Journal: bioRxiv

Article Title: A novel pan class-I glucose transporter inhibitor DRB18 exhibits synergistic effects in vitro and in vivo with paclitaxel against human non-small cell lung cancer

doi: 10.1101/2024.12.15.628558

Figure Lengend Snippet: 2 μM of DRB18 combined with or without other drugs to treat A549 cells for 48 hours. The inhibition of cell proliferation was compared to single compound treatment to identify anti-proliferation synergism. 4A. DRB18 combined with 10 µM V9302 showed more inhibition against A549 cells. 4B . DRB18 combined with 100 nM or 300 nM of CB839 showed more cytotoxicity against A549. 4C . DRB18 combined with 5 µM or 10 µM of sunitinib (Sutent) showed more inhibition against A549 cells. 4D . DRB18 combined with 10 µM V9302 showed more inhibition against Panc1 cells. 4E . DRB18 combined with 100 nM or 300 nM of CB839 showed more inhibition against Panc1 cells. 4F . DRB18 combined with 5 µM or 10 µM of sunitinib showed more inhibition against Panc1 cells. 4G . 5 µM of DRB18 combined with 0.75 µM of brigatinib showed more cytotoxicity against A549 cells. 4H . 5 µM of DRB18 combined with 15 µM of trametinib showed more inhibition against A549 cells.

Article Snippet: V9302 (HY-112683) and CB839 (HY-12248) were purchased from MedChemExpress.

Techniques: Inhibition

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: High LAMC2 associated with EGFR signaling activation and LAMC2 promoted EGFR translation. A) GSEA analysis with significantly altered genes ( p < 0.01) between LAMC2 ‐high and LAMC2 ‐low iCCA patients. The top 20 enriched signatures were listed. B) The enrichment of LAMC2 ‐high patients in EGFR signaling activation and non‐activation groups subclassified by an EGF/EGFR signaling gene set. C) Western blot analysis in RBE and HUCCT1 cells transfected with control siRNA, si LAMC2 #1, or si LAMC2 #2 and treated with EGF. D) Western blot analysis in RBE and HUCCT1 cells transfected with control siRNA as well as si LAMC2 #1 and #2. E) Western blot analysis of RBE and HUCCT1 cells transfected with Ctrl vector or LAMC2 ‐HA, along with EGFR‐flag. F) iCCA tumor formation in AKT/YapS127A‐induced iCCA mouse model with or without silencing LAMC2 by shRNAs, upon with or without LAMC2 /EGFR L858R overexpression. Student's t ‐test was used. G) The flow chart of Boncat Assay with L‐AHA to detect the newly synthesized proteins. H) Boncat assay in RBE and HUCCT1 cells with LAMC2 overexpression. I) Boncat assay in RBE and HUCCT1 cells with LAMC2 silencing.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Activation Assay, Western Blot, Transfection, Control, Plasmid Preparation, Over Expression, Synthesized

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 interacted with unglycosylated EGFR. A) Cells were co‐transfected with indicated vectors and IP was performed with anti‐HA beads. B) IP assay with anti‐ LAMC2 antibody in iCCA cells. C) N‐linked glycosylation sites of EGFR (up panel) and the flow chart of the N‐glycosylation process (bottom panel). The related N‐glycosylation inhibitors and N‐glycan removing enzyme were indicated in red color. D) IP assay with anti‐HA beads in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag. Both cell lysates and IP products were treated with or without PNGase F for 1 h before analysis. E) Anti‐HA IP in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag and treated with or without tunicamycin (0.5 µg mL −1 ) for 24 h. F) Anti‐HA IP in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag and treated with or without NGI‐1 (10 µ m ) for 24 h. G) Anti‐ LAMC2 IP in RBE and HUCCT1 cells treated with tunicamycin (0.5 µg mL −1 ) or NGI‐1 (10 µ m ) for 24 h. H) Confocal microscopy images of endogenous LAMC2 , EGFR, and ER marker Calreticulin and their co‐localization in iCCA cells. I) Construction of EGFR 13Q ‐flag and anti‐HA IP assay in cells co‐transfected with LAMC2 ‐HA and EGFR 13Q ‐flag. J) Boncat assay in cells co‐transfected with LAMC2 ‐HA and EGFR 13Q ‐flag. SP, signal peptide; TM, transmembrane; N, Asparagine; Q, Glutamine.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Transfection, Glycoproteomics, Confocal Microscopy, Marker

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 N‐terminus interacted with the extracellular domain of EGFR, promoting EGFR translation. A) Mapping LAMC2 regions involved in EGFR binding via IP in cells co‐transfected with EGFR‐flag and LAMC2 ‐HA deletion mutants. B) Mapping LAMC2 N‐terminus regions involved in EGFR binding via IP in cells co‐transfected with EGFR‐flag and LAMC2 ‐HA deletion mutants. C) Mapping EGFR regions involved in LAMC2 binding via IP in cells co‐transfected with LAMC2 ‐HA and different EGFR‐flag vectors. D) Boncat assay in cells co‐transfected with LAMC2 ‐HA deletion mutants and EGFR‐flag. E) Boncat assay in cells co‐transfected with LAMC2 ‐HA deletion mutants and EGFR 13Q ‐flag. F) iCCA tumor formation in AKT/YapS127A‐induced iCCA mouse model with or without silencing LAMC2 by shRNAs, upon with or without C‐ LAMC2 /N‐ LAMC2 overexpression. Student's t ‐test was used.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Binding Assay, Transfection, Over Expression

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 promoting EGFR translation was partially dependent on BiP. A) The flow chart of tandem IP followed by MS, and the top 4 candidates were listed based on the quantity of unique peptides. B) Anti‐HA IP in cells co‐transfected with LAMC2 ‐HA and BiP‐flag. C) Anti‐flag IP in cells co‐transfected with LAMC2 ‐HA and BiP‐flag. D) EGFR‐flag expression in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag with or without silencing BiP. E) EGFR 13Q ‐flag expression in cells co‐transfected with LAMC2 ‐HA and EGFR 13Q ‐flag with or without silencing BiP. F) Boncat assay in cells co‐transfected with LAMC2 ‐HA and EGFR‐flag with or without silencing BiP. G) Boncat assay in cells co‐transfected with LAMC2 ‐HA and EGFR 13Q ‐flag with or without silencing BiP. H) Boncat assay in cells transfected with BiP‐flag and EGFR‐flag (left panel) or EGFR 13Q ‐flag (right panel). I) Boncat assay in RBE and HUCCT1 cells upon BiP silencing. J,K) Boncat assay in cells co‐transfected with BiP‐HA and EGFR‐flag (J) or EGFR 13Q ‐flag (K) with or without silencing LAMC2 .

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Transfection, Expressing

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 , EGFR, and BiP interacted with each other, contributing to EGFR translation. A) Anti‐flag IP in cells co‐transfected with BiP‐flag and EGFR‐HA with or without tunicamycin treatment. B) Anti‐HA IP in cells co‐transfected with BiP‐HA and EGFR 13Q ‐flag. C) Schematic diagram of BiP functional domain and a group of BiP truncations. D) Mapping BiP regions involved in LAMC2 binding via IP in cells co‐transfected with LAMC2 ‐HA and different BiP‐flag vectors. E) Mapping BiP regions involved in EGFR binding via IPs in cells co‐transfected EGFR‐HA and different BiP‐flag vectors with tunicamycin treatment. F) Anti‐HA IP in cells co‐transfected with BiP‐flag and different LAMC2 vectors. G) An illustrated interacting model of LAMC2 , BiP, and nascent EGFR in promoting EGFR translation in ER. H) Boncat assay in cells co‐transfected with BiP‐flag/EGFR‐flag and an intact LAMC2 , or N‐ LAMC2 . I) Boncat assay in cells co‐transfected with LAMC2 ‐HA /EGFR‐flag and an intact BiP, or BiP ΔSBD and BiP Δ501‐650 . SP, signal peptide; NBD, nucleotide‐binding domain; SBD, substrate binding domain.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Transfection, Functional Assay, Binding Assay

Journal: Advanced Science

Article Title: Oncogenic Roles of Laminin Subunit Gamma‐2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation

doi: 10.1002/advs.202309010

Figure Lengend Snippet: LAMC2 ‐high iCCA tumors had poor prognosis, but were sensitive to EGFR TKIs treatment. A) Kaplan–Meier survival analysis of iCCA patients from Cohort 4 based on LAMC2 IHC staining score and LAMC2 copy number. B) Kaplan–Meier survival analysis of iCCA patients from Cohort 5 based on LAMC2 protein level (the median cut‐off). C) EGFR IHC staining and the spearman correlation of EGFR and LAMC2 in Cohort 4. Student's t ‐test was used for group comparison. D,E) Cell viability of iCCA cells with different LAMC2 levels under treatment of EGFR TKI Gefitinib (C) or EGFR neutralizing antibody Cetuximab (D). Two‐way ANOVA analysis was used. F) Representative images and quantitative analysis of orthoptic iCCA tumor formation in AKT/YapS127A‐induced iCCA mouse model with or without LAMC2 , upon with or without Gefitinib treatment. Student's t ‐test was used. NS, not significant. G) The schematic model summarized LAMC2 as a key oncogenic event in iCCA.

Article Snippet: Cells were also treated with 20 μg mL −1 CHX and 100 n m bafilomycin A1 (Cat# HY‐100558, MedChemExpress) for 12 h to measure EGFR degradation, as well as with 20 μg mL −1 CHX and 2 μ m CB‐5083 (Cat# HY‐12861, MedChemExpress) to examine the degradation of unglycosylated EGFR.

Techniques: Immunohistochemistry, Comparison

Journal: The FASEB Journal

Article Title: Inhibition of FAAH suppresses RANKL ‐induced osteoclastogenesis and attenuates ovariectomy‐induced bone loss partially through repressing the IL17 pathway

doi: 10.1096/fj.202200911R

Figure Lengend Snippet: PF inhibits osteoclast differentiation – CB1 or CB2 independence. (A–C) BMMs were infected with the lentivirus carrying FAAH‐specific shRNA or the control lentivirus and then treated with RANKL for 3 days, and the mRNA and protein expression of CB1 and CB2 were detected by qPCR and Western blot respectively. (D–F) BMMs were treated with indicated concentrations of PF (0, 0.5, 1, and 2.5 μM) with or without stimulation by 75 ng/ml RANKL for 3 days, and the mRNA and protein expression of CB1 or CB2 were determined using qPCR and Western blot respectively. # p < .05, ## p < .01, compared with the control groups; ns, non‐significant, compared with the RANKL groups. (G and H) BMMs were treated with PF (2.5 μM), AM251 (0.1 μM), and AM630 (0.1 μM) as indicated in the presence of 75 ng/ml RANKL. After 5–7 d, trap staining was conducted, and multinucleated (>3 nuclei) cells were quantified (scale bar = 400 μm). (I–K) BMMs were treated with PF (2.5 μM), AM251 (0.1 μM), and AM630 (0.1 μM) as indicated in the presence of 75 ng/ml RANKL for 3 days, and the mRNA and protein expression of osteoclast‐specific genes were determined using qPCR and Western blot respectively. # p < .05, ## p < .01, compared with the RANKL groups; ns, non‐significant, compared with the RANKL + PF groups. All data are presented as the mean ± SD. N = 3 per group.

Article Snippet: Primary antibodies targeting FAAH, CB1, and CB2 were purchased from Abclonal (Wuhan, Hubei, China).

Techniques: Infection, shRNA, Control, Expressing, Western Blot, Staining