Journal: Gut Microbes

Article Title: Fusobacterium nucleatum induces chemoresistance in colorectal cancer by inhibiting pyroptosis via the Hippo pathway

doi: 10.1080/19490976.2024.2333790

Figure Lengend Snippet: BCL2 knockdown affects the inhibitory effect of F. nucleatum on chemotherapy-induced pyroptosis. (a-b) the effect of F. nucleatum on the expression of pyroptosis-related proteins in chemotherapy-treated CRC cells after inhibition of BCL2 expression was detected by Western blotting. (c) The morphological changes in F. nucleatum- treated CRC cells after BCL2 inhibition were observed under a light microscope. (d) LDH release from chemotherapy-treated CRC cells after F. nucleatum intervention after BCL2 inhibition was detected. All experiments were performed in biological triplicates.

Article Snippet: The sections were blocked with QuickBlock blocking buffer (P0260, Beyotime, China) for 15 min at RT for subsequent immunostaining, followed by incubation with primary antibodies against YAP (1:200, CST, USA 14,074), BCL2 (1:400, CST, USA 15,071), Cleaved-Caspase-3 (1:200, CST, USA, 9664), N-GSDME (1:200, Abcam, USA, ab222408) at 4°C overnight.

Techniques: Knockdown, Expressing, Inhibition, Western Blot, Light Microscopy

Journal: Gut Microbes

Article Title: Fusobacterium nucleatum induces chemoresistance in colorectal cancer by inhibiting pyroptosis via the Hippo pathway

doi: 10.1080/19490976.2024.2333790

Figure Lengend Snippet: F. nucleatum induces chemotherapy resistance by regulating BCL2. (a) Survival of chemotherapy-treated CRC cells after F. nucleatum intervention and BCL2 inhibition was determined by CCK8 assay. (b) Representative images of CRC patient-derived organoids with F. nucleatum or BCL2 shRNA. (c) The size of proliferation on CRC patient-derived organoids was measured. (d) LDH release from CRC patient-derived organoids with F. nucleatum after BCL2 downregulation was detected. (e) Xenograft tumors in the nude mouse model were used to examine the effect of BCL2 inhibition on the chemoresistance of F. nucleatum -treated CRC cells. (f) Tumor volumes were calculated after injection every 3 days. (g) Tumor weights are represented as the means of tumor weights ± S.D. (h) The release of serum LDH in nude mice was measured. Bars indicate S.D.

Article Snippet: The sections were blocked with QuickBlock blocking buffer (P0260, Beyotime, China) for 15 min at RT for subsequent immunostaining, followed by incubation with primary antibodies against YAP (1:200, CST, USA 14,074), BCL2 (1:400, CST, USA 15,071), Cleaved-Caspase-3 (1:200, CST, USA, 9664), N-GSDME (1:200, Abcam, USA, ab222408) at 4°C overnight.

Techniques: Inhibition, CCK-8 Assay, Derivative Assay, shRNA, Injection

Journal: Gut Microbes

Article Title: Fusobacterium nucleatum induces chemoresistance in colorectal cancer by inhibiting pyroptosis via the Hippo pathway

doi: 10.1080/19490976.2024.2333790

Figure Lengend Snippet: F. nucleatum affects BCL2-mediated pyroptosis by regulating YAP. (a) ChIP assay to detect the enrichment of YAP in the BCL2 promoter region in chemotherapeutic drug-treated CRC cells after F. nucleatum intervention. (b-c) CRC cells were transfected with si-YAP. After coculturing with F. nucleatum and chemotherapy drugs, pyroptosis target proteins were detected by western blotting. (d) Representative bright-field images were observed under a light microscope. Yellow arrows indicate large bubbles emerging from the cell membrane. (e) The effect of YAP knockdown on LDH release in CRC cells infected with F. nucleatum was detected. All experiments were performed in biological triplicates.

Article Snippet: The sections were blocked with QuickBlock blocking buffer (P0260, Beyotime, China) for 15 min at RT for subsequent immunostaining, followed by incubation with primary antibodies against YAP (1:200, CST, USA 14,074), BCL2 (1:400, CST, USA 15,071), Cleaved-Caspase-3 (1:200, CST, USA, 9664), N-GSDME (1:200, Abcam, USA, ab222408) at 4°C overnight.

Techniques: Transfection, Western Blot, Light Microscopy, Membrane, Knockdown, Infection

Journal: Gut Microbes

Article Title: Fusobacterium nucleatum induces chemoresistance in colorectal cancer by inhibiting pyroptosis via the Hippo pathway

doi: 10.1080/19490976.2024.2333790

Figure Lengend Snippet: YAP mediates F. nucleatum -induced chemoresistance. (a) CCK8 assay was used to detect the effect of si-YAP on the viability of CRC cells treated with chemotherapy drugs after F. nucleatum intervention. (b) Representative images of chemotherapy-treated CRC patient-derived organoids with F. nucleatum , YAP shRNA or BCL2 adenovirus. (c) The size of proliferation on chemotherapy-treated CRC cells CRC patient-derived organoids was measured. (d) LDH release from CRC patient-derived organoids with F. nucleatum after YAP downregulation or BCL2 upregulation was detected. (e) Representative data of xenograft tumors in the nude mouse model bearing HCT116 cells in different groups. (f-g) statistical analysis of mouse tumor volumes (f) and weights (g). (h) The release of LDH in the serum of nude mice was detected. Bars indicate S.D.

Article Snippet: The sections were blocked with QuickBlock blocking buffer (P0260, Beyotime, China) for 15 min at RT for subsequent immunostaining, followed by incubation with primary antibodies against YAP (1:200, CST, USA 14,074), BCL2 (1:400, CST, USA 15,071), Cleaved-Caspase-3 (1:200, CST, USA, 9664), N-GSDME (1:200, Abcam, USA, ab222408) at 4°C overnight.

Techniques: CCK-8 Assay, Derivative Assay, shRNA

Journal: Gut Microbes

Article Title: Fusobacterium nucleatum induces chemoresistance in colorectal cancer by inhibiting pyroptosis via the Hippo pathway

doi: 10.1080/19490976.2024.2333790

Figure Lengend Snippet: F. nucleatum , YAP, BCL2, caspase-3 and GSDME were clinically relevant. (a) Protein levels of YAP and BCL2 in CRC tissues from non-recurrent and recurrent patients were detected by immunohistochemistry. (b and c) the mRNA expression of YAP and BCL2 was statistically analyzed by qRT‒PCR. (d and e) correlation of the expression levels of F. nucleatum , YAP and BCL2 in human CRC tissues. (f) Protein levels of cleaved caspase-3 and N-GSDME in CRC tissues from non-recurrent and recurrent patients were detected by immunohistochemistry. (g and h) statistical analysis of immunohistochemical immunoreactive score of Remmele and Stegner (IRS) scores of cleaved caspase-3 and N-GSDME proteins. (i and j) correlation of the expression levels of F. nucleatum , cleaved caspase-3 and N-GSDME in human CRC tissues. (k) The schematic model shows that F. nucleatum may affect chemoresistance in CRC cells by inhibiting chemotherapy-induced pyroptosis.

Article Snippet: The sections were blocked with QuickBlock blocking buffer (P0260, Beyotime, China) for 15 min at RT for subsequent immunostaining, followed by incubation with primary antibodies against YAP (1:200, CST, USA 14,074), BCL2 (1:400, CST, USA 15,071), Cleaved-Caspase-3 (1:200, CST, USA, 9664), N-GSDME (1:200, Abcam, USA, ab222408) at 4°C overnight.

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Pitavastatin protects against neomycin-induced ototoxicity through inhibition of endoplasmic reticulum stress

doi: 10.3389/fnmol.2022.963083

Figure Lengend Snippet: Impacts of PTV on neomycin-triggered ER stress in cochlear hair cells. (A) Immunostaining with myosin 7a and GRP78 in middle turns in cochlear explants. Scale bars = 20 μm. (B) Statistics of the proportions of GRP78 highlighted hair cells in (A) . (C) Immunostaining with myosin 7a and CHOP in middle turns in cochlear explants. Scale bars = 20 μm. (D) Statistics of the proportions of CHOP highlighted hair cells in (C) . **** p < 0.0001.

Article Snippet: After blocking with QuickBlockTM buffer (Beyotime, P0260) for 1 h, and the primary antibodies against myosin 7a (Abcam, ab150386, 1:200 dilution), cleaved caspase-3 (Abcam, ab32042, 1:150 dilution), GRP78 (Proteintech, 11587-1-AP, 1:200 dilution), and CHOP (Proteintech, 15204-1-AP, 1:200 dilution) were added to the samples at 4°C overnight.

Techniques: Immunostaining

Journal: Frontiers in Molecular Neuroscience

Article Title: Pitavastatin protects against neomycin-induced ototoxicity through inhibition of endoplasmic reticulum stress

doi: 10.3389/fnmol.2022.963083

Figure Lengend Snippet: PTV alleviates neomycin-induced ER stress by inhibiting PERK/eIF2α/ATF4 signaling. (A,C) Immunofluorescence of HEI-OC1 cells with anti-GRP78 and anti-CHOP antibodies. Scale bars = 20 μm. (B,D) Statistics of the mean intensity of GRP78 and CHOP in HEI-OC1 cells in (A,C) . (E) Western blot analysis of GRP78, CHOP, p-PERK, PERK, p-eIF2α, eIF2α, ATF4, ATF6, IRE1α, and GAPDH in HEI-OC1 cells with PTV pretreatment followed by neomycin exposure. (F–L) Quantification of the protein expression in (E) with ImageJ ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: After blocking with QuickBlockTM buffer (Beyotime, P0260) for 1 h, and the primary antibodies against myosin 7a (Abcam, ab150386, 1:200 dilution), cleaved caspase-3 (Abcam, ab32042, 1:150 dilution), GRP78 (Proteintech, 11587-1-AP, 1:200 dilution), and CHOP (Proteintech, 15204-1-AP, 1:200 dilution) were added to the samples at 4°C overnight.

Techniques: Immunofluorescence, Western Blot, Expressing