Journal: Gels

Article Title: Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

doi: 10.3390/gels9030243

Figure Lengend Snippet: The production yield, activity, and purification of recombinant Cripto produced in the 3D microcarriers as compared to the 2D method. ( A ) SDS-PAGE analysis of the purification steps of recombinant Cripto: lane M is the protein molecular weight marker; lane 1 is the protein solution from ultrafiltration; lane 2 is the flowthrough after the first passage through the His-tag affinity Ni-NTA resin; lane 3 is the flowthrough after the second passage through the same resin; lane 4 is the first wash step; lane 5 is the second wash step; lane 6 is the first elution from the His-tag affinity Ni-NTA resin; and lane 7 is the second elution from the same resin. The band at approximately 27 kDa is the Cripto protein (indicated by the red arrow). ( B ) Quantitative amounts of Cripto protein produced in the 3D batch (Cripto (3D) ) with HEK293 cells encapsulated in PF microcarriers and incubated in bioreactors after three rounds of harvesting are compared to the maximum amount of Cripto produced in the 2D batch (Cripto (2D) ) with HEK293 cells adherent to cell culture plates and cultured to their density threshold limits. An initial cell seeding of 3.2 × 10 6 cells was used for both techniques. ( C ) The biological activity of recombinant Cripto was compared for Cripto produced in PF microcarriers versus the 2D method by measuring binding affinity to the AlK4 receptor. Four independent experiments were carried out for the 3D system and three independent experiments were performed for the 2D cultivation method. Results are shown as mean ± S.D. *** indicates p < 0.001.

Article Snippet: Serial dilutions of the concentrated protein were put into 96-well plates coated with either mouse Cripto antibody (for total concentration) or recombinant mouse activin receptor IB/Fc (for active protein concentration) (R&D systems AF1538 and 1477-AR, respectively).

Techniques: Activity Assay, Purification, Recombinant, Produced, SDS Page, Molecular Weight, Marker, Incubation, Cell Culture, Binding Assay

Journal: Gels

Article Title: Biomanufacturing Recombinantly Expressed Cripto-1 Protein in Anchorage-Dependent Mammalian Cells Growing in Suspension Bioreactors within a Three-Dimensional Hydrogel Microcarrier

doi: 10.3390/gels9030243

Figure Lengend Snippet: The effect of Cripto produced in 3D microcarriers on C2C12 cell proliferation detected by the BrdU incorporation assay. ( A ) C2C12 myoblast proliferation was evaluated by the BrdU incorporation assay after being cultured for 48 h in serum-free medium containing Cripto produced in 3D microcarriers (Cripto (3D) ) or commercially available Cripto (Cripto (R&D) ). Also evaluated were a bFGF medium positive control and a serum-free medium negative control. ( B ) The recombinant Cripto induces myoblast proliferation in a dose-dependent pattern; increasing concentrations of Cripto (3D) and Cripto (R&D) were added to C2C12 cells, and proliferation was quantified by the BrdU incorporation assay. ( C ) Cell proliferation was further evaluated by counting the total number of live cells. The proliferative effect of Cripto (3D) was compared with that of commercial Cripto (R&D) . The data are presented as mean ± S.D. from at least three independent experiments. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

Article Snippet: Serial dilutions of the concentrated protein were put into 96-well plates coated with either mouse Cripto antibody (for total concentration) or recombinant mouse activin receptor IB/Fc (for active protein concentration) (R&D systems AF1538 and 1477-AR, respectively).

Techniques: Produced, BrdU Incorporation Assay, Cell Culture, Positive Control, Negative Control, Recombinant

Journal: Frontiers in oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: FIGURE 1 | CRIPTO (CR-1) is dynamically expressed in stem cell-enriched cultures of adenocarcinoma (AC) and squamous cell carcinoma (SCC). (A) Hematoxylin/Eosin staining of paraffin-embedded xenograft sections obtained from two SCC and two AC spheroid cultures (upper panels) and of the parental patient tumor (lower panels), 20X magnification, scale bar 50 mm. Pictures in the upper panels are representative of xenograft sections derived from three different mice. (B) Flow cytometry analysis of surface CRIPTO expression on two SCC and two AC spheroid cultures derived from patients represented in (A) and in Table S1. (C) Immunofluorescence staining for CRIPTO in combination with E-Cadherin or Prolyl 4-Hydroxylase subunit beta (P4HB) to show respectively plasmamembrane and endoplasmic reticulum localization on AC1 cells, 60X magnification, 1.8X zoom, scale bar 10 mm. (D) Values obtained from flow cytometry analysis of surface CRIPTO (indicated as percentage of positive cells) in AC1 (light purple rhombus) and SCC1 (light blue square) at the indicated days. The histogram represents the percentage of FACS-positive cells for 7-amino actinomycin D (7-AAD). FACS plots are shown in Supplemental Figure 1.

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Staining, Derivative Assay, Flow Cytometry, Expressing, Cytometry

Journal: Frontiers in oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: FIGURE 2 | CRIPTO (CR-1) expression regulates cells proliferation and stem cell gene expression in NSCLC spheroids. (A) Immunoblot analysis of CRIPTO and bActin in SCC1 spheroids transduced with the empty vector (Vector), with CRIPTO shRNA vector (CRIPTO KO) or with exogenous CRIPTO (CRIPTO-over). (B) CRIPTO and stem cell genes (OCT3/4, SOX2, NANOG) mRNA expression in SCC1 transduced with vector, CRIPTO KO or CRIPTO-over sequences. (C) ATP assay performed on SCC1 transduced with the vector, CRIPTO KO or CRIPTO-over sequences, performed 4 days after transduction (D). Cell cycle analysis of SCC1 transduced as above, performed 3 days after transduction. (E) Soft agar pictures (left; two technical replicates for each sample) and graph (right) of colony forming assay performed on control (Vector), CRIPTO KO and CRIPTO overexpressing SCC1 spheroids. The results are evaluated as colony formation in semisolid culture and expressed as normalized colony size/percentage over plated cells. (F) ELISA assay performed on SCC1 cells transduced with empty vector, CRIPTO KO sequences and CRIPTO overexpression vector. (G) Invasion/migration assay performed on vector-transduced, CRIPTO KO and CRIPTO overexpressing SCC1 cells. *P < 0.05; **P < 0.01, ***P < 0.001 by unpaired student’s t test (transduced vs vector).

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, Gene Expression, Western Blot, Transduction, Plasmid Preparation, shRNA, ATP Assay, Cell Cycle Assay, Control, Enzyme-linked Immunosorbent Assay, Over Expression, Migration

Journal: Frontiers in oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: FIGURE 3 | NSCLC subpopulations expressing high or low CRIPTO (CR-1) levels are interconvertible in vitro and in vivo. (A) FACS-based separation of CRIPTOhigh

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, In Vitro, In Vivo

Journal: Frontiers in oncology

Article Title: CRIPTO Is a Marker of Chemotherapy-Induced Stem Cell Expansion in Non-Small Cell Lung Cancer.

doi: 10.3389/fonc.2022.830873

Figure Lengend Snippet: FIGURE 4 | Chemotherapy treatment increases CRIPTO (CR-1) expression and tumor progression in NSCLC xenografts. (A) Flow cytometry analysis of CRIPTO on SCC1 cells treated with vehicle only (Vehicle) or with chemotherapeutic agents (Cis+Gem early and Cis+Gem late). Cells in the Cis+Gem samples were treated with Cisplatin 5 mM plus Gemcitabine 25 mM for 4 days then washed, replated and analyzed after 3 additional days (Cis+Gem early) or 7 additional days (Cis+Gem late). The graph shows the mean ± SD of two independent experiments. Ns, non-significant, ***P < 0.001. (B) Left: Xenograft volume of SCC1 spheroids cells treated with Vehicle (Vehicle, light blue square) or with Cisplatin plus Gemcitabine (Cis+Gem, Dark pink dots). Mean ± SEM, 6 mice/group. **P < 0.01 (two-tailed t test). Middle: representative confocal images of CRIPTO (red) and TUNEL (green) staining of Vehicle and Cis+Gem treated tumors. 40X magnification, scale bar 50 mm. Right: quantification of CRIPTO and TUNEL performed on 3 sets composed of 5 fields/group. **P < 0.01. AU, arbitrary units. (C) Tumor variation after treatment withdrawal of SCC1 spheroids treated with vehicle (Vehicle, light blue square), and Cis plus Gemcitabine (Cis+Gem, dark pink dots). Mean ± SEM, 4 mice/group. (D) qRT-PCR analysis of CRIPTO and Cyclin E mRNA expression of SCC1-derived xenografts, monitored during treatment and after treatment withdrawal at the indicated times. *P < 0.05; **P < 0.01, ***P < 0.001.

Article Snippet: Sheep anti-mouse CRIPTO Ab (R&D System, #AF1538) and mouse CRIPTO biotinylated Ab (R&D System, #BAF 1538) were used for coating and detection, respectively.

Techniques: Expressing, Flow Cytometry, Two Tailed Test, TUNEL Assay, Staining, Quantitative RT-PCR, Derivative Assay

Journal: Developmental cell

Article Title: CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

doi: 10.1016/j.devcel.2023.11.009

Figure Lengend Snippet: Figure 1. CRIPTO micro-heterogeneity in ASC population (A) Schematic representation of the experimental design. (B) Representative FACS plots showing the gating strategy. The percentage of CRIPTO-positive cells above the threshold is indicated (bottom panel).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Cripto Allophycocyanin MAb Biotechne - R&D systems Cat# FAB1538A Cripto Mouse specific Cell Signaling Technology Cat# 2818; RRID: AB_2240508 Anti-P4HB antibody [RL90] abcam Cat# ab2792; RRID: AB_303304 Pax7 Antibody Developmental Studies Hybridoma Bank (DHSB) Cat# PAX7; RRID: AB_528428 Anti-MyoD (C-20) Santa Cruz Biotechnology, Inc Cat# sc-304; RRID: AB_631992 Anti-GFP antibody abcam Cat# ab13970; RRID: AB_300798 Anti-human Ki67 Monosan Cat# MONX10283 Myogenin Antibody (F5D) Developmental Studies Hybridoma Bank (DHSB) Cat# F5D, RRID: AB_2146602 BrdU antibody [BU1/75 (ICR1)] GeneTex Cat# GTX26326; RRID: AB_1081056 Ki-67 Recombinant Rabbit Monoclonal Antibody (SP6) ThermoFisher Scientific Cat# MA5-14520; RRID: AB_10979488 Anti-Laminin Sigma-Aldrich L9393; RRID: AB_477163 Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, Fcg subclass 1 specific Jackson ImmunoResearch Cat# 115-065-205, RRID: AB_2338571 Anti-mouse Alexa Fluor 549 ThermoFisher Scientific Cat# A-21203; RRID: AB_2535789 Anti-rabbit Alexa Fluor 488 ThermoFisher Scientific Cat# A-11001; RRID: AB_2534069 Anti-rabbit Alexa Fluor 555 ThermoFisher Scientific Cat# A-31572; RRID: AB_162543 Anti-rabbit Alexa Fluor 549 ThermoFisher Scientific Cat# A-21207; RRID: AB_141637 Alexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L) ThermoFisher Scientific Cat# 703-545-155, RRID: AB_2340375 Cy 3 AffiniPure Donkey Anti-Rat IgG (H+L) Jackson ImmunoResearch Cat# 712-165-153, RRID: AB_2340667 Chemicals, peptides, and recombinant proteins Sambucus Nigra Lectin (SNA, EBL), Biotinylated Vector Laboratories Cat# B-1305-2 Rhodamine Phalloidin ThermoFisher Scientific Cat# R415 Streptavidin-Cy3 conjugated Jackson ImmunoResearch Cat# 016-160-084 Streptavidin-488 conjugated Invitrogen Cat# S32354 Streptavidin-647 conjugated Invitrogen Cat# 532357 Tamoxifene Sigma-Aldrich Cat# T5648 Cardiotoxin Latoxan Laboratory Cat# L8102 Dispase II Roche Cat# 10887800 Collagenase A Roche Cat# 10103586001 DNAse I Roche Cat# 11284932001 Hank’s Balanced Salt Solution (HBSS) Gibco Cat# 14170-088 Bovine Serum Albumin (BSA) Sigma-Aldrich Cat# A4503 Penicillin-Streptomycin Gibco Cat# 15140-122 Collagenase Sigma-Aldrich Cat# C0130 Dulbecco’s Modified Eagle’s medium (DMEM) Gibco Cat# 41965-039 Ham’s F-12 Nutrient Mix, GlutaMAX Gibco Cat# 31765-027 Pyruvate Sodium Gibco Cat# 11360-039 L-Glutamine 200mM (100x) Gibco Cat# 25030-024 (Continued on next page) e1 Developmental Cell 58, 2896–2913.e1–e6, December 18, 2023

Techniques:

Journal: Developmental cell

Article Title: CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

doi: 10.1016/j.devcel.2023.11.009

Figure Lengend Snippet: Figure 2. Transcriptional profiles of CRIPTONeg and CRIPTOPos cells and correlation between CRIPTO and PAX7 expression levels (A) Principal-component analysis (PCA) of CRIPTONeg and CRIPTOPos cells. Each dot represents individual mice. (B) Heatmap of the differential gene expression genes from individual mice (M1–M4). The color scale represents the normalized expression values of each gene.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Cripto Allophycocyanin MAb Biotechne - R&D systems Cat# FAB1538A Cripto Mouse specific Cell Signaling Technology Cat# 2818; RRID: AB_2240508 Anti-P4HB antibody [RL90] abcam Cat# ab2792; RRID: AB_303304 Pax7 Antibody Developmental Studies Hybridoma Bank (DHSB) Cat# PAX7; RRID: AB_528428 Anti-MyoD (C-20) Santa Cruz Biotechnology, Inc Cat# sc-304; RRID: AB_631992 Anti-GFP antibody abcam Cat# ab13970; RRID: AB_300798 Anti-human Ki67 Monosan Cat# MONX10283 Myogenin Antibody (F5D) Developmental Studies Hybridoma Bank (DHSB) Cat# F5D, RRID: AB_2146602 BrdU antibody [BU1/75 (ICR1)] GeneTex Cat# GTX26326; RRID: AB_1081056 Ki-67 Recombinant Rabbit Monoclonal Antibody (SP6) ThermoFisher Scientific Cat# MA5-14520; RRID: AB_10979488 Anti-Laminin Sigma-Aldrich L9393; RRID: AB_477163 Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, Fcg subclass 1 specific Jackson ImmunoResearch Cat# 115-065-205, RRID: AB_2338571 Anti-mouse Alexa Fluor 549 ThermoFisher Scientific Cat# A-21203; RRID: AB_2535789 Anti-rabbit Alexa Fluor 488 ThermoFisher Scientific Cat# A-11001; RRID: AB_2534069 Anti-rabbit Alexa Fluor 555 ThermoFisher Scientific Cat# A-31572; RRID: AB_162543 Anti-rabbit Alexa Fluor 549 ThermoFisher Scientific Cat# A-21207; RRID: AB_141637 Alexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L) ThermoFisher Scientific Cat# 703-545-155, RRID: AB_2340375 Cy 3 AffiniPure Donkey Anti-Rat IgG (H+L) Jackson ImmunoResearch Cat# 712-165-153, RRID: AB_2340667 Chemicals, peptides, and recombinant proteins Sambucus Nigra Lectin (SNA, EBL), Biotinylated Vector Laboratories Cat# B-1305-2 Rhodamine Phalloidin ThermoFisher Scientific Cat# R415 Streptavidin-Cy3 conjugated Jackson ImmunoResearch Cat# 016-160-084 Streptavidin-488 conjugated Invitrogen Cat# S32354 Streptavidin-647 conjugated Invitrogen Cat# 532357 Tamoxifene Sigma-Aldrich Cat# T5648 Cardiotoxin Latoxan Laboratory Cat# L8102 Dispase II Roche Cat# 10887800 Collagenase A Roche Cat# 10103586001 DNAse I Roche Cat# 11284932001 Hank’s Balanced Salt Solution (HBSS) Gibco Cat# 14170-088 Bovine Serum Albumin (BSA) Sigma-Aldrich Cat# A4503 Penicillin-Streptomycin Gibco Cat# 15140-122 Collagenase Sigma-Aldrich Cat# C0130 Dulbecco’s Modified Eagle’s medium (DMEM) Gibco Cat# 41965-039 Ham’s F-12 Nutrient Mix, GlutaMAX Gibco Cat# 31765-027 Pyruvate Sodium Gibco Cat# 11360-039 L-Glutamine 200mM (100x) Gibco Cat# 25030-024 (Continued on next page) e1 Developmental Cell 58, 2896–2913.e1–e6, December 18, 2023

Techniques: Expressing, Gene Expression

Journal: Developmental cell

Article Title: CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

doi: 10.1016/j.devcel.2023.11.009

Figure Lengend Snippet: Figure 4. Regenerative potential of CRIPTONeg and CRIPTOPos cells and reconstitution of naı¨ve CRIPTO micro-heterogeneity following in vivo cell transplantation (A) Schematic representation of the experimental design. (B) Representative images of mosaic recipient muscles showing tdTomato-positive myofibers (RFP-red) and double staining with GFP (green) and LAMININ (white). (C and D) Quantification of tdTomato+ myofibers number/area (C) and GFP+ cells from CRIPTONeg and CRIPTOPos donor cells normalized per mm2 (D). Scale bars: 100 mm. Data are mean ± SEM. Nuclei were counter-stained with DAPI. (n = 3). (E) Schematic representation of the experimental design. (F and G) Representative dot plots of surface CRIPTO in GFP-positive CRIPTONeg and CRIPTOPos cells used for transplantation (F) and GFP-positive ASCs (3 dpi) derived from CRIPTONeg and CRIPTOPos -transplanted mice (G). The percentage of CRIPTO-positive cells above the threshold is indicated in the dot plots.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Cripto Allophycocyanin MAb Biotechne - R&D systems Cat# FAB1538A Cripto Mouse specific Cell Signaling Technology Cat# 2818; RRID: AB_2240508 Anti-P4HB antibody [RL90] abcam Cat# ab2792; RRID: AB_303304 Pax7 Antibody Developmental Studies Hybridoma Bank (DHSB) Cat# PAX7; RRID: AB_528428 Anti-MyoD (C-20) Santa Cruz Biotechnology, Inc Cat# sc-304; RRID: AB_631992 Anti-GFP antibody abcam Cat# ab13970; RRID: AB_300798 Anti-human Ki67 Monosan Cat# MONX10283 Myogenin Antibody (F5D) Developmental Studies Hybridoma Bank (DHSB) Cat# F5D, RRID: AB_2146602 BrdU antibody [BU1/75 (ICR1)] GeneTex Cat# GTX26326; RRID: AB_1081056 Ki-67 Recombinant Rabbit Monoclonal Antibody (SP6) ThermoFisher Scientific Cat# MA5-14520; RRID: AB_10979488 Anti-Laminin Sigma-Aldrich L9393; RRID: AB_477163 Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, Fcg subclass 1 specific Jackson ImmunoResearch Cat# 115-065-205, RRID: AB_2338571 Anti-mouse Alexa Fluor 549 ThermoFisher Scientific Cat# A-21203; RRID: AB_2535789 Anti-rabbit Alexa Fluor 488 ThermoFisher Scientific Cat# A-11001; RRID: AB_2534069 Anti-rabbit Alexa Fluor 555 ThermoFisher Scientific Cat# A-31572; RRID: AB_162543 Anti-rabbit Alexa Fluor 549 ThermoFisher Scientific Cat# A-21207; RRID: AB_141637 Alexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L) ThermoFisher Scientific Cat# 703-545-155, RRID: AB_2340375 Cy 3 AffiniPure Donkey Anti-Rat IgG (H+L) Jackson ImmunoResearch Cat# 712-165-153, RRID: AB_2340667 Chemicals, peptides, and recombinant proteins Sambucus Nigra Lectin (SNA, EBL), Biotinylated Vector Laboratories Cat# B-1305-2 Rhodamine Phalloidin ThermoFisher Scientific Cat# R415 Streptavidin-Cy3 conjugated Jackson ImmunoResearch Cat# 016-160-084 Streptavidin-488 conjugated Invitrogen Cat# S32354 Streptavidin-647 conjugated Invitrogen Cat# 532357 Tamoxifene Sigma-Aldrich Cat# T5648 Cardiotoxin Latoxan Laboratory Cat# L8102 Dispase II Roche Cat# 10887800 Collagenase A Roche Cat# 10103586001 DNAse I Roche Cat# 11284932001 Hank’s Balanced Salt Solution (HBSS) Gibco Cat# 14170-088 Bovine Serum Albumin (BSA) Sigma-Aldrich Cat# A4503 Penicillin-Streptomycin Gibco Cat# 15140-122 Collagenase Sigma-Aldrich Cat# C0130 Dulbecco’s Modified Eagle’s medium (DMEM) Gibco Cat# 41965-039 Ham’s F-12 Nutrient Mix, GlutaMAX Gibco Cat# 31765-027 Pyruvate Sodium Gibco Cat# 11360-039 L-Glutamine 200mM (100x) Gibco Cat# 25030-024 (Continued on next page) e1 Developmental Cell 58, 2896–2913.e1–e6, December 18, 2023

Techniques: In Vivo, Transplantation Assay, Muscles, Double Staining, Staining, Derivative Assay

Journal: Developmental cell

Article Title: CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

doi: 10.1016/j.devcel.2023.11.009

Figure Lengend Snippet: Figure 5. Cellular mechanisms of CRIPTO micro-heterogeneity reconstitution (A) Schematic representation of the experimental design. GFP-positive CRIPTONeg and CRIPTOPos ASCs were incubated at RT ± aCRIPTO-APC antibody (line a) or ± U73122 (line b) and stained with aCRIPTO-APC.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Cripto Allophycocyanin MAb Biotechne - R&D systems Cat# FAB1538A Cripto Mouse specific Cell Signaling Technology Cat# 2818; RRID: AB_2240508 Anti-P4HB antibody [RL90] abcam Cat# ab2792; RRID: AB_303304 Pax7 Antibody Developmental Studies Hybridoma Bank (DHSB) Cat# PAX7; RRID: AB_528428 Anti-MyoD (C-20) Santa Cruz Biotechnology, Inc Cat# sc-304; RRID: AB_631992 Anti-GFP antibody abcam Cat# ab13970; RRID: AB_300798 Anti-human Ki67 Monosan Cat# MONX10283 Myogenin Antibody (F5D) Developmental Studies Hybridoma Bank (DHSB) Cat# F5D, RRID: AB_2146602 BrdU antibody [BU1/75 (ICR1)] GeneTex Cat# GTX26326; RRID: AB_1081056 Ki-67 Recombinant Rabbit Monoclonal Antibody (SP6) ThermoFisher Scientific Cat# MA5-14520; RRID: AB_10979488 Anti-Laminin Sigma-Aldrich L9393; RRID: AB_477163 Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, Fcg subclass 1 specific Jackson ImmunoResearch Cat# 115-065-205, RRID: AB_2338571 Anti-mouse Alexa Fluor 549 ThermoFisher Scientific Cat# A-21203; RRID: AB_2535789 Anti-rabbit Alexa Fluor 488 ThermoFisher Scientific Cat# A-11001; RRID: AB_2534069 Anti-rabbit Alexa Fluor 555 ThermoFisher Scientific Cat# A-31572; RRID: AB_162543 Anti-rabbit Alexa Fluor 549 ThermoFisher Scientific Cat# A-21207; RRID: AB_141637 Alexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L) ThermoFisher Scientific Cat# 703-545-155, RRID: AB_2340375 Cy 3 AffiniPure Donkey Anti-Rat IgG (H+L) Jackson ImmunoResearch Cat# 712-165-153, RRID: AB_2340667 Chemicals, peptides, and recombinant proteins Sambucus Nigra Lectin (SNA, EBL), Biotinylated Vector Laboratories Cat# B-1305-2 Rhodamine Phalloidin ThermoFisher Scientific Cat# R415 Streptavidin-Cy3 conjugated Jackson ImmunoResearch Cat# 016-160-084 Streptavidin-488 conjugated Invitrogen Cat# S32354 Streptavidin-647 conjugated Invitrogen Cat# 532357 Tamoxifene Sigma-Aldrich Cat# T5648 Cardiotoxin Latoxan Laboratory Cat# L8102 Dispase II Roche Cat# 10887800 Collagenase A Roche Cat# 10103586001 DNAse I Roche Cat# 11284932001 Hank’s Balanced Salt Solution (HBSS) Gibco Cat# 14170-088 Bovine Serum Albumin (BSA) Sigma-Aldrich Cat# A4503 Penicillin-Streptomycin Gibco Cat# 15140-122 Collagenase Sigma-Aldrich Cat# C0130 Dulbecco’s Modified Eagle’s medium (DMEM) Gibco Cat# 41965-039 Ham’s F-12 Nutrient Mix, GlutaMAX Gibco Cat# 31765-027 Pyruvate Sodium Gibco Cat# 11360-039 L-Glutamine 200mM (100x) Gibco Cat# 25030-024 (Continued on next page) e1 Developmental Cell 58, 2896–2913.e1–e6, December 18, 2023

Techniques: Incubation, Staining

Journal: Developmental cell

Article Title: CRIPTO-based micro-heterogeneity of mouse muscle satellite cells enables adaptive response to regenerative microenvironment.

doi: 10.1016/j.devcel.2023.11.009

Figure Lengend Snippet: Figure 7. CRIPTO micro-heterogeneity preserves the balance between self-renewal and myogenic commitment (A) Representative pictures of PAX7 (magenta) and GFP (green) staining (upper panel) and quantification of PAX7+/GFP± cells (bottom panel) in Criptowt and CriptocKO muscle sections. Inset shows higher magnification (103) of the boxed area. Scale bars: 75 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse Cripto Allophycocyanin MAb Biotechne - R&D systems Cat# FAB1538A Cripto Mouse specific Cell Signaling Technology Cat# 2818; RRID: AB_2240508 Anti-P4HB antibody [RL90] abcam Cat# ab2792; RRID: AB_303304 Pax7 Antibody Developmental Studies Hybridoma Bank (DHSB) Cat# PAX7; RRID: AB_528428 Anti-MyoD (C-20) Santa Cruz Biotechnology, Inc Cat# sc-304; RRID: AB_631992 Anti-GFP antibody abcam Cat# ab13970; RRID: AB_300798 Anti-human Ki67 Monosan Cat# MONX10283 Myogenin Antibody (F5D) Developmental Studies Hybridoma Bank (DHSB) Cat# F5D, RRID: AB_2146602 BrdU antibody [BU1/75 (ICR1)] GeneTex Cat# GTX26326; RRID: AB_1081056 Ki-67 Recombinant Rabbit Monoclonal Antibody (SP6) ThermoFisher Scientific Cat# MA5-14520; RRID: AB_10979488 Anti-Laminin Sigma-Aldrich L9393; RRID: AB_477163 Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, Fcg subclass 1 specific Jackson ImmunoResearch Cat# 115-065-205, RRID: AB_2338571 Anti-mouse Alexa Fluor 549 ThermoFisher Scientific Cat# A-21203; RRID: AB_2535789 Anti-rabbit Alexa Fluor 488 ThermoFisher Scientific Cat# A-11001; RRID: AB_2534069 Anti-rabbit Alexa Fluor 555 ThermoFisher Scientific Cat# A-31572; RRID: AB_162543 Anti-rabbit Alexa Fluor 549 ThermoFisher Scientific Cat# A-21207; RRID: AB_141637 Alexa Fluor 488 AffiniPure Donkey Anti-Chicken IgY (IgG) (H+L) ThermoFisher Scientific Cat# 703-545-155, RRID: AB_2340375 Cy 3 AffiniPure Donkey Anti-Rat IgG (H+L) Jackson ImmunoResearch Cat# 712-165-153, RRID: AB_2340667 Chemicals, peptides, and recombinant proteins Sambucus Nigra Lectin (SNA, EBL), Biotinylated Vector Laboratories Cat# B-1305-2 Rhodamine Phalloidin ThermoFisher Scientific Cat# R415 Streptavidin-Cy3 conjugated Jackson ImmunoResearch Cat# 016-160-084 Streptavidin-488 conjugated Invitrogen Cat# S32354 Streptavidin-647 conjugated Invitrogen Cat# 532357 Tamoxifene Sigma-Aldrich Cat# T5648 Cardiotoxin Latoxan Laboratory Cat# L8102 Dispase II Roche Cat# 10887800 Collagenase A Roche Cat# 10103586001 DNAse I Roche Cat# 11284932001 Hank’s Balanced Salt Solution (HBSS) Gibco Cat# 14170-088 Bovine Serum Albumin (BSA) Sigma-Aldrich Cat# A4503 Penicillin-Streptomycin Gibco Cat# 15140-122 Collagenase Sigma-Aldrich Cat# C0130 Dulbecco’s Modified Eagle’s medium (DMEM) Gibco Cat# 41965-039 Ham’s F-12 Nutrient Mix, GlutaMAX Gibco Cat# 31765-027 Pyruvate Sodium Gibco Cat# 11360-039 L-Glutamine 200mM (100x) Gibco Cat# 25030-024 (Continued on next page) e1 Developmental Cell 58, 2896–2913.e1–e6, December 18, 2023

Techniques: Staining

Journal: Cell stem cell

Article Title: Cripto regulates hematopoietic stem cells as a hypoxic-niche-related factor through cell surface receptor GRP78.

doi: 10.1016/j.stem.2011.07.016

Figure Lengend Snippet: Figure 1. Cripto Is Highly Expressed in LT-HSCs and Maintains the Reconstitution Ability of HSCs after Ex Vivo Culture (A) Quantitative Real-Time PCR analysis for Cripto expression in LT-HSC (CD34Flt3KSL), ST-HSC (CD34+Flt3KSL), LMPP (CD34+Flt3+KSL), Lineage, and Lineage+ cells. Each value is normalized to HPRT expression and mean ± SD are shown. Significance is calculated against LT-HSC (n = 3). (B) Cell count for cultured cells with or without rmCripto. Thirty CD34KSL cells were directly sorted into SFEM media supplemented with 100 ng/ml of mSCF and hTPO with or without 500 ng/ml of rmCripto in 96-well plates. Absolute cell number was counted at day 7 (left) and day 14 (right) using Trypan blue (mean ± SD, n = 4). (C) Colony forming unit assay (CFU assay) for cultured cells. All colony assays were initiated by plating 30 CD34KSL cells and scoring colonies at day 7 (left) and day 14 (right) (mean ± SD, n = 8). The number of GEMM mixed colonies was significantly increased after treatment with rmCripto (day 7: p = 0.0473, day 14: p = 0.0422). GEMM: granulocytes, erythrocytes, macrophages, with or without megakaryocytes; E: erythrocytes; GM: granulocytes and macrophages; G: granulocytes; M: macrophages. (D) Competitive repopulation assay using fresh or cultured cells. Results from 4 weeks (left) and 16 weeks (right) after transplantation are shown (n = 10). Each circle indicates one mouse and red bars indicate average chimerism. (E) Secondary transplantation. Each circle indicates one mouse and red bars indicate average chimerism. Results from 12 weeks after transplantation are shown (n = 8).

Article Snippet: 342 Cell Stem Cell 9, 330–344, October 7, 2011 a2011 Elsevier Inc. Recombinant Protein and Blocking Antibody Recombinant mouse Cripto was obtained from R&D Systems .

Techniques: Ex Vivo, Real-time Polymerase Chain Reaction, Expressing, Cell Counting, Cell Culture, Colony-forming Unit Assay, Transplantation Assay

Journal: Cell stem cell

Article Title: Cripto regulates hematopoietic stem cells as a hypoxic-niche-related factor through cell surface receptor GRP78.

doi: 10.1016/j.stem.2011.07.016

Figure Lengend Snippet: Figure 2. GRP78 Is a Functional Receptor for Cripto and Can Distinguish between Different Types of HSC Subsets (A) GRP78 expression on CD34KSL cells. Flow cytometry analysis revealed two clearly separated populations in the CD34KSL compartment. A representative profile is shown. (B) CFU assay for GRP78+ and GRP78HSCs cultured with or without rmCripto and/or N-20. All colony assays were initiated by plating 30 GRP78+ or GRP78HSCs and colonies were scored at day 7 (mean ± SD, n = 4). The number of GEMM mixed colonies was significantly increased only when GRP78+HSCs were treated with rmCripto (p = 0.0013). (C) Representative pictures of cultured GRP78+HSCs from (B). Black bars indicate 1 mm. (D) Competitive reconstitution assay using GRP78+ and GRP78HSCs. Chimerism in PB is shown 1 to 4 months after transplantation from engrafted mice with fresh cells or cultured cells (mean ± SD, *p < 0.001, **p < 0.05, n = 8). Cr: rmCripto; N: N-20. (E) Lineage distribution in mice transplanted with freshly isolated GRP78+ or GRP78HSCs. Results from 4 weeks (above) and 16 weeks (below) are shown. Each bar represents analysis from one mouse.

Article Snippet: 342 Cell Stem Cell 9, 330–344, October 7, 2011 a2011 Elsevier Inc. Recombinant Protein and Blocking Antibody Recombinant mouse Cripto was obtained from R&D Systems .

Techniques: Functional Assay, Expressing, Flow Cytometry, Colony-forming Unit Assay, Cell Culture, Reconstitution Assay, Transplantation Assay, Isolation

Journal: Cell stem cell

Article Title: Cripto regulates hematopoietic stem cells as a hypoxic-niche-related factor through cell surface receptor GRP78.

doi: 10.1016/j.stem.2011.07.016

Figure Lengend Snippet: Figure 3. Cripto Signaling Is Involved in Akt Pathway and Upregulates Glycolysis-Related Proteins (A) Intracellular staining of phosphorylated Akt (Ser473) in GRP78+HSCs and GRP78HSCs treated with or without rmCripto. Representative flow cytometry patterns are shown. Black lines in Fresh panel mean starting materials. Blue lines show control condition (mSCF and hTPO) and red lines show rmCripto-treated samples. Each faint-colored line represents isotype controls. Analyses were performed after 15 min, 90 min, 12 hr, and 36 hr in culture. (B) Intracellular staining of phosphorylated 4E-BP1 (Thr37/46). (C) Intracellular staining of phosphorylated S6 (Ser235/236). (D) Intracellular staining of phosphorylated Src (Tyr416). (E) Intracellular staining of Smad2/3 (Ser423/425). (F) Phosphorylated protein levels in Cripto-treated or nontreated GRP78+/GRP78HSCs. All data represent mean fluorescence intensity (MFI) ± SD (n = 4, *p < 0.05, **p < 0.01).

Article Snippet: 342 Cell Stem Cell 9, 330–344, October 7, 2011 a2011 Elsevier Inc. Recombinant Protein and Blocking Antibody Recombinant mouse Cripto was obtained from R&D Systems .

Techniques: Staining, Cytometry, Control

Journal: Cell stem cell

Article Title: Cripto regulates hematopoietic stem cells as a hypoxic-niche-related factor through cell surface receptor GRP78.

doi: 10.1016/j.stem.2011.07.016

Figure Lengend Snippet: Figure 5. Endosteal Niche Component Cells Express Cripto and Play a Critical Role for Maintenance of Dormant HSCs (A) Flow cytometry analysis of cell surface Cripto on bone-associated niche cells. Cells isolated from bone fragments were subdivided into three different populations based on the expression of ALCAM and Sca-I within the CD31CD45Ter119 fraction, and the cell-membrane-associated form of Cripto protein on each subpopulation was detected using anti-Cripto antibody. (B) Experimental plan for experiments using N-20 antibody injection in vivo. N-20 or control goat IgG (500 mg/kg) was injected into 8-week-old C57BL/6 female mice five times in 2 weeks. (C) Flow cytometry analysis of GRP78+ and GRP78HSCs in cHSCs from injected mice. The means ± SD percentage of GRP78+CD34 or GRP78CD34 cells in the KSL population are shown (n = 4). (D) Flow cytometry analysis for GRP78+ and GRP78HSCs in eHSCs of injected mice. The means ± SD percentage of GRP78+CD34 or GRP78CD34 cells in the KSL population are shown (n = 4). See also Figure S4.

Article Snippet: 342 Cell Stem Cell 9, 330–344, October 7, 2011 a2011 Elsevier Inc. Recombinant Protein and Blocking Antibody Recombinant mouse Cripto was obtained from R&D Systems .

Techniques: Flow Cytometry, Isolation, Expressing, Membrane, Injection, In Vivo, Control

Journal: Cell stem cell

Article Title: Cripto regulates hematopoietic stem cells as a hypoxic-niche-related factor through cell surface receptor GRP78.

doi: 10.1016/j.stem.2011.07.016

Figure Lengend Snippet: Figure 6. HIF-1a Knockout Mice Contain Reduced Frequency of GRP78+HSCs and Impaired Expression of Cripto (A) Comparable frequency of the central GRP78+HSCs in HIF-1a cKO mice and controls. Representative FACS pattern of HIF-1a+/+ (left) and HIF-1aD/D (right) mice are shown. All data represent gating on the KSL population. (B) Reduced frequency of the endosteal GRP78+HSCs in HIF-1a cKO mice. Representative FACS pattern of HIF-1a+/+ (left) and HIF-1aD/D (right) mice are shown. All data represent gating on the KSL population. (C) Reduced frequency of GRP78+HSCs in HIF-1a cKO mice. The graph shows mean ± SD (n = 3). (D) Decreased expression of Cripto mRNA in KSL population of HIF-1a cKO mice. The graph shows mean ± SD (n = 3). (E)DecreasedfrequencyoftheendostealnichepopulationsofHIF-1acKOmice.RepresentativeFACSpatternsofHIF-1a+/+(left)andHIF-1aD/D(right)miceareshown.

Article Snippet: 342 Cell Stem Cell 9, 330–344, October 7, 2011 a2011 Elsevier Inc. Recombinant Protein and Blocking Antibody Recombinant mouse Cripto was obtained from R&D Systems .

Techniques: Knock-Out, Expressing