of in situ liver metastatic tumor formation. Data are presented as mean ± SD (n = 5). P < 0.05 was considered statistically significant

Article Snippet: The sections were incubated with primary antibodies: HSP90B1 (ab3674, Abcam) and EGFR (ab52894, Abcam) at 4 degrees Celsius overnight, and subsequently with secondary antibodies for 90 min at ambient temperature.

Techniques: Western Blot, Expressing, Imaging, Luciferase, Inhibition, In Situ

Journal: Journal of hepatology

Article Title: Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis

doi: 10.1016/j.jhep.2014.11.010

Figure Lengend Snippet: (A) Immunoblot for gp96 in the whole hepatic lysates of 6 week-old, WT and liver-specific gp96 KO mice (WT, n=3; KO, n=4). (B) Total bilirubin in the serum of 3 month-old mice (WT, n=8; KO n=7). (C) Masses of livers from WT and KO mice at different ages (6 weeks: 3 WT and 3 KO. 3 months: 5 WT and 5 KO. 1 year: 6 WT and 7 KO). (D) Gross appearance of WT and KO livers (Representative of >10 mice). (E) H&E staining of liver section of 3 month-old mice (Representative of 5 mice per group). The margin between steatotic lesion and normal appearing hepatocyte regions was indicted. (F) Oil red O staining of hepatic section from WT and KO mice (Representative of 5 mice per group). * p<0.05, ** p<0.01, ns: non-significant by 2-tailed student t-test.

Article Snippet: Primary antibodies, such as gp96 antibody (9G10, Stressgen) and Ki67 antibody (NB110-89717, Novus Biologicals), were applied for 1 hour at room temperature, followed by incubation with secondary antibodies and avidin-HRP from the ABC Systems of Vector BioLabs.

Techniques: Western Blot, Staining

Journal: Journal of hepatology

Article Title: Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis

doi: 10.1016/j.jhep.2014.11.010

Figure Lengend Snippet: (A) Immunohistochemical staining of gp96 on the liver sections from WT and KO mice. (B) Quantification of gp96+ hepatocytes with age (n=5/group). (C) Quantification of Ki67+ cells per high power field in the livers of 3 month-old mice (n=5/group). (D) Immunohistochemical staining Ki67 as in (C) (n=5/group). Arrows indicate Ki67+ cells. * p<0.05, ** p<0.01, *** p<0.0001, ns: non-significant by 2-tailed student t-test.

Article Snippet: Primary antibodies, such as gp96 antibody (9G10, Stressgen) and Ki67 antibody (NB110-89717, Novus Biologicals), were applied for 1 hour at room temperature, followed by incubation with secondary antibodies and avidin-HRP from the ABC Systems of Vector BioLabs.

Techniques: Immunohistochemical staining, Staining

Journal: Journal of hepatology

Article Title: Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis

doi: 10.1016/j.jhep.2014.11.010

Figure Lengend Snippet: (A) cDNA microarray analysis shows mRNA levels of gp96 in human hepatocellular carcinoma. (B,C) Surface tumors (B) and tumor burden (C) in diethylnitrosamine (DENA)-treated mice 32 weeks post-injection (WT males, n=12; KO males, n=7; WT females, n=6; KO females, n=11). (D) Post-DENA weight curve in mice of both sexes described in (B). (E) Serum ALT levels in untreated and tumor-bearing mice (n=6 per group). (F) Reticulin and gp96 stains of DENA-induced tumors, and quantification of gp96+ cells in tumors from WT and KO mice (n=5 per group). (G) Immunoblot of HGF in adjacent normal tissue of WT and KO livers at 32 weeks post induction (n=4 per group). (H) HGF expression in untreated WT and KO mice at 6 weeks of age, when the KO liver is mostly gp96− (n=4 per group). * p<0.05, **p<0.01, *** p<0.0001, ns: non-significant by 2-tailed student t-test.

Article Snippet: Primary antibodies, such as gp96 antibody (9G10, Stressgen) and Ki67 antibody (NB110-89717, Novus Biologicals), were applied for 1 hour at room temperature, followed by incubation with secondary antibodies and avidin-HRP from the ABC Systems of Vector BioLabs.

Techniques: Microarray, Injection, Western Blot, Expressing

Journal: Journal of hepatology

Article Title: Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis

doi: 10.1016/j.jhep.2014.11.010

Figure Lengend Snippet: (A,B) HepG2 (A) and PLC (B) HCC cells were treated with different doses of WS13 for 5 days, followed by MTT assay (representative of 2 experiments). (C) Immunoblot for β-catenin, PARP, Beclin1, HGF, Cyclin D1, p-AKT, p-ERK1/2, and p-STAT3 in hepatoma cells after 3 days of treatment with WS13 (representative of 2 experiments). (D) Cellular proliferation quantified by CFSE dilution in gp96 knockdown and control cells, with or without recombinant human HGF treatment. (E) Quantification of cell death by propidium iodide nuclear staining. (F,G) d18 (F) and d16 (G) base ceramides in HepG2 cells treated with WS13 (10 µM) for 3 days. (H,I) qRT-PCR of ceramide-generating enzymes and alkaline ceramidase in PLC and HepG2 cells treated with 2 µM (PLC) or 6 µM (HepG2) WS13 for 36 hours. Error bars in (H) and (I) represent standard deviation. (J,K) HepG2 cells were treated with WS13 (10 µM) for 24 hours and RNA was harvested for cDNA gene expression analysis. * p<0.05, ** p<0.01, ns: non-significant by 2-tailed student t-test.

Article Snippet: Primary antibodies, such as gp96 antibody (9G10, Stressgen) and Ki67 antibody (NB110-89717, Novus Biologicals), were applied for 1 hour at room temperature, followed by incubation with secondary antibodies and avidin-HRP from the ABC Systems of Vector BioLabs.

Techniques: MTT Assay, Western Blot, Knockdown, Control, Recombinant, Staining, Quantitative RT-PCR, Standard Deviation, Gene Expression

Journal: Journal of hepatology

Article Title: Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis

doi: 10.1016/j.jhep.2014.11.010

Figure Lengend Snippet: The asynchronous deletion of gp96 in this study uncovered that gp96 promotes hepatic steatosis and oncogenesis via empowering cell proliferation and survival via multiple pathways including sphingolipid biosynthesis.

Article Snippet: Primary antibodies, such as gp96 antibody (9G10, Stressgen) and Ki67 antibody (NB110-89717, Novus Biologicals), were applied for 1 hour at room temperature, followed by incubation with secondary antibodies and avidin-HRP from the ABC Systems of Vector BioLabs.

Techniques:

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: HSP90B1 and HSPA5 genes and GP96 are induced in ALD. (A) mRNA abundance of HSP90B1 and HSPA5 genes (in transcripts per million) was evaluated by RNA sequencing in patients with normal liver (n = 10), early ASH (n = 12), severe_AH (n = 17), explants_AH (n = 10), and livers from NAFLD (n = 10), HCV (n = 10), and comp_Cirrhosis (n = 9). (B) Representative immunohistochemistry of GP96 in human normal and alcoholic liver (magnification × 200). WT mice were fed with control liquid diet (pair‐fed) or diet containing 5% alcohol for 4 weeks. The mRNA expression of GP96 and GRP78 was measured by RT‐PCR in livers (C) and isolated hepatocytes (E) and liver macrophages (n = 4‐10). (D) Protein level of GP96 and GRP78 in liver lysate was analyzed by western blotting (n = 6). (F) BMDMs were treated with 25 mM ethanol and mRNA expression of GP96 and GRP78 (n = 6). (G) The protein level of GP96 was analyzed at different time intervals (n = 3). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: EtOH, alcohol; comp, compensated; tpm, transcripts per million.

Article Snippet: For immunohistochemistry, liver sections were incubated with GP96 primary antibodies (2104; Cell Signaling Technology, Danvers, MA), followed by incubation with secondary antibody.

Techniques: RNA Sequencing, Immunohistochemistry, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Western Blot

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Myeloid‐specific GP96 deficiency alleviates chronic alcohol‐induced liver injury. Female WT and M‐GP96KO mice were fed with isocaloric control liquid diet (pair‐fed) or 5% alcohol‐containing Leiber‐DeCarli diet (alcohol‐fed) for 4 weeks, and liver‐to–body weight ratio (A), serum levels of ALT (n = 10‐16) (B), and liver triglycerides (C) were analyzed. Representative photomicrographs of hepatic injury and steatosis assessed by H&E (D) and Oil Red O (E) staining in livers of mice of the indicated genotype (magnification × 100). Percentage of Oil Red O–positive area was quantitated by ImageJ (n = 3‐6). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: EtOH, alcohol; wt, weight.

Article Snippet: For immunohistochemistry, liver sections were incubated with GP96 primary antibodies (2104; Cell Signaling Technology, Danvers, MA), followed by incubation with secondary antibody.

Techniques: Control, Staining

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Mice lacking myeloid‐specific GP96 exhibit altered lipid metabolism after 4 weeks of alcohol consumption. (A) PPAR‐α protein was detected in nuclear extracts of livers by western blot, and expression was quantitated and normalized to pair‐fed group (n = 4). TBP was used as loading control. The mRNA expression of genes involved in fatty acid β‐oxidation (CPT1a, ACOX1, LCAD, and MCAD) (B‐E) and lipogenesis (SREBPF1, SCD1, and FAS) (F‐H) was analyzed in WT and M‐GP96KO hepatocytes by RT‐PCR and compared with pair‐fed control (n = 3‐6). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For immunohistochemistry, liver sections were incubated with GP96 primary antibodies (2104; Cell Signaling Technology, Danvers, MA), followed by incubation with secondary antibody.

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

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Myeloid‐specific GP96 deficiency prevents chronic alcohol‐induced endotoxin and pro‐inflammatory cytokine production. (A) Endotoxin level was measured in serum (n = 8‐12). (B) CyP2e1 was detected in liver microsomal fraction by western blot, and calnexin was used as an internal loading control (n = 3). Total RNA from liver tissue was subjected to quantitative RT‐PCR for analysis of pro‐inflammatory cytokines TNF‐α, IL‐6, MCP‐1, and IL‐1β; NLRP3; anti‐inflammatory markers IL‐10, TGF‐β, and ATF3 (C); and macrophage markers (n = 6‐10) (F). (D) Total liver protein level for TNF‐α was measured in tissue extracts of pair‐fed and alcohol‐fed mice by ELISA (n = 6‐10). (E) ATF3 protein level was analyzed by western blot in whole‐liver extracts using tubulin as an internal control (n = 3). The mRNA expression profile of (G) pro‐inflammatory, and (H) anti‐inflammatory and restorative macrophage markers, Trem2 and ATF3, were analyzed in liver macrophages of pair‐fed and alcohol‐fed mice (n = 5). (I) The mRNA level of pro‐inflammatory cytokines was analyzed in BMDMs isolated from WT and M‐GP96KO mice and stimulated with 100 ng/mL LPS for 2 hours (n = 9). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For immunohistochemistry, liver sections were incubated with GP96 primary antibodies (2104; Cell Signaling Technology, Danvers, MA), followed by incubation with secondary antibody.

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

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Loss of myeloid‐specific GP96 prevents LPS‐induced liver injury and inflammation. Female WT and M‐GP96KO mice were injected intraperitoneally with LPS (0.5 mg/kg body weight or saline (n = 4‐8). Serum was collected after 18 hours and subjected to analysis of ALT (A) and AST (B) and compared with the control group. (C) Livers were collected 2 hours after LPS injection, and liver cytokine mRNA was analyzed by RT‐PCR. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: For immunohistochemistry, liver sections were incubated with GP96 primary antibodies (2104; Cell Signaling Technology, Danvers, MA), followed by incubation with secondary antibody.

Techniques: Injection, Saline, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Inhibition of GP96 using specific inhibitor PU‐WS13 and siRNA reduces LPS‐induced pro‐inflammatory cytokine production. BMDMs were isolated from C57BL/6J mice and stimulated with LPS (100 ng/mL) for 2 hours and treated with PU‐WS13 (0.5 μM) either alone for 2 hours or before LPS for 1 hour. DMSO‐treated cells served as control group (n = 8). RT‐PCR was carried out to evaluate the expression of TNF‐α (A), IL‐6 (B), IL‐1β (C), and MCP‐1 (D) and compared with the untreated group. (E) Culture supernatant was evaluated for secreted TNF‐α by ELISA. (F) Murine macrophage RAW 264.7 cell line was transiently transfected with GP96 siRNA (100 nM) or negative control siRNA for 48 hours and stimulated with LPS for the final 6 hours. Secreted TNF‐α level was measured in culture supernatant by ELISA. Data are presented as mean ± SEM. *** P < 0.001, **** P < 0.0001. Abbreviation: ND, not detected.

Article Snippet: For immunohistochemistry, liver sections were incubated with GP96 primary antibodies (2104; Cell Signaling Technology, Danvers, MA), followed by incubation with secondary antibody.

Techniques: Inhibition, Isolation, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control

Journal: Hepatology Communications

Article Title: Myeloid Endoplasmic Reticulum Resident Chaperone GP96 Facilitates Inflammation and Steatosis in Alcohol‐Associated Liver Disease

doi: 10.1002/hep4.1713

Figure Lengend Snippet: Schematic representation depicting pathophysiological significance of macrophage‐specific GP96 during chronic alcohol‐mediated liver inflammation and injury.

Article Snippet: For immunohistochemistry, liver sections were incubated with GP96 primary antibodies (2104; Cell Signaling Technology, Danvers, MA), followed by incubation with secondary antibody.

Techniques: