Alkbh1

technology roadmap

Experimental content

cells ：BMSCs Adipose differentiation 1 μg/ml mouse optineurin (optn)-plasmid
dyeing ： Alkaline phosphatase (ALP) dyeing 、 Alizarin red staining 、Red O dyeing
qRT-PCR Western blot
Dot blot hybridization (Dotblotting)
µCT analysis
Immunofluorescence ：Alkbh1
Histochemistry and immunohistochemistry
Calcein double label analysis
ChIP assays：ChIP-qPCR assay

Image Reading 1：Alkbh1 Down regulated expression in BMSCs during aging

A： come from GTEx Of 31 In an individual's normal organization Alkbh1 Expression patterns
B： Immunofluorescence showed 3 In the bone marrow of month old rats Aklbh1（ green ） And nestin （ Red ） Co location of . scale ：50μm
C： Immunofluorescence showed that in cultured bone marrow cells Aklbh1（ green ） And nestin （ Red ） overlap . scale ：100μm
D： Microarray data before 500 individual deg Heat map of （GSE35955）
E and F：qRT-PCR and western blot Analyze youth （3 Month old ） And old age （18 Month old ） Mice （n=3）BMSCs in Alkbh1 The expression of

Image Reading 2：BMSCs in Alkbh1 The absence of leads to impaired osteogenic differentiation and enhanced adipogenic differentiation

A： Transgenic mice PCR Representative images of genotyping .
B and C： come from Prx1-Cre Of BMSCs in Alkbh1 Express qRT-PCR（B） and western blot（C） analysis ;Alkbh1fl/fl and Alkbh1fl/fl Mice （n=3）.
D： come from Prx1-Cre Of BMSCs Of ARS and ALP Representative images and quantification of staining ; Osteogenic differentiation Alkbh1fl/fl and Alkbh1fl/fl Mice （n=3）.
E–H：qRT-PCR The analysis shows that it comes from Prx1-Cre Of BMSCs in Runx2、ALP、SP7 and Bglap Transcriptional reduction of ; Osteogenic differentiation Alkbh1fl/fl Mice （n=3）.
I：Prx1-Cre Bone marrow mesenchymal stem cells oil red O Representative images and quantification of staining ;Alkbh1fl/fl and Alkbh1fl/fl Mice are differentiated by fat （n=3）. scale ：50μm.
J and K：qRT-PCR The analysis shows that it comes from Prx1-Cre Of BMSCs in Pparg and Fabp4 Increased transcription ; Adipose differentiation Alkbh1fl/fl Mice （n=3）.

Image Reading 3： Knock out BMSCs Medium Alkbh1 Lead to decreased bone mass and increased bone marrow fat

A–E：3 Months old and 15 Month old male mice （n=6–8） Representativeness of femur μCT Image and quantitative analysis .
F and G：HE Representative images of staining （F） And the quantity of adipocytes in femur （G）（n=5）. scale ：100μm.
H and I： Immunohistochemical staining of osteocalcin positive cells in femur （H） And quantitative analysis （I） A representative image of （n=5）. scale ：50μm. J and K： Representative image of trabecular bone calcein double labeling （J） and MAR（K） Quantitative analysis of （n=5）. scale ：20μm.

Image Reading 4：Alkbh1 Deletion inhibition optn The transcription of

A： Immunofluorescence showed Alkbh1 The protein is mainly located in the nucleus of mouse bone marrow mesenchymal stem cells .DAPI For blue ,Aklbh1 It's green ,nestin In red . scale ：25μm.
B： Dot blot analysis showed , come from Prx1-Cre Of BMSCs Metagenome DNA 6mA Level increase ;Alkbh1fl/fl Mice .
C： Heat map of selected genes related to bone marrow mesenchymal stem cell differentiation （GSE30561）.
D： ChIP-seq The atlas is shown in optn Promoter region Alkbh1-Flag-ChIP-seq enrichment .
E： Logo chip qPCR.Alkbh1 Signs combine optn Promoter region （n=3）.
F：N6 mA Chip qPCR. Knock out Alkbh1 Can increase the optn Promoter region N6-mA enrichment （n=3）.
G and H：optn Express qRT PCR（G） and western blot（H） analysis （n=3）.

Image Reading 5：optn The overexpression of saved Alkbh1 Knock out BMSCs Abnormal pedigree assignment

A：optn and Alkbh1 Of Western blot analysis .
B and C：ARS and ALP Representative images of staining （B） And quantitative （C）（n=3）.
D–G：qRT-PCR Analyze osteogenic differentiation BMSCs in Runx2、ALP、SP7 and Bglap The transcription of （n=3）.
H： Adipose differentiated bone marrow mesenchymal stem cells oil red O Representative images and quantification of staining （n=3）. scale ：50μm.
I and J：qRT-PCR Analyze fat differentiation BMSCs in Pparg and Fabp4 The transcription of （n=3）.

Image Reading 6： Overexpression Alkbh1 It can reduce bone loss and bone marrow fat accumulation in elderly mice

A and B：Alkbh1 Express qRT PCR（A） and western blot（B） analysis （n=3）.
C–G： Representation of the distal femur μCT Image and quantitative analysis （n=5）.
H and I：HE Representative images of staining （H） And the quantification of the number of adipocytes in the femur （I）（n=5）. scale ：100μm.
J and K： Immunohistochemical staining of osteocalcin positive cells in femur （J） And quantitative analysis （K） A representative image of （n=5）. scale ：50μm.
L and M： Representative image of trabecular bone calcein double labeling （L） and MAR（M） Quantitative analysis of （n=5）.