lenti crisprv2 52961 addgene backbone  (Addgene inc)

Journal: Cell reports

Article Title: Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration.

doi: 10.1016/j.celrep.2022.110473

Figure Lengend Snippet: Figure 1. STAT3 is expressed in tubular epithelial cells in AKI and also in interstitial cells in CKD of human and mouse kidneys (A) pSTAT3 immunostaining (red) in healthy (n = 10), AKI (acute tubular necrosis [ATN]; n = 5), and CKD (diabetic nephropathy [DN]; n = 10) paraffin sections of human kidneys. Na+ K+ ATPase (green) for tubular staining. DAPI staining (blue) for nuclei. Images were captured on a confocal microscope using 603 objective. Scale bars represent 10 mm. Percentage of pSTAT3-positive tubular epithelial cells normalized to total nuclei (n = 5–10) is shown. Percentage of pSTAT3-positive interstitial cells normalized to total nuclei (n = 5–10) is shown. p values are shown above the bars as determined by two-tailed unpaired t test. (B) STAT3 immunostaining (green) in healthy and CKD paraffin sections of human kidneys (n = 10). Images were captured on a confocal microscope using 603 objective. Scale bars represent 10 mm. White arrows show tubular and gray arrows show interstitial staining. Scale bars represent 10 mm. Fold STAT3 intensity as compared with healthy kidneys plotted after normalization to number of total nuclei (n = 10) is shown. p values are shown above the bars as determined by two-tailed unpaired t test. (C) Co-immunostaining for a-SMA (green) and pSTAT3 (red) staining on paraffin sections of human healthy and CKD kidneys (n = 10). Arrows show double- positive cells. Scale bars represent 10 mm. Images were captured on a confocal microscope using 603 objective. Fold of a-SMA and pSTAT3 double-positive cells were plotted as compared with healthy kidneys (n = 10). p values are shown above the bars as determined by two-tailed unpaired t test. (D) a-SMA (green) and pSTAT3 (red) in normal (day 0) and folic-acid-induced AKI (day 2) and fibrotic (day 14) paraffin kidney sections. Double-positive cells are shown by arrows (n = 5). Scale bars represent 10 mm. Quantitation of percentage of tubular pSTAT3-positive cells normalized to total nuclei (n = 5) is shown. Quantitation of percentage of interstitial pSTAT3-positive cells normalized to total nuclei (n = 5) is shown. Quantitation of percentage of a-SMA and pSTAT3- positive cells (n = 5). p values are shown above the bars as determined by two-tailed unpaired t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Stattic Millipore Sigma Cat#R415 IL-6 R&D Systems Cat#7270-IL-025/CF DAPI Vector laboratories Cat#H-1200-10 F-Actin Invitrogen Cat#A-12381 Cell mask Thermo Fisher Scientific Cat#C2925 Eagle’s Basal medium Thermo Fisher Scientific Cat#21010-046 BrdU Thermo Fisher Scientific Cat#B5002-5G Heparin Fisher Scientific Cat#H19 LTL Vector Laboratories Cat#NC0127927 Dual luciferase assay kit Promega Corporation Cat#E1910 Glutamine ATCC Cat#30-2214 Critical commercial assays Lightning-Link Rapid Alexa Fluor 488 Antibody Labeling Kit Novus Biologicals Cat#ab236553 Dual-Luciferase Reporter assay Promega Cat#E1910 BrdU assay kit Abcam Cat#ab126556 RNeasy Mini Kit Qiagen Cat#74104 miRNeasy Mini Kit Qiagen Cat#217084 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 TaqManTM Universal PCR Master Mix ThermoFisher Scientific Cat#4304437 Kappa Genotyping Kit Roche Cat#KK7352 Quantitative Colorimetric Urea Determination Kit Bio Assay Systems Cat#DIUR-100 PureLink Genomic DNA Mini Kit Thermo Fisher Scientific Cat#K182002 Deposited data GSM3176738 NCBI https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSM3176738 GSM3176739 NCBI https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSM3176739 Experimental models: Cell lines HEK 293 ATCC, USA Cat#CRL-1573, RRID:CVCL_0045 10T1/2 ATCC, USA Cat#CCL-226, RRID:CVCL_0190 Experimental models: Organisms/strains Stat3flox The Jackson Laboratory Cat#016923 Foxd1GC (Kobayashi et al., 2014) N/A Oligonucleotides Primers for quantitative PCR: listed in Table S1 This paper N/A Primers and sgRNAs for the construction of CRISPR plasmids: listed in Table S1 This paper N/A Recombinant DNA GAPDH Luciferase Dr. Matthias Brock N/A STAT3 Luciferase Dr. David Frank N/A lenti-CRISPRV2 Addgene Cat#52961 pCMV-VSV-G Addgene Cat#8454 pRSV-REV Addgene Cat#12253 pMDLg/pRRE Addgene Cat#12251 Software and algorithms GraphPad Prism 9 GraphPad Software RRID:SCR_002798 FIJI v1.0 (Schindelin et al., 2012) RRID:SCR_003070 (Continued on next page) Cell Reports 38, 110473, March 8, 2022 e2

Techniques: Immunostaining, Staining, Microscopy, Two Tailed Test, Quantitation Assay

Journal: Cell reports

Article Title: Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration.

doi: 10.1016/j.celrep.2022.110473

Figure Lengend Snippet: Figure 3. Foxd1 Cre-mediated depletion of Stat3 shows decreased macrophage infiltration, inflammation, decreased DNA damage signaling, and reduced cell proliferation in fibrotic kidneys (A) Co-immunostaining for F4/80 (green) and Na+ K+ ATPase (red) on the paraffin sections of mouse kidneys at 14 days post-AA treatment. Scale bars represent 10 mm. Fold change in the intensity of F4/80 normalized to nuclei and plotted in comparison with control kidneys at day 14 post-AA treatment (n = 4) is shown. p values are shown above the bars as determined by two-tailed unpaired t test. (B) FACS strategy for macrophages from control and Stat3 KO mouse kidneys at 14 days post-AA treatment. (C and D) TaqMan base quantitative RT-PCR for (C) inflammatory mediators and M1 markers and (D) M2 markers from macrophages isolated from mouse kidneys at 14 days post-AA treatment. Fold changes were plotted in comparison with control mice at 14 days post-AA treatment (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (E) Co-immunostaining of F4/80 (green) and IL-10 (red) and IL-10 (red) alone on the paraffin sections of mouse kidneys 14 days post-AA treatment. Scale bars represent 10 mm. Fold change in the intensity of IL-10 normalized to nuclei and plotted in comparison with control kidneys at day 14 post-AA treatment (n = 4) is shown. p values are shown above the bars as determined by two-tailed unpaired t test. (F) Representative immunostaining for pH2AX (green) in the control and Stat3 KO mouse paraffin kidney sections at 14 days post-AA injection. Scale bars represent 10 mm. Images were captured on a wide-field immunofluorescence microscope using 603 objective. Quantitation of interstitial and tubular pH2AX positive cells (n = 4) is shown. Fold changes were calculated by normalizing the number of pH2AX cells to number of nuclei and plotted in comparison with control mice at 14 days post-AA treatment. p values are shown above the bars as determined by two-tailed unpaired t test. (G) Co-immunostaining for PDGFRb (green) and pH2AX (red) on the paraffin sections of control mouse kidneys at 14-days post AA treatment. Scale bar rep- resents 10 mm. PDGFRb and pH2AX double-positive cells are shown by white arrows. Numbers of PDGFRb and pH2AX double-positive cells were counted and plotted (n = 4). (H) Immunostaining for Ki67 (cyan) in the mouse kidneys 14 days post-AA injection. Images were captured on a wide-field immunofluorescence microscope using 603 objective. Quantitation of interstitial and tubular Ki67-positive cells (n = 4) is shown. Scale bars represent 10 mm. Fold changes were calculated by normalizing the number of pH2AX cells to number of nuclei and plotting in comparison with control mice at 14 days post-AA treatment. p values are shown above the bars as determined by two-tailed unpaired t test. (I) Co-immunostaining for PDGFRb (green) and Ki67 (red) on the paraffin sections of control mouse kidneys at 14 days post-AA treatment. Scale bar represents 10 mm. PDGFRb and Ki67 double-positive cells are shown by white arrows. Numbers of PDGFRb and Ki67 double-positive cells were counted and plotted (n = 4).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Stattic Millipore Sigma Cat#R415 IL-6 R&D Systems Cat#7270-IL-025/CF DAPI Vector laboratories Cat#H-1200-10 F-Actin Invitrogen Cat#A-12381 Cell mask Thermo Fisher Scientific Cat#C2925 Eagle’s Basal medium Thermo Fisher Scientific Cat#21010-046 BrdU Thermo Fisher Scientific Cat#B5002-5G Heparin Fisher Scientific Cat#H19 LTL Vector Laboratories Cat#NC0127927 Dual luciferase assay kit Promega Corporation Cat#E1910 Glutamine ATCC Cat#30-2214 Critical commercial assays Lightning-Link Rapid Alexa Fluor 488 Antibody Labeling Kit Novus Biologicals Cat#ab236553 Dual-Luciferase Reporter assay Promega Cat#E1910 BrdU assay kit Abcam Cat#ab126556 RNeasy Mini Kit Qiagen Cat#74104 miRNeasy Mini Kit Qiagen Cat#217084 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 TaqManTM Universal PCR Master Mix ThermoFisher Scientific Cat#4304437 Kappa Genotyping Kit Roche Cat#KK7352 Quantitative Colorimetric Urea Determination Kit Bio Assay Systems Cat#DIUR-100 PureLink Genomic DNA Mini Kit Thermo Fisher Scientific Cat#K182002 Deposited data GSM3176738 NCBI https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSM3176738 GSM3176739 NCBI https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSM3176739 Experimental models: Cell lines HEK 293 ATCC, USA Cat#CRL-1573, RRID:CVCL_0045 10T1/2 ATCC, USA Cat#CCL-226, RRID:CVCL_0190 Experimental models: Organisms/strains Stat3flox The Jackson Laboratory Cat#016923 Foxd1GC (Kobayashi et al., 2014) N/A Oligonucleotides Primers for quantitative PCR: listed in Table S1 This paper N/A Primers and sgRNAs for the construction of CRISPR plasmids: listed in Table S1 This paper N/A Recombinant DNA GAPDH Luciferase Dr. Matthias Brock N/A STAT3 Luciferase Dr. David Frank N/A lenti-CRISPRV2 Addgene Cat#52961 pCMV-VSV-G Addgene Cat#8454 pRSV-REV Addgene Cat#12253 pMDLg/pRRE Addgene Cat#12251 Software and algorithms GraphPad Prism 9 GraphPad Software RRID:SCR_002798 FIJI v1.0 (Schindelin et al., 2012) RRID:SCR_003070 (Continued on next page) Cell Reports 38, 110473, March 8, 2022 e2

Techniques: Immunostaining, Comparison, Control, Two Tailed Test, Quantitative RT-PCR, Isolation, Injection, Microscopy, Quantitation Assay

Journal: Cell reports

Article Title: Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration.

doi: 10.1016/j.celrep.2022.110473

Figure Lengend Snippet: Figure 4. IL-6-mediated STAT3 phosphorylation regulates proliferation, migration, and profibrotic signaling in pericyte-like 10T1/2 cells (A) Representative western blots for pSTAT3 and STAT3 following treatment with IL-6 or IL-6 in combination with stattic in 10T1/2 cells. Fold changes were calculated after normalization to b-actin and plotted in comparison with control (ctrl) cells (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (B) Nuclear translocation of STAT3 in 10T1/2 cells following IL-6 and or stattic treatment. Scale bars represent 10 mm. Intensity of pSTAT3 was normalized to total number of cells, and fold change in comparison with control cells was plotted (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (C and D) Proliferation of 10T1/2 cells following IL-6 and stattic treatments using BrdU incorporation assay in a (C) time- and (D) STAT3-dependent manner (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (E) Immunofluorescence staining for Ki67 with or without IL-6 and stattic treatments in 10T1/2 cells. Scale bars represent 10 mm. Intensity of Ki67 was normalized to total number of cells, and fold change in comparison with control cells was plotted (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (F) Migration of 10T1/2 cells following IL-6 and stattic treatment using scratch assay. Scale bars represent 50 mm. Percent cell migration was calculated as compared with gap area in the control cells at 24 h (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (G) Transwell migration of 10T1/2 cells treated with IL-6 or IL-6 in combination with stattic. Scale bars represent 50 mm. Cell area normalized to cell number calculated for CellMask stain (green) and fold change were calculated as compared with control cells (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (H–K) Immunostaining for (H) pSMAD2 (green), (I) collagen 1 (green), (J) fibronectin (green), and (K) a-SMA (green) following IL-6 alone or in combination with stattic on 10T1/2 cells. Scale bars represent 10 mm. Intensity of pSMAD2, collagen 1, fibronectin, and a-SMA was normalized with number of cells, and fold change was plotted in comparison with control cells (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Stattic Millipore Sigma Cat#R415 IL-6 R&D Systems Cat#7270-IL-025/CF DAPI Vector laboratories Cat#H-1200-10 F-Actin Invitrogen Cat#A-12381 Cell mask Thermo Fisher Scientific Cat#C2925 Eagle’s Basal medium Thermo Fisher Scientific Cat#21010-046 BrdU Thermo Fisher Scientific Cat#B5002-5G Heparin Fisher Scientific Cat#H19 LTL Vector Laboratories Cat#NC0127927 Dual luciferase assay kit Promega Corporation Cat#E1910 Glutamine ATCC Cat#30-2214 Critical commercial assays Lightning-Link Rapid Alexa Fluor 488 Antibody Labeling Kit Novus Biologicals Cat#ab236553 Dual-Luciferase Reporter assay Promega Cat#E1910 BrdU assay kit Abcam Cat#ab126556 RNeasy Mini Kit Qiagen Cat#74104 miRNeasy Mini Kit Qiagen Cat#217084 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 TaqManTM Universal PCR Master Mix ThermoFisher Scientific Cat#4304437 Kappa Genotyping Kit Roche Cat#KK7352 Quantitative Colorimetric Urea Determination Kit Bio Assay Systems Cat#DIUR-100 PureLink Genomic DNA Mini Kit Thermo Fisher Scientific Cat#K182002 Deposited data GSM3176738 NCBI https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSM3176738 GSM3176739 NCBI https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSM3176739 Experimental models: Cell lines HEK 293 ATCC, USA Cat#CRL-1573, RRID:CVCL_0045 10T1/2 ATCC, USA Cat#CCL-226, RRID:CVCL_0190 Experimental models: Organisms/strains Stat3flox The Jackson Laboratory Cat#016923 Foxd1GC (Kobayashi et al., 2014) N/A Oligonucleotides Primers for quantitative PCR: listed in Table S1 This paper N/A Primers and sgRNAs for the construction of CRISPR plasmids: listed in Table S1 This paper N/A Recombinant DNA GAPDH Luciferase Dr. Matthias Brock N/A STAT3 Luciferase Dr. David Frank N/A lenti-CRISPRV2 Addgene Cat#52961 pCMV-VSV-G Addgene Cat#8454 pRSV-REV Addgene Cat#12253 pMDLg/pRRE Addgene Cat#12251 Software and algorithms GraphPad Prism 9 GraphPad Software RRID:SCR_002798 FIJI v1.0 (Schindelin et al., 2012) RRID:SCR_003070 (Continued on next page) Cell Reports 38, 110473, March 8, 2022 e2

Techniques: Phospho-proteomics, Migration, Western Blot, Comparison, Control, Two Tailed Test, Translocation Assay, BrdU Incorporation Assay, Staining, Wound Healing Assay, Immunostaining

Journal: Cell reports

Article Title: Deletion of STAT3 from Foxd1 cell population protects mice from kidney fibrosis by inhibiting pericytes trans-differentiation and migration.

doi: 10.1016/j.celrep.2022.110473

Figure Lengend Snippet: Figure 5. STAT3 directly regulates attachment, spreading, migration, proliferation, and profibrotic signaling in 10T1/2 cells (A) Scheme for STAT3 activation using synergistic activation mediators (SAMs) and activation mutants (STAT3-C). (B–F) Immunostaining for (B) pSTAT3 (green), (C) pSMAD2 (green), (D) collagen 1, (E) fibronectin, and (F) a-SMA (green) on STAT3-activated 10T1/2 cells. Scale bars represent 10 mm. Intensity of staining for pSTAT3, pSMAD2, collagen 1, fibronectin, and a-SMA were normalized with cell numbers and plotted in com- parison with control (ctrl) cells (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (G) Transwell migration of 10T1/2 cells in STAT3-activated cells. Scale bars represent 50 mm. Cell area normalized to cell number was calculated by CellMask, and fold change was plotted in comparison with control cells (n = 3). Total number of migrated cells was counted on the lower side of the polycarbonate membrane by counting the nuclei (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (H) Proliferation of Stat3 WT and KO 10T1/2 cells as assayed by BrdU incorporation assay for 5 days. Percent change was calculated in comparison with day-1 Stat3 WT cells (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (I) Adherence assay as shown by staining for F-actin (red) of Stat3 WT and Stat3 KO 10T1/2 cells at 1 h after plating. Scale bars represent 10 mm. Intensity of F-actin normalized to total cell number was calculated, and fold change were calculated in comparison with Stat3 WT cells (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test. (J) Spreading assay as shown staining for F-actin (red) of Stat3 WT and Stat3 KO 10T1/2 cells at 3 h after plating. Scale bars represent 10 mm. Cell area of F-actin normalized to total cell number and fold change were calculated in comparison with Stat3 WT cells (n = 3). p values are shown above the bars as determined by two-tailed unpaired t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins Stattic Millipore Sigma Cat#R415 IL-6 R&D Systems Cat#7270-IL-025/CF DAPI Vector laboratories Cat#H-1200-10 F-Actin Invitrogen Cat#A-12381 Cell mask Thermo Fisher Scientific Cat#C2925 Eagle’s Basal medium Thermo Fisher Scientific Cat#21010-046 BrdU Thermo Fisher Scientific Cat#B5002-5G Heparin Fisher Scientific Cat#H19 LTL Vector Laboratories Cat#NC0127927 Dual luciferase assay kit Promega Corporation Cat#E1910 Glutamine ATCC Cat#30-2214 Critical commercial assays Lightning-Link Rapid Alexa Fluor 488 Antibody Labeling Kit Novus Biologicals Cat#ab236553 Dual-Luciferase Reporter assay Promega Cat#E1910 BrdU assay kit Abcam Cat#ab126556 RNeasy Mini Kit Qiagen Cat#74104 miRNeasy Mini Kit Qiagen Cat#217084 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 TaqManTM Universal PCR Master Mix ThermoFisher Scientific Cat#4304437 Kappa Genotyping Kit Roche Cat#KK7352 Quantitative Colorimetric Urea Determination Kit Bio Assay Systems Cat#DIUR-100 PureLink Genomic DNA Mini Kit Thermo Fisher Scientific Cat#K182002 Deposited data GSM3176738 NCBI https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSM3176738 GSM3176739 NCBI https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSM3176739 Experimental models: Cell lines HEK 293 ATCC, USA Cat#CRL-1573, RRID:CVCL_0045 10T1/2 ATCC, USA Cat#CCL-226, RRID:CVCL_0190 Experimental models: Organisms/strains Stat3flox The Jackson Laboratory Cat#016923 Foxd1GC (Kobayashi et al., 2014) N/A Oligonucleotides Primers for quantitative PCR: listed in Table S1 This paper N/A Primers and sgRNAs for the construction of CRISPR plasmids: listed in Table S1 This paper N/A Recombinant DNA GAPDH Luciferase Dr. Matthias Brock N/A STAT3 Luciferase Dr. David Frank N/A lenti-CRISPRV2 Addgene Cat#52961 pCMV-VSV-G Addgene Cat#8454 pRSV-REV Addgene Cat#12253 pMDLg/pRRE Addgene Cat#12251 Software and algorithms GraphPad Prism 9 GraphPad Software RRID:SCR_002798 FIJI v1.0 (Schindelin et al., 2012) RRID:SCR_003070 (Continued on next page) Cell Reports 38, 110473, March 8, 2022 e2

Techniques: Migration, Activation Assay, Immunostaining, Staining, Control, Two Tailed Test, Comparison, Membrane, BrdU Incorporation Assay