Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 6. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against the HuCCT-1 cell line. A): peripheral NK cell degranulation, evaluated as frequency of CD107a+ NK cells, in iCCA patients (n = 13) and HC (n = 16) in the presence of 7C6 mAb or IgG1-Fc. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56+ CD107a+ NK cells in a HC and a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): the proportion of circulating IFNγ+ NK cells in patients (n = 10) and HC (n = 11) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, the parametric t test and the non-parametric Wilcoxon t test were used. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 7. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against patient-derived iCCA cell lines. A): peripheral NK cell degranulation, evaluated as CD107a+NK frequency, in iCCA patients (n = 12) and HC (n = 8) in the presence of 7C6 mAb or IgG1-Fc using patient- derived primary tumor cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56 + CD107a+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): proportion of circulating IFNγ+NK cells in patients (n = 10) and in HC (n = 8) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, we used the parametric t test and the non-parametric Wilcoxon t test. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ +NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay, MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 8. 7C6 mAb enhances the anti-tumor effect of liver- and tumor-infiltrating NK cells in iCCA patients. A): Frequency of degranulating CD107a+NK cells in LIL (n = 13) and TIL (n = 10) of iCCA patients in the presence of anti-MICA/B 7C6 mAb or IgG1-Fc using autologous tumor-derived cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): representative dot plots showing the frequency of CD3-CD56+ CD107a+ LIL- and TIL-NK cells in the presence of 7C6 mAb or IgG1-Fc. C): proportion of IFNγ+ NK cells in LIL (n = 10) and TIL (n = 8) of iCCA patients in the presence of 7C6 mAb compared with IgG1-Fc using autologous tumor-derived cell lines as targets. The parametric t test and non-parametric Wilcoxon t test were used to compare paired data. The parametric t test was used to compare unpaired data.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 9. Cytotoxicity assay of HC PBMC, patient PBMC, LIL and TIL cells. A, B): Frequency of CFSE+LIVE/DEAD (LD)+ HuCCT-1 cell line targets when HC PBMC (n = 5), patient PBMC (n = 10), LIL (n = 8) and TIL (n = 5) were used as effector cells in the presence of 7C6 mAb and isotype control (IgG1). The parametric paired t tests were used to compare data. C, D): Frequency of CFSE+LD+ patient-derived cell line targets when HC PBMC (n = 5), patient PBMC (n = 8), LIL (n = 8) and TIL (n = 4) were used as effectors in the presence of 7C6 and isotype control. The parametric paired t test was used to compare data in panel C. The non-parametric Wilcoxon t test was used to compare paired data in panel D. Target cell death was determined as frequency of CFSE+LD+ cells.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Cytotoxicity Assay, Control, Derivative Assay

Journal: Advanced Science

Article Title: GLS1‐RNA Polymerase II Axis Mediates Glutamine‐Dependent Hepatoprotective Effects on Alcoholic Liver Disease in High‐Protein Diets

doi: 10.1002/advs.202502738

Figure Lengend Snippet: GLS1 interacts with POLR2E or POLR2H. A) Schematic of immunoprecipitation using mass spectrometry (IP–MS) to identify proteins associated with GLS1 in LO2 cells. B) CoIP analysis of the interaction of endogenous GLS1 with endogenous POLR2E or POLR2H in the primary hepatocytes. β‐actin served as the loading control. C–F) CoIP analysis of the interaction of GAC–Myc and POLR2H‐HA, KGA‐Flag and POLR2H‐HA, GAC–Myc and POLR2E‐HA and KGA‐Flag and POLR2E‐HA in HEK293T cells. β‐actin served as the loading control. G) CoIP analysis of the interaction of endogenous GLS1 with endogenous POLR2E or POLR2H in the primary hepatocytes treated with ethanol (600 m m ) for 24 h. β‐actin served as the loading control. H,I) CoIP analysis of the interaction of GAC–Myc or KGA‐Flag with endogenous POLR2H, GAC–Myc or KGA‐Flag with endogenous POLR2E treated with ethanol (400 m m ) for 24 h in HEK293T cells. β‐actin served as the loading control.

Article Snippet: For analysis of endogenous proteins, the primary hepatocytes lysed by cell lysis IP buffer were incubated overnight at 4 °C with GLS1 (12855‐1; Proteintech) or POLR2E (sc‐390902; santa cruz) or POLR2H (sc‐398512; santa cruz) antibody and protein A+G Agarose beads.

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Control

Journal: Advanced Science

Article Title: GLS1‐RNA Polymerase II Axis Mediates Glutamine‐Dependent Hepatoprotective Effects on Alcoholic Liver Disease in High‐Protein Diets

doi: 10.1002/advs.202502738

Figure Lengend Snippet: GLS1 interacts with POLR2H or POLR2E to reduce the activity of RNA pol II. A,B) The protein complex structure of GLS1, POLR2H and POLR2E performed on the GROMACS platform. The corresponding location of the putative binding motif on GLS1 and POLR2H protein, GLS1 and POLR2E protein. (C) The truncated GLS1 variants. D,E) CoIP analysis of the interaction of truncated GLS1 proteins and POLR2E‐HA or POLR2H‐HA in lysates of HEK293T cells. β‐actin served as the loading control. F) RNA pol II activity of AML12 cells expressing pGL3‐Basic or pGL3‐Promoter with KGA WT or ΔKGA‐3. Cells were serum starved overnight followed by ethanol (400 m m ) stimulation for 1 h. n = 10. G,H) The truncated POLR2E or POLR2H variants. I,J) CoIP analysis of the interaction of truncated POLR2E or POLR2H proteins and KGA‐Flag in lysates of HEK293T cells. β‐actin served as the loading control. K) RNA pol II activity of AML12 cells expressing pGL3‐Basic or pGL3‐Promoter with GLS1 and POLR2E WT or ΔPOLR2E‐3 or POLR2H WT or ΔPOLR2H‐1. Cells were serum starved overnight followed by ethanol (400 m m ) stimulation for 1 h. n = 7–10. Data in (F) and (K) are presented as the mean ± SEM, determined by one‐way ANOVA and Fisher's LSD test.

Article Snippet: For analysis of endogenous proteins, the primary hepatocytes lysed by cell lysis IP buffer were incubated overnight at 4 °C with GLS1 (12855‐1; Proteintech) or POLR2E (sc‐390902; santa cruz) or POLR2H (sc‐398512; santa cruz) antibody and protein A+G Agarose beads.

Techniques: Activity Assay, Binding Assay, Control, Expressing

Journal: Advanced Science

Article Title: GLS1‐RNA Polymerase II Axis Mediates Glutamine‐Dependent Hepatoprotective Effects on Alcoholic Liver Disease in High‐Protein Diets

doi: 10.1002/advs.202502738

Figure Lengend Snippet: GLS1 reduces hepatic alcoholic steatosis through interacting with POLR2E or POLR2H. A) TG levels of the primary hepatocytes expressing with KGA WT or ΔKGA‐3. Cells were serum starved overnight followed by ethanol (600 m m ) stimulation for 24 h. The amount was normalized to the protein content. n = 5‐6. B) Western blots of ACLY, ACC, FASN levels in the primary hepatocytes described in (A); β‐actin serves as a loading control. C) TG levels of the primary hepatocytes expressing with GLS1 and POLR2E WT or ΔPOLR2E‐3 or POLR2H WT or ΔPOLR2H‐1. Cells were serum starved overnight followed by ethanol (600 m m ) stimulation for 24 h. The amount was normalized to the protein content. n = 4–6. D) Western blots of ACLY, ACC, FASN levels in the primary hepatocytes described in (C); β‐actin serves as a loading control. E) Schematic illustrating the groups and procedures of the mouse model. C57BL/6J mice were injected with AAV8‐TBG‐GLS1 with POLR2E WT or POLR2H WT or their truncated variants (ΔPOLR2E‐3 or ΔPOLR2H‐1) via the tail vein for additional 4‐week pair or ethanol feeding. n = 8–10 biologically independent mice per group. F) Change curves of body weight of mice. G) Liver TG levels of mice in the indicated group in (E); n = 6‐7. H) Representative H&E staining of liver sections in the indicated group in (E). Scale bars, 100 and 200 µm. I) Western blots of ACLY, ACC, FASN levels in the primary hepatocytes described in (E); β‐actin serves as a loading control. Data in (A), (C), (G) are presented as the mean ± SEM, determined by one‐way ANOVA and Fisher's LSD test.

Article Snippet: For analysis of endogenous proteins, the primary hepatocytes lysed by cell lysis IP buffer were incubated overnight at 4 °C with GLS1 (12855‐1; Proteintech) or POLR2E (sc‐390902; santa cruz) or POLR2H (sc‐398512; santa cruz) antibody and protein A+G Agarose beads.

Techniques: Expressing, Western Blot, Control, Injection, Staining

Journal: Advanced Science

Article Title: GLS1‐RNA Polymerase II Axis Mediates Glutamine‐Dependent Hepatoprotective Effects on Alcoholic Liver Disease in High‐Protein Diets

doi: 10.1002/advs.202502738

Figure Lengend Snippet: Schematic diagram of glutamine‐regulated GLS1 coordinates RNA polymerase II manipulates AFLD. High‐protein diet reduces alcoholic hepatic steatosis through glutamine stabilizing GLS1 to regulate RNA pol II activity by interacting with POLR2E or POLR2H.

Article Snippet: For analysis of endogenous proteins, the primary hepatocytes lysed by cell lysis IP buffer were incubated overnight at 4 °C with GLS1 (12855‐1; Proteintech) or POLR2E (sc‐390902; santa cruz) or POLR2H (sc‐398512; santa cruz) antibody and protein A+G Agarose beads.

Techniques: Activity Assay

Journal: EMBO Molecular Medicine

Article Title: Identification of the HSPB4/TLR2/NF-κB axis in macrophage as a therapeutic target for sterile inflammation of the cornea

doi: 10.1002/emmm.201200221

Figure Lengend Snippet: A,B. Dose-dependent suppression by TSG-6 of macrophages activated by HSPB4. n = 6 per group. p values, versus PBS treatment. Error bars represent means + s.e.m. C. TSG-6 had no effect on NF-κB signalling in HEK-TLR2 cells that did not express CD44. n = 8 per group. Error bars represent means + s.e.m. D. Inhibition by TSG-6 of NF-κB signalling in HEK-TLR2 cells expressing CD44. n = 8 per group. Error bars represent means + s.e.m. E. TSG-6 did not suppress the inflammatory response at 24 h after injury in transgenic mice with null alleles for CD44 ( CD44 −/− ), whereas TSG-6 significantly inhibited inflammation in the cornea of wild-type mice expressing CD44 (C57B/6). n = 10 per group. Error bars represent means + s.e.m.

Article Snippet: The CD44 coding sequence (Origene, Rockville, MD) was inserted into the plasmid pcDNA 3.1 (Invitrogen, Carlsbad, CA) using Not1/Xba1 sites and the plasmid (pcDNA 3.1-CD44) transformed into E. coli (Subcloning EfficiencyTM DH5aTM Competent Cells; Invitrogen) for cloning.

Techniques: Inhibition, Expressing, Transgenic Assay

Journal: EMBO Molecular Medicine

Article Title: Identification of the HSPB4/TLR2/NF-κB axis in macrophage as a therapeutic target for sterile inflammation of the cornea

doi: 10.1002/emmm.201200221

Figure Lengend Snippet: Immediately after injury, SN is released from nerve endings in the cornea to recruit circulating neutrophils and induce an initial inflammatory response ( Phase I ). Then necrotic or injured keratocytes release HSPB4 in response to injury and oxidative stress. The HSPB4 activates resident macrophages in the cornea via TLR2/NF-κB signalling pathway to produce pro-inflammatory cytokines including IL-1 and IL-6. The pro-inflammatory signals released by resident macrophages are amplified by keratocytes that produce chemokines to further recruit large amount of neutrophils ( Phase II ). TSG-6 inhibits the initial activation of resident macrophages by modulating TLR2/CD44/NF-κB signalling and thereby decreases the Phase II inflammatory response.

Article Snippet: The CD44 coding sequence (Origene, Rockville, MD) was inserted into the plasmid pcDNA 3.1 (Invitrogen, Carlsbad, CA) using Not1/Xba1 sites and the plasmid (pcDNA 3.1-CD44) transformed into E. coli (Subcloning EfficiencyTM DH5aTM Competent Cells; Invitrogen) for cloning.

Techniques: Amplification, Activation Assay

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: ( A ) Schematic of mass spectrometry workflow. TN1 patient-derived xenograft (PDX) tumor was dissociated and sorted based on CD44 and CD24 expression and analyzed by mass spec. Four hundred and ninety-seven differential proteins were identified. TN1 PDX tumor was dissociated and transfected with si CD44 and si Con and analyzed by mass spec. Three hundred and forty-four differential proteins were identified. Thirty-eight differential proteins overlapped in both screens, one of which was CD81. ( B, C ) Immunoblots and quantification of CD44 and CD81 in dissociated PDX (WT and CD44KO) tumor cells in suspension and MDA-MB-231 cells, in suspension and adherent (WT, CD44KO, CD81KO, and dKO). N = 3. Error bars represent standard deviation. *p < 0.05, **p = 0.018, ***p = 0.0026, ****p = <0.0008. ( D ) Mammosphere formation and immunoblot of mouse 4T1 cells (WT and Cd81 KO via CRISPR/Cas9). Cells were seeded 2000 cells/well in 6-well plate in four replicate.Two-tailed Student T -test was used. Repeated twice. Representative images ( E ) and quantifications ( F ) of mammospheres derived from 4T1 cells in suspension, including WT cells and Cd81KO cells. 2000 cells were seeded in 6-cm plates in mammosphere media, and the images were captured on day 4. N = 3. Error bars represent standard deviation. Two-tailed Student T -test was used to calculate p values. Figure 1—figure supplement 1—source data 1. Uncropped blots associated with . Figure 1—figure supplement 1—source data 2. Uncropped blots associated with . Figure 1—figure supplement 1—source data 3. Uncropped blots associated with .

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Mass Spectrometry, Derivative Assay, Expressing, Transfection, Western Blot, Suspension, Standard Deviation, CRISPR, Two Tailed Test

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: Representative images ( A ) and bar graphs ( B ) of the mammospheres of MDA-MB-231 cell groups (WT, CD44KO, CD81KO, dKO pool populations), 17 days after seeded at 2000 cells per well (6-well plate) in serum-free mammosphere formation media. N = 4 replicates. Error bars represent standard deviation. Repeated three times. Scale bar = 100 µm. One-tailed Student T -test *p < 0.05. Bar graphs ( C ) and representative images ( D ) of membrane and intracellular CD44 and CD81 localization in WT, CD44KO, and CD81KO MDA-MB-231 cells (adherent and in suspension), detected by immunofluorescence staining with anti-CD44-Texas Red, anti-CD81-Alexa488, and DAPI (4’, 6-diamidino-2-phenylindole) for DNA staining. Sample size N = 20 (WT), 14 (CD44KO), and 20 (CD81KO) adherent cells and N = 31 (WT), 31 (CD44KO), and 28 CD81KO cells in suspension. Error bars represent standard deviation. Scale bar = 5 µm. Two-tailed Student T -test p = 0.03 for intracellular CD44 levels between adherent and suspension cells (both WT and CD81KO cells). Compared to WT cells, T -test p values for adherent CD44KO/CD81KO cells are 9.52e−06/0.22 (membrane CD44), 0.81/0.65 (intracellular CD44), 0.009/0.006 (membrane CD81), 0.15/0.002 (intracellular CD81); and p values for cells in suspension are 1.21e−11/0.003 (membrane CD44), 0.002/0.72 (intracellular CD44), 3.08e−06/2.64e−07 (membrane CD81), and 0.04/0.004 (intracellular CD81). Significant pair comparison differences are marked in the graph. ANOVA analyses among three groups with p values for adherent and in-suspension cells, respectively: 0.0004 and 7.357E−08 (membrane CD44), 0.9119 and 0.0024 (intracellular CD44), 0.0022 and 1.079E−09 (membrane CD81), and 0.0255 and 0.0103 (intracellular CD81). ( E ) Predictive modeling of CD44 and CD81 interaction with hot spots shown in red and yellow. Top-ranked structural models of predictive interactions between CD44/CD81 (estimated binding energy: −12.23 kcal/mol) and between CD44/CD81d (deletion mutant) (estimated binding energy: −9.41 Kcal/mol). ( F ) Immunoblots of endogenous CD44 and CD81 immunoprecipitated by anti-CD44 from the lysates (membrane fraction) of WT/44KO MDA-MB-231 and (total) WT TN1 patient-derived xenograft (PDX) cells. ( G ) Predictive modeling of CD44 and CD81d (deletion mutant) interaction with hot spots shown in red and yellow. Top-ranked structural models of predictive interactions between CD44/CD81d (deletion mutant) (estimated binding energy: −9.41 kcal/mol). ( H ) Representative immunoblots of CD81-HA (CD81d) immunoprecipitated by anti-CD44-Flag (CD44 mutants CD44.1 and CD44.2) from the lysates of HEK293ft cells ( N = 3 biological replicates; Con, control). Cells were transfected with either Flag-CD44 or mutants (Flag-CD44.1, Flag-CD44.2) and Con HA, HA-CD81, or HA-CD81d with deletion at amino acids 159–187 (CD81d). Forty-eight hours after transfection the cells were harvested for Flag IP. Figure 1—source data 1. Uncropped blots associated with . Figure 1—source data 2. Uncropped blots associated with .

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Standard Deviation, One-tailed Test, Membrane, Suspension, Immunofluorescence, Staining, Two Tailed Test, Comparison, Binding Assay, Mutagenesis, Western Blot, Immunoprecipitation, Derivative Assay, Control, Transfection

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: ( A, B ) IncuCyte images and curve analyses of cell confluence of human MDA-MB-231 WT, CD44KO, and CD81KO cells ( C ) and mouse 4T1 WT and Cd81KO cells ( D ) over time. Scale bar is 300 µm. N = 3. Error bars represent standard deviation. Two-tailed Student T -test ******p = 1.97E−08. ( C ) Immunoblot analysis of OCT4, Notch1, STAT3, and pSTAT3 expression in WT, CD81KO, and CD44KO MDA-MB-231 cells (pooled KO cells). ( D ) % of CD44 and CD81 colocalization shown in 50% of adherent (14 out of 26 cells) and 70% of suspension (22 out of 31 cells) MDA-MB-231 cells (WT). Pearson coefficient r = 0.57 (average from three experimental replicates r 1 = 0.52, r 2 = 0.54, r 3 = 0.64). ( E ) Immunoblots of exogenously expressed CD44-Flag and CD81-HA immunoprecipitated by anti-Flag (CD44) from the lysates of transfected HEK293ft cells. Repeated three times. ( F ) Immunofluorescence of HA tagged CD81d in HEK293ft cells showing its membrane localization. ( G ) Alignment of CD44 and CD81 amino acids showing deletion in CD81 and point mutations in CD44 domains I and II. ( H ) Representative images and bar graphs of the mammospheres of MDA-MB-231 cell groups (WT HACon, CD81KO HACon, CD81KO HACD81, CD81KO HACD81m), 3 days after seeded at 1000 cells per well (12-well plate) in serum-free mammosphere formation media. N = 4 replicates. Repeated two times. Error bars represent standard deviation. Scale bar = 100 µm. Two-tailed Student T -test *******p = 0.00000001, ****p = 0.0004, **p = 0.005. ( I ) Representative images and bar graphs of the mammospheres of MDA-MB-231 cell groups (WT, CD81KO, CD81KO GFPCD81) overexpression (OE), CD81KO GFPCon, 3 days after seeded at 1000 cells per well (12-well plate) in serum-free mammosphere formation media. N = 4 replicates. Repeated two times. Error bars represent standard deviation. Scale bar = 100 µm. Two-tailed Student T -test *******p = 0.00000005, ******p = 0.0000009, ****p = 0.00004, ***p < 0.004, **p = 0.005. Figure 1—figure supplement 2—source data 1. Uncropped blots associated with . Figure 1—figure supplement 2—source data 2. Uncropped blots associated with .

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Standard Deviation, Two Tailed Test, Western Blot, Expressing, Suspension, Immunoprecipitation, Transfection, Immunofluorescence, Membrane, Over Expression

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: ( A ) The Volcano plots of different abundance in global proteome and phosphoproteome between adherent and clustered tumor cells. ( B ) Immunoblots and bar graphs of CD44 and CD81 in MDA-MB-231 cells after transient knockdowns after si CD81 and si CD44 transfections. ( C ) The protein–protein interactions network of altered phosphoproteome in the endocytosis pathway across four phosphoproteome clusters P2, P3, P5, and P6. The enrichment of KEGG pathway ( D ) and Kinase Enrichment Analysis (KEA) ( E ) from expressed phosphopeptides in the different clusters P1–P6 comparing siControl with si CD81 and si CD44 groups. Figure 2—figure supplement 2—source data 1. Uncropped blots associated with .

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Western Blot, Transfection, Protein-Protein interactions

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: Global mass spectrometry heatmap ( A ) and KEGG pathway analysis ( B ) of altered top pathways with significantly expressed proteome within six different clusters (G1–G6) in siControl, si CD81 , and si CD44 cells at 3 hr in suspension ( N = 3 replicates, ANOVA T -test false discovery rate (FDR) <0.01, p = <0.05). Phosphoproteomic mass spectrometry heatmap ( C ) and KEGG pathway analysis ( D ) of altered top pathways with significantly changed phosphoproteome within six different clusters (P1–P6) in si Control , si CD81 , and si CD44 cells at 3 hr in suspension ( N = 3 replicates, ANOVA T -test FDR <0.01, p = <0.05).

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Mass Spectrometry, Suspension, Control

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: The MaxQuant kinase reactome networks based on the downregulated ( A ) and upregulated ( B ) phosphorylation sites in both si CD81 - and si CD44 -transfected cells and known curation of kinase–substrate interactions in the literature. ( C ) Immunoblot validation of mass spectrometry analysis targets including Rab11FIP1, Rab7a, Rab8a, and Rab10. N = 3. Error bars represent standard deviation. *p = <0.05. Figure 2—figure supplement 3—source data 1. Uncropped blots associated with .

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Phospho-proteomics, Transfection, Western Blot, Biomarker Discovery, Mass Spectrometry, Standard Deviation

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: ( A ) GO Localization analysis of downregulated proteins in pooled CD44KO cells compared to WT MDA-MB-231 cells ( N = 3 replicates, p < 0.05). ( B ) GO Processes analyses of global mass spectrometry-based upregulated proteins in the CD44KO cells in comparison to CD44 WT MDA-MB-231 cells. ( C ) Transmission electron microscopy image and quantification of lysosomes (red arrows) in MB-MDA-231 cells. Scale bar = 500 nm. N = 4. Error bars represent standard deviation. Student two-tailed T -test *p = 0.02, ***p = 5e−04, ****p = 2e−09. The purple arrow points to a multivesicular body (MVB) which is not significant different among three types of cells (1 or 0 observed per cell). ( D ) Left panel: nanoparticle tracking analysis (NTA) counts of EVs per cell in purified EVs (100,000 × g x 70 min) from WT, 44KO, and 81KO cells, measured by NanoSight ( N = 3). Error bars represent standard deviation. Two-tailed Student T -test ****p = 0.0001, ***p = 0.0003, **p = 0.006. Right panel: NTA-based size distributions (repeated three times) of the three types of EVs. ( E ) Schematic of EV isolation by ultracentrifugation steps and characterization.

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Mass Spectrometry, Comparison, Transmission Assay, Electron Microscopy, Standard Deviation, Two Tailed Test, Purification, Isolation

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: ( A ) Immunoblot analyses for OCT 4, STAT3, phosphoSTAT3 (pSTAT3), FAK, pFAK, CD44, and β-actin using CD81KO MDA-MB-231 cells educated with phosphate-buffered saline (PBS) or extracellular vesicles (EVs) derived from WT, CD81KO, and CD44KO, and CD81KO cells. ( B, C ) Representative images ( B ) and quantifications of mammospheres derived from 4T1 cells in suspension, including WT cells and Cd81KO cells, the latter of which were educated with PBS or exosomes (10 µg for 1 week). 2000 cells were seeded in 6-cm plates in mammosphere media, and the images were captured on day 4. N = 4. Error bars represent standard deviation. Two-tailed Student T -test was used *p<0.05, **p<0.005, ***p<0.001. Figure 3—figure supplement 3—source data 1. Uncropped blots associated with .

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Western Blot, Saline, Derivative Assay, Suspension, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: Kaplan–Meier plots of CD81 protein expression in patients with triple negative breast cancer (TNBC) correlate with an unfavorable overall survival ( A ), relapse-free survival ( B ), and distant-metastasis-free survival ( C ). Representative images ( D ) and quantified % ( E ) of CD81 + and CD81 − CTC events in the blood of six patients with metastatic breast cancer, analyzed on CellSearch. Scale bar = 5 µm. N = 3. Error bars represent standard deviation. Two-tailed Student T -test *****p = 9E−11. Representative images of flow cytometry gated singlets and clusters ( F , scale bar = 25 µm) and bar graphs of proportion of CD81 + ( G ) and CD44 + CD81 + ( H ) in putative Lin-CD45- CTC events (622,509) in the blood ( N = 50 patients). N = 50. Error bars represent standard deviation. Two-tailed Student T -test ***p = 0.005, ****p = 0.00001. IncuCyte images (top panels) and quantified tumor cell aggregation curves of TN1 patient-derived xenograft (PDX) ( I ), MDA-MB-231 ( J ) cells upon CD81 KD (repeated at least three times). Scale bar = 300 µm. N = 5. Error bars represent standard deviation. Two-tailed Student T -test *****p = 1E−12, ******p = 8E−19.

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Expressing, Standard Deviation, Two Tailed Test, Flow Cytometry, Derivative Assay

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: ( A ) Table of tumor TMA breast cancer patient characteristics. ( B ) Immunohistochemical staining of CD81 and CD44 expression (in brown) and hemotoxylin (in blue) in TMA of breast tumors ( N = 77 with CD81 IHC and N = 75 with CD44 IHC). ( C ) A scatter plot of CD44 and CD81 expression on tumor microarray. CD81 and CD44 expression levels were quantified using ImageJ and assessed in all subtypes of breast tumors ( N = 77, N = 75, patient tumors, respectively). ( D ) Percentage (%) of tumor regions with overlapped CD81 and CD44 expression within each tumor ( N = 75 with both CD81 and CD44 IHC). Error bars represent standard deviation. Two-tailed Student T -test p = 0.03 and 0.001 showing triple negative breast cancer (TNBC) with a higher overlap between CD81/CD44 than the average level of all subtypes as well as that of the Luminal A/B subtype.

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Immunohistochemical staining, Staining, Expressing, Microarray, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: ( A ) Forward scatter channel (FSC)-gated singles and clusters of WT and CD44KO MDA-MB-231 cells in suspension ( n = 3 replicates). N = 3. Error bars represent standard deviation. T -Test p = 0.0038. ( B ) Gating strategies for patient blood-isolated CD45 − single cells and clusters for CD81 and CD44 analyses. ( C ) Plots of proportion of putative CD44 + CTC events (EpCAM +/− clusters and singles in the blood of 50 patients with metastatic breast cancer, analyzed on flow cytometer). N = 50. Error bars represent standard deviation. Two-tailed Student T -test *******p = 2.79e-13. ( D ) mRNA expression of CD81 in single/clusters of CTCs from a publicly available dataset ( and ). Error bars represent standard deviation. Wilcoxon **p for CTC-single versus CTC-cluster = 0.0034.

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Suspension, Standard Deviation, Isolation, Flow Cytometry, Two Tailed Test, Expressing

Journal: eLife

Article Title: Machine learning-assisted elucidation of CD81–CD44 interactions in promoting cancer stemness and extracellular vesicle integrity

doi: 10.7554/eLife.82669

Figure Lengend Snippet: Schematic summary of CD81 in interacting with CD44 on the cytoplasmic membrane of tumor-initiating cells (TIC), facilitating the exosome cargo packaging with CD44 and CD81, promoting exosome-induced self-renewal in recipient cells via phosphoreactome pathways, and strengthening CD44-mediated circulating tumor cell (CTC) cluster formation and lung metastasis.

Article Snippet: For overexpression experiments in HEK293ft cells, pCMV6-FLAG- CD44 (OriGene), pCMV3-HA- CD81 (Sino Biological HG14244-NY), pCMV3-SP-N-HA (Sino Biological CV021), pDUAL GFP (addgene #86980; RRID: Addgene_86980 ), pDUAL e-GFP (addgene #63215; RRID: Addgene_63215 ) plasmids were transfected into cells using Fugene HD (Promega E231A) or jetPRIME (Polyplus 114-01).

Techniques: Membrane

Journal: Frontiers in Pharmacology

Article Title: Effect of gentiopicroside on endogenous formaldehyde homocysteine–pathway related proteins in rats with non-alcoholic steatohepatitis

doi: 10.3389/fphar.2025.1700101

Figure Lengend Snippet: Expression levels of CBS, ALDH2, AHCY, MAT1A and MTHFR proteins in liver tissues of rats in each group. (A) Expression levels of MAT1A protein in liver tissues of rats in each group (n = 3); (B) Expression levels of AHCY protein in liver tissues of rats in each group (n = 3); (C) Expression levels of MYHFR protein in liver tissues of rats in each group (n = 3); (D) Expression levels of ALDH2 protein in liver tissues of rats in each group (n = 3); (E) Expression levels of CBS protein in liver tissues of rats in each group (n = 3). Note: Data are expressed as the mean ± SD. “#” indicates that P < 0.05 compared with the normal group; “*” indicates that P < 0.05 compared with the model group.

Article Snippet: ROS, GSH, MDA, SOD, SAH, SAM, GST, CAT, GSH, HCY were obtained from Jiangsu Enzyme Immunoassay Biotechnology Co., LTD. Their item numbers are MM-88564O1, MM-20251R1, MM-2037H1, MM-20387R1, MM-50456H1, MM-0248H1, MM-21254R1, MM-20447R1, MM-20251R1 and MM-50456H1. β-actin antibody (Proteintech, 20536-1-AP); ALDH2 antibody (Proteintech, 15310-1-AP); MAT1A antibody (Proteintech, 12395-1-AP); MTHFR antibody (Proteintech, 26591-1-AP); CBS antibody (Proteintech, 14787-1-AP); AHCY antibody (Proteintech, 10757-2-AP); PVDF membrane with a thickness of 0.45 μm (IPVH00010, Millipore); Universal total RNA extraction reagent (U7431, Suzhou Youyi Landi Biotechnology Co., LTD.) Taq SYBR® Green qPCR Premix (Universal) (EG0117M, Jiangsu Bishi Mei Biotechnology Co., LTD.) All-in-One First-Strand Synthesis MasterMix (with dsDNase) was obtained from Jiangsu Bestmate Biotechnology Co., LTD. (item number (EG15133S).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Effect of gentiopicroside on endogenous formaldehyde homocysteine–pathway related proteins in rats with non-alcoholic steatohepatitis

doi: 10.3389/fphar.2025.1700101

Figure Lengend Snippet: Expression levels of CBS, ALDH2, AHCY, MAT1A and MTHFRmRNA in liver tissues of rats in each group (A) Expression levels of ALDH2 mRNA in liver tissues of rats in each group (n = 3); (B) Expression levels of MTHFRmRNA in liver tissues of rats in each group (n = 3); (C) Expression levels of CBS mRNA in liver tissues of rats in each group (n = 3); (D) Expression levels of AHCY mRNA in liver tissues of rats in each group (n = 3); (E) Expression levels of MAT1A mRNA in liver tissues of rats in each group (n = 3). Note: “#” indicates that P < 0.05 compared with the normal group; “*” indicates that P < 0.05 compared with the model group.

Article Snippet: ROS, GSH, MDA, SOD, SAH, SAM, GST, CAT, GSH, HCY were obtained from Jiangsu Enzyme Immunoassay Biotechnology Co., LTD. Their item numbers are MM-88564O1, MM-20251R1, MM-2037H1, MM-20387R1, MM-50456H1, MM-0248H1, MM-21254R1, MM-20447R1, MM-20251R1 and MM-50456H1. β-actin antibody (Proteintech, 20536-1-AP); ALDH2 antibody (Proteintech, 15310-1-AP); MAT1A antibody (Proteintech, 12395-1-AP); MTHFR antibody (Proteintech, 26591-1-AP); CBS antibody (Proteintech, 14787-1-AP); AHCY antibody (Proteintech, 10757-2-AP); PVDF membrane with a thickness of 0.45 μm (IPVH00010, Millipore); Universal total RNA extraction reagent (U7431, Suzhou Youyi Landi Biotechnology Co., LTD.) Taq SYBR® Green qPCR Premix (Universal) (EG0117M, Jiangsu Bishi Mei Biotechnology Co., LTD.) All-in-One First-Strand Synthesis MasterMix (with dsDNase) was obtained from Jiangsu Bestmate Biotechnology Co., LTD. (item number (EG15133S).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Effect of gentiopicroside on endogenous formaldehyde homocysteine–pathway related proteins in rats with non-alcoholic steatohepatitis

doi: 10.3389/fphar.2025.1700101

Figure Lengend Snippet: The mechanism diagram of endogenous formaldehyde participating in one-carbon metabolism leading to homocysteine accumulation and causing NASH. Note: formaldehyde (Endogenous FA); Glutathione (GSH) Mitochondrial aldehyde dehydrogenase 2 (ALDH2); Formate; Tetrahydrofolate (THF); 5, 10-methylenetetrahydrofolate (5, 10-CH2-THF); Methylenetetrahydrofolate reductase (MTHFR); Homocysteine (HCY); S-adenosine homocysteine (SAH); Reactive oxygen species (ROS); Non-alcoholic steatohepatitis (NASH); Methionine Cystathionine -β -synthase (CBS); 5-methyltetrahydrofolate (5-CH3-THF); S-adenosylmethionine (SAM); Methionine adenosyltransferase 1A (MAT1A); S-adenosine homocysteine hydrolase (AHCY).

Article Snippet: ROS, GSH, MDA, SOD, SAH, SAM, GST, CAT, GSH, HCY were obtained from Jiangsu Enzyme Immunoassay Biotechnology Co., LTD. Their item numbers are MM-88564O1, MM-20251R1, MM-2037H1, MM-20387R1, MM-50456H1, MM-0248H1, MM-21254R1, MM-20447R1, MM-20251R1 and MM-50456H1. β-actin antibody (Proteintech, 20536-1-AP); ALDH2 antibody (Proteintech, 15310-1-AP); MAT1A antibody (Proteintech, 12395-1-AP); MTHFR antibody (Proteintech, 26591-1-AP); CBS antibody (Proteintech, 14787-1-AP); AHCY antibody (Proteintech, 10757-2-AP); PVDF membrane with a thickness of 0.45 μm (IPVH00010, Millipore); Universal total RNA extraction reagent (U7431, Suzhou Youyi Landi Biotechnology Co., LTD.) Taq SYBR® Green qPCR Premix (Universal) (EG0117M, Jiangsu Bishi Mei Biotechnology Co., LTD.) All-in-One First-Strand Synthesis MasterMix (with dsDNase) was obtained from Jiangsu Bestmate Biotechnology Co., LTD. (item number (EG15133S).

Techniques: