Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: The genome sequence flanking the gRNA targeting sites were sequenced and the most abundant indel mutations and their frequency is listed. The ALDH3B2 gRNA targeting site is labelled in red.

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: (a) , Western blot shows that ALDH3B2 protein level is reduced in ALDH3B2 mutant PANC-1 cells compared to control NTC-PANC-1 cells. (b) , quantification of (a) . (c), Western blot shows that ALDH3B2 protein level is reduced only in PANC-1 cells with short hairpin RNA against ALDH3B2 (shALDH3B2) when treated with Doxycycline. (d) , quantification of (c) . (e), Western blot shows that ALDH3B2 protein level is reduced in ALDH3B2 mutant HPPD cells compared to control NTC-HPPD cells. (b) , quantification of (a) .

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Western Blot, Mutagenesis, Control, shRNA

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: a-d: qPCR analysis of human beta cell and duct signature genes. Data show mean ± SEM of n=4 biological replicates per condition and are representative of 2–3 independent experiments. *p < 0.05, **p < 0.01, calculated by two-way ANOVA with Sidak’s multiple comparisons test. a, Genes related to pancreatic endocrine hormones. b, Genes related to beta cell key transcription factors. c, Genes related to beta cell function genes. d, Genes related to pancreatic duct cell markers. e, Insulin and C-peptide immunofluorescence in NTC cells and ALDH3B2 mut -PANC-1 cells. f: Insulin content in NTC cells and ALDH3B2 mut -PANC-1 cells were measured by insulin ELISA, normalized by cells genomic DNA content. n=7 biological replicates per condition and genotype. Data show mean ± SEM, **p < 0.01. g , Electron microscopy (EM) analysis of NTC-PANC-1 cells and ALDH3B2 mut -PANC-1 cells. Insulin secretion vesicles are indicated by arrow heads. h , An illustration of the in vivo function evaluation of trans-differentiated PANC-1 cells, and random blood glucose levels in the NTC and ALDH3B2 mut -PANC-1 cells transplanted diabetic NSG mice over 8 weeks of monitoring. Data show mean ± SEM of n=5 mice (NTC-PANC-1) and n=6 mice (ALDH3B2 mut -PANC-1) per group and are representative of three experiments, calculated by two-way ANOVA with Tukey’s multiple comparisons test. i, An Intraperitoneal glucose tolerance test (IPGTT) in NTC or ALDH3B2 mut -PANC-1 cells transplanted NSG mice 6 weeks post-transplantation. j, Serum human insulin levels measurement in the NTC and ALDH3B2 mut -PANC-1 cells transplanted diabetic NSG mice. k, Pdx1 and Insulin immunofluorescence in transplanted NTC cells and ALDH3B2 mut -PANC-1 cells. l, Nkx6.1 and Insulin immunofluorescence in transplanted NTC cells and ALDH3B2 mut -PANC-1 cells. m-o, Percentage of Pdx1 + (m) , Insulin + (n) , Nkx6.1 + (o) cells in all CK19 + cells. p, Percentage of Insulin + cells in all Pdx1 + cells.

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Cell Function Assay, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Electron Microscopy, In Vivo, Transplantation Assay

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: (a) Compared to control NTC-PANC-1 cells, the ALDH3B2 mutant PANC-1 cells has higher basal level insulin secretion (2.8mM), and increased insulin secretion upon high glucose treatment (16.7mM). Data show mean ± SEM of n=4. * p<0.05, ****p < 0.001. (b) ALDH3B2 mutant HPPD cells has higher basal level insulin secretion (2.8mM) than control NTC-HPPD cells, and mildly responds to high glucose treatment by increasing their insulin secretion (16.7mM). Human islets are included as a reference. Data show mean ± SEM of n=3. * p<0.05, ****p < 0.001.

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Control, Mutagenesis

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: Pdx1 and C-Peptide immunofluorescence (a) , Nkx6.1 and C-Peptide immunofluorescence (b) , Insulin, Glucagon and Somatostatin immunofluorescence (c) and Insulin Amylase and Sox9 immunofluorescence (d) in transplanted NTC and ALDH3B2 mut -PANC-1 cells.

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: a, Generation of inducible non-targeting (shControl) cells and shALDH3B2-PANC-1 cells, and ALDH3B2 expression is significantly reduced in shALDH3B2 PANC-1 cells with Doxycycline treatment shown by qPCR analysis. b-e: qPCR analysis of human beta cell and duct signature genes. Data show mean ± SEM of n=4 biological replicates per condition and are representative of 2–3 independent experiments. *p < 0.05, **p < 0.01, calculated by two-way ANOVA with Sidak’s multiple comparisons test. b, Genes related to beta cell key transcription factors. c, Genes related to pancreatic endocrine hormones. d, Genes related to beta cell function genes. e, Genes related to pancreatic duct cell markers. f, Insulin and C-peptide immunofluorescence imaging in shControl and shALDH3B2 PANC-1 cells with or without Doxycycline treatment.

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Expressing, Cell Function Assay, Immunofluorescence, Imaging

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: a-c: qPCR analysis of human Insulin (a) , KRT19 (b) and ALDH3B2 (c) expression in purified human primary pancreatic duct cells (HPPD) and human primary islets. Data show mean ± SEM of n=3. ***p < 0.005. d-g , qPCR analysis of human beta cell and duct signature genes. Data show mean ± SEM of n=3 biological replicates per condition and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, calculated by two-way ANOVA with Sidak’s multiple comparisons test. d, Genes related to beta cell key transcription factors. e, Genes related to pancreatic endocrine hormones. f, Genes related to beta cell function genes. g, Genes related to pancreatic duct cell markers. h, Insulin and CK19 immunofluorescence in NTC-HPPD and ALDH3B2 mut -HPPD cells. i , Electron microscopy (EM) analysis of NTC-HPPD cells, ALDH3B2 mut -HPPD cells and human primary islet cells. Arrow heads points to insulin secretion vesicles.

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Expressing, Purification, Cell Function Assay, Immunofluorescence, Electron Microscopy

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: a, An illustration of the in vivo function evaluation of NTC-HPPD cells and ALDH3B2 mut -HPPD cells, and random blood glucose levels in the NTC-HPPD cells and ALDH3B2 mut -HPPD cells transplanted diabetic NSG mice for 60 days of monitoring. Data show mean ± SEM of n=5 mice per group, calculated by two-way ANOVA with Tukey’s multiple comparisons test. b and c, Serum human insulin levels in non-transplanted NSG mice (Normal NSG), NTC-HPPD cells and ALDH3B2 mut -HPPD cells transplanted NSG mice 5 minutes after intraperitoneal (IP) glucose injection at 1 week post-transplantation (b) and 3 weeks post-transplantation (c). Calculated by two-way ANOVA with Sidak’s multiple comparisons test. * NSG vs. NTC-HPPD; # NSG vs. ALDH3B2 mut -HPPD. d , Insulin, CK19 and Cas9 (Flag) immunofluorescence of transplanted NTC-HPPD and ALDH3B2 mut -HPPD cells. e , Pdx1, CK19 and Cas9 (Flag) immunofluorescence of transplanted NTC-HPPD and ALDH3B2 mut -HPPD cells. f-i , percentage of Cas9 lentivirus transduced cells in all transplanted duct cells (f) , Insulin + cells in all Cas9 lentivirus transduced cells (g) , Pdx1 + cells in all Cas9 lentivirus transduced cells (h) and Insulin + cells in all Pdx1 + cells (i) .

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: In Vivo, Injection, Transplantation Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: a, An Intraperitoneal glucose tolerance test (IPGTT) in NTC or ALDH3B2 mut -HPPD cells transplanted NSG mice 6 weeks post-transplantation. *p < 0.05, **p < 0.01, ***p < 0.001, calculated by two-way ANOVA with Sidak’s multiple comparisons test. b, Serum human insulin levels in non-transplanted NSG mice (Normal NSG), NTC-HPPD cells and ALDH3B2 mut -HPPD cells transplanted NSG mice 5 minutes after intraperitoneal (IP) glucose injection at 3 week post-transplantation. **p < 0.01, calculated by two-way ANOVA with Sidak’s multiple comparisons test. c, Human insulin secretion index (0-5 minutes) calculated from b .

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Transplantation Assay, Injection

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: Pdx1 and Insulin immunofluorescence (a) , Pdx1 and C-Peptide immunofluorescence (b) , Nkx6.1 and insulin immunofluorescence (c) , Nkx6.1 and C-Peptide immunofluorescence (d) , Insulin Amylase and Sox9 immunofluorescence (e) , and Insulin, Glucagon and Somatostatin immunofluorescence (f) in transplanted NTC and ALDH3B2 mut -HPPD cells.

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: a, Pattern and percentage of the DNA methylation at the position +63, +127 and +139 of the human insulin gene locus in NTC or ALDH3B2 mut -PANC-1 cells. b , quantification of the DNA methylation percentage in a . c , Pattern and percentage of the DNA methylation at the position +63, +127 and +139 of the human insulin gene locus in HPPD-NTC, HPPD-ALDH3B2 mut and human primary pancreatic islet cells. d , quantification of the DNA methylation percentage in c .

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: DNA Methylation Assay

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: a-c: 13 unique cell clusters with a total of 3701 cells for the control and 4517 cells for the ALDH3B2 UMPA plots showing all identified cell cluster ( a ), insulin (INS) expression level of non-target control (NTC) ( b ) and ALDH3B2 mut ( c ) human primary pancreatic duct cells (HPPD). d , violin plot showing INS expression level of each individual cell grouped by cell cluster for NTC (left) and ALDH3B2 mut HPPD cells (right). In the ALDH3B2 mut 817 insulin-expressing cells were found, most prominently in clusters 5, 10, 11 and 12; in the NTC cells there were only 21. e , Heatmap showing indicated endocrine cell and duct cell maker gene expression of control (blue) and ALDH3B2 mut (red) HPPD cells at single cell level grouped by cell cluster. f , volcano plot showing differential expressed genes in ALDH3B2 mut HPPD cells comparing cells with high INS expression (intensity >1) vs. low INS expression (intensity <1). g , Gene set enrichment analysis showing the upregulated reactome of ALDH3B2 mut HPPD cells using the top 300 most significant upregulated genes of high INS-expressing (intensity >1) vs. low INS-expressing (intensity <1) cells. h and i , Dot plots showing average gene expression of indicated endocrine, progenitor, and duct cell marker genes in all identified cell cluster of NTC ( h ) or ALDH3B2 mut HPPD cells ( i ).

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Control, Expressing, Gene Expression, Marker

Journal: bioRxiv

Article Title: Loss-of-function of ALDH3B2 transdifferentiates human pancreatic duct cells into beta-like cells

doi: 10.1101/2024.05.13.593941

Figure Lengend Snippet: a , proportion of all identified cell cluster for non-target control (NTC, left) and ALDH3B2 mut (right) HPPD cells. UMAP plot showing all 13 identified cell cluster. b , proportion of INS and duct cell marker keratin 19 (KRT19) high (intensity >1) vs. low (intensity <1) expressing cells. c , Trajectory analysis showing calculated pseudotime (top) of indicated cell cluster (center) in comparison with insulin (INS) expression level (bottom). Black open circles represent calculated root nodes and red colored root nodes where defined as starting points. d and e, re-analysis of variable INS-expressing cluster 5 of ALDH3B2 mut -HPPD cells showing UMAP plot of INS high (intensity >1, yellow dots) and INS low (intensity <1, pink dots) on the bottom and the trajectory analysis of the transition from INS low to INS high expressing cells in calculated pseudotime on the top ( d ). Gene ontology biological processes (GOBP) analysis of INS high vs. INS low ALDH3B2 mut -HPPD cells from cluster 5 using the top 200 most significant upregulated genes ( e ). f - k , comparison of gene expression profiles and indicated pathway analysis between cluster 2 (f-h) and cluster 3 (i-k) showing high potential in beta-like cell transdifferentiation vs. cluster 4 showing no or low beta-like cell transdifferentiation capacity.

Article Snippet: Primary antibodies include ALDH3B2 (Proteintech, #15746-1-AP) and β-Actin (8H10D10) Mouse mAb (HRP Conjugate) (Cell signaling, #12262S).

Techniques: Control, Marker, Expressing, Comparison, Gene Expression

Journal: Frontiers in Pharmacology

Article Title: Dobutamine promotes the clearance of erythrocytes from the brain to cervical lymph nodes after subarachnoid hemorrhage in mice

doi: 10.3389/fphar.2022.1061457

Figure Lengend Snippet: Experimental design of the present study and gross appearances of the SAH model and dCLN drainage of Evans blue and erythrocytes. (A) We first collected dCLNs from the sham and SAH groups for hematoxylin–eosin staining (HE staining). We then compared the Evans blue concentration of dCLNs among the control, Evans blue, Evans blue + saline, and Evans blue + dobutamine groups. Finally, cerebrospinal fluid, brain tissue, dura mater, and dCLNs were isolated and collected for different analyses of the sham, SAH, SAH + saline, SAH + dobutamine, ligation + SAH, and ligation + SAH + dobutamine groups. (B) Gross appearances of the skull base in the sham group (upper left insert) and SAH model (lower left insert). The right panel shows the gross observation of dCLNs after Evans blue staining (blue arrow) and RBC injection (red arrow).

Article Snippet: A femoral venous catheter was inserted for systemic dobutamine (40 μg/kg in saline, HY-15746, MCE) administration.

Techniques: Staining, Concentration Assay, Control, Saline, Isolation, Ligation, Injection

Journal: Frontiers in Pharmacology

Article Title: Dobutamine promotes the clearance of erythrocytes from the brain to cervical lymph nodes after subarachnoid hemorrhage in mice

doi: 10.3389/fphar.2022.1061457

Figure Lengend Snippet: Dobutamine promoted the drainage of Evans blue from SAS to dCLNs. (A) Gross appearance of dCLNs (upper) and the skull base views of the brains (lower) in the control, Evan blue, Evans blue + saline, and Evans blue + dobutamine groups. (B) dCLN sections after Evans blue injection. (C) Quantitative analysis of the concentration of Evans blue in dCLNs. Data are presented as the mean ± SD ( n = 6 mice and ** p < 0.01)

Article Snippet: A femoral venous catheter was inserted for systemic dobutamine (40 μg/kg in saline, HY-15746, MCE) administration.

Techniques: Control, Saline, Injection, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Dobutamine promotes the clearance of erythrocytes from the brain to cervical lymph nodes after subarachnoid hemorrhage in mice

doi: 10.3389/fphar.2022.1061457

Figure Lengend Snippet: Dobutamine promoted the drainage of RBCs from SAS to dCLNs. (A) HE staining of RBC accumulation in the SAS in the sham, SAH, SAH + saline, SAH + dobutamine, ligation + SAH, and ligation + SAH + dobutamine groups. (B) HE staining of dCLNs showed decreased RBC accumulation in dCLNs after dobutamine administration. (C) Quantitative analysis of erythrocyte count in CSF. Data are presented as the mean ± SD ( n = 6 mice, ** p < 0.01, and *** p < 0.001). (D) Hemoglobin content of dCLNs after dobutamine administration was detected. Data are presented as the mean ± SD ( n = 6 mice and ** p < 0.01).

Article Snippet: A femoral venous catheter was inserted for systemic dobutamine (40 μg/kg in saline, HY-15746, MCE) administration.

Techniques: Staining, Saline, Ligation

Journal: Frontiers in Pharmacology

Article Title: Dobutamine promotes the clearance of erythrocytes from the brain to cervical lymph nodes after subarachnoid hemorrhage in mice

doi: 10.3389/fphar.2022.1061457

Figure Lengend Snippet: Dobutamine promoted the drainage of RBCs from the SAS to dCLNs via mLVs. (A) Representative immunofluorescence staining of RBCs and mLVs in different groups. (B) Quantitative analysis of the number of lymphatic erythrocytes per field in mLVs. Data are presented as the mean ± SD ( n = 6 mice, ** p < 0.01, and *** p < 0.001).

Article Snippet: A femoral venous catheter was inserted for systemic dobutamine (40 μg/kg in saline, HY-15746, MCE) administration.

Techniques: Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Dobutamine promotes the clearance of erythrocytes from the brain to cervical lymph nodes after subarachnoid hemorrhage in mice

doi: 10.3389/fphar.2022.1061457

Figure Lengend Snippet: Dobutamine alleviated neuronal damage and improved cognitive function after SAH. (A) Representative Nissl staining of the dobutamine-treated SAH model. (B) Quantitative analysis of the morphologically normal neuron count. Data are presented as the mean ± SD ( n = 3 mice, * p < 0.05, and *** p < 0.001). (C) Representative swimming tracks of mice in the Morris water maze test. The green point indicates the starting point, and the blue point indicates the end point. The small circle in the lower right quadrant shows the hidden platform. (D) Escape latencies in the first 4 days were recorded. Data are presented as the mean ± SD ( n = 4 mice, *** p < 0.001 when the SAH + saline group was compared to the sham group, # p < 0.05 when the SAH + dobutamine group was compared to the SAH + saline group, ## p < 0.01 when the SAH + dobutamine group was compared to the SAH + saline group, and $$$ p < 0.001 when the SAH group was compared to the ligation + SAH group). (E) Then, the time spent in the target quadrant was analyzed. Data are presented as the mean ± SD ( n = 4 mice, * p < 0.05, and ** p < 0.01). (F) Frequency of mice platform crossing was recorded ( n = 4 mice and * p < 0.05).

Article Snippet: A femoral venous catheter was inserted for systemic dobutamine (40 μg/kg in saline, HY-15746, MCE) administration.

Techniques: Staining, Saline, Ligation